Compositions and Methods for TTR Gene Editing and Treating ATTR Amyloidosis

Abstract

Compositions and methods for editing, e.g., introducing double-stranded breaks, within the TTR gene are provided. Compositions and methods for treating subjects having amyloidosis associated with transthyretin (ATTR), are provided.

Description

[0001] This application is a Continuation of International Application No. PCT/US2018/053382, which was filed on Sep. 28, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/556,236, which was filed on Sep. 29, 2017, and U.S. Provisional Application No. 62/671,902, which was filed on May 15, 2018, the contents of each of which are incorporated by reference in their entirety.

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 23, 2020, is named 2020-03-23_01155-0013-US_ST25.txt and is 417,533 bytes in size.

[0003] Transthyretin (TTR) is a protein produced by the TTR gene that normally functions to transport retinol and thyroxine throughout the body. TTR is predominantly synthesized in the liver, with small fractions being produced in the choroid plexus and retina. TTR normally circulates as a soluble tetrameric protein in the blood.

[0004] Pathogenic variants of TTR, which may disrupt tetramer stability, can be encoded by mutant alleles of the TTR gene. Mutant TTR may result in misfolded TTR, which may generate amyloids (i.e., aggregates of misfolded TTR protein). In some cases, pathogenic variants of TTR can lead to amyloidosis, or disease resulting from build-up of amyloids. For example, misfolded TTR monomers can polymerize into amyloid fibrils within tissues, such as the peripheral nerves, heart, and gastrointestinal tract. Amyloid plaques can also comprise wild-type TTR that has deposited on misfolded TTR.

[0005] Misfolding and deposition of wild-type TTR has also been observed in males aged 60 or more and is associated with heart rhythm problems, heart failure, and carpal tunnel.

[0006] Amyloidosis characterized by deposition of TTR may be referred to as "ATTR," "TTR-related amyloidosis," "TTR amyloidosis," or "ATTR amyloidosis," "ATTR familial amyloidosis" (when associated with a genetic mutation in a family), or "ATTRwt" or "wild-type ATTR" (when arising from misfolding and deposition of wild-type TTR).

[0007] ATTR can present with a wide spectrum of symptoms, and patients with different classes of ATTR may have different characteristics and prognoses. Some classes of ATTR include familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and wild-type TTR amyloidosis (wt-TTR amyloidosis). FAP commonly presents with sensorimotor neuropathy, while FAC and wt-TTR amyloidosis commonly present with congestive heart failure. FAP and FAC are usually associated with a genetic mutation in the TTR gene, and more than 100 different mutations in the TTR gene have been associated with ATTR. In contrast, wt-TTR amyloidosis is associated with aging and not with a genetic mutation in TTR. It is estimated that approximately 50,000 patients worldwide may be affected by FAP and FAC.

[0008] While more than 100 mutations in TTR are associated with ATTR, certain mutations have been more closely associated with neuropathy and/or cardiomyopathy. For example, mutations at T60 of TTR are associated with both cardiomyopathy and neuropathy; mutations at V30 are more associated with neuropathy; and mutations at V122 are more associated with cardiomyopathy.

[0009] A range of treatment approaches have been studied for treatment of ATTR, but there are no approved drugs that stop disease progression and improve quality of life. While liver transplant has been studied for treatment of ATTR, its use is declining as it involves significant risk and disease progression sometimes continues after transplantation. Small molecule stabilizers, such as diflunisal and tafamidis, appear to slow ATTR progression, but these agents do not halt disease progression.

[0010] Approaches using small interfering RNA (siRNA) knockdown, antisense knockdown, or a monoclonal antibody targeting amyloid fibrils for destruction are also currently being investigated, but while results on short-term suppression of TTR expression show encouraging preliminary data, a need exists for treatments that can produce long-lasting suppression of TTR.

[0011] Accordingly, the following embodiments are provided. In some embodiments, the present invention provides compositions and methods using a guide RNA with an RNA-guided DNA binding agent such as the CRISPR/Cas system to substantially reduce or knockout expression of the TTR gene, thereby substantially reducing or eliminating the production of TTR protein associated with ATTR. The substantial reduction or elimination of the production of TTR protein associated with ATTR through alteration of the TTR gene can be a long-term reduction or elimination.

SUMMARY

[0012] Embodiment 1 is a method of inducing a double-stranded break (DSB) within the TTR gene, comprising delivering a composition to a cell, wherein the composition comprises

a. a guide RNA comprising a guide sequence selected from SEQ ID NOs: 5-82; b. a guide RNA comprising at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

[0013] Embodiment 2 is a method of modifying the TTR gene comprising delivering a composition to a cell, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

[0014] Embodiment 3 is a method of treating amyloidosis associated with TTR (ATTR), comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby treating ATTR.

[0015] Embodiment 4 is a method of reducing TTR serum concentration, comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby reducing TTR serum concentration.

[0016] Embodiment 5 is a method for reducing or preventing the accumulation of amyloids or amyloid fibrils comprising TTR in a subject, comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby reducing accumulation of amyloids or amyloid fibrils.

[0017] Embodiment 6 is a composition comprising a guide RNA comprising:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

[0018] Embodiment 7 is a composition comprising a vector encoding a guide RNA, wherein the guide RNA comprises:

a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

[0019] Embodiment 8 is the composition of embodiment 6 or 7, for use in inducing a double-stranded break (DSB) within the TTR gene in a cell or subject.

[0020] Embodiment 9 is the composition of embodiment 6 or 7, for use in modifying the TTR gene in a cell or subject.

[0021] Embodiment 10 is the composition of embodiment 6 or 7, for use in treating amyloidosis associated with TTR (ATTR) in a subject.

[0022] Embodiment 11 is the composition of embodiment 6 or 7, for use in reducing TTR serum concentration in a subject.

[0023] Embodiment 12 is the composition of embodiment 6 or 7, for use in reducing or preventing the accumulation of amyloids or amyloid fibrils in a subject.

[0024] Embodiment 13 is the method of any one of embodiments 1-5 or the composition for use of any one of embodiments 8-12, wherein the composition reduces serum TTR levels.

[0025] Embodiment 14 is the method or composition for use of embodiment 13, wherein the serum TTR levels are reduced by at least 50% as compared to serum TTR levels before administration of the composition.

[0026] Embodiment 15 is the method or composition for use of embodiment 13, wherein the serum TTR levels are reduced by 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-98%, 98-99%, or 99-100% as compared to serum TTR levels before administration of the composition.

[0027] Embodiment 16 is the method or composition for use of any one of embodiments 1-5 or 8-15, wherein the composition results in editing of the TTR gene.

[0028] Embodiment 17 is the method or composition for use of embodiment 16, wherein the editing is calculated as a percentage of the population that is edited (percent editing).

[0029] Embodiment 18 is the method or composition for use of embodiment 17, wherein the percent editing is between 30 and 99% of the population.

[0030] Embodiment 19 is the method or composition for use of embodiment 17, wherein the percent editing is between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the population.

[0031] Embodiment 20 is the method of any one of embodiments 1-5 or the composition for use of any one of embodiments 8-19, wherein the composition reduces amyloid deposition in at least one tissue.

[0032] Embodiment 21 is the method or composition for use of embodiment 20, wherein the at least one tissue comprises one or more of stomach, colon, sciatic nerve, or dorsal root ganglion.

[0033] Embodiment 22 is the method or composition for use of embodiment 20 or 21, wherein amyloid deposition is measured 8 weeks after administration of the composition.

[0034] Embodiment 23 is the method or composition for use of any one of embodiments 20-22, wherein amyloid deposition is compared to a negative control or a level measured before administration of the composition.

[0035] Embodiment 24 is the method or composition for use of any one of embodiments 20-23, wherein amyloid deposition is measured in a biopsy sample and/or by immunostaining.

[0036] Embodiment 25 is the method or composition for use of any one of embodiments 20-24, wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the amyloid deposition seen in a negative control.

[0037] Embodiment 26 is the method or composition for use of any one of embodiments 20-25, wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the amyloid deposition seen before administration of the composition.

[0038] Embodiment 27 is the method or composition for use of any one of embodiments 1-5 or 8-26, wherein the composition is administered or delivered at least two times.

[0039] Embodiment 28 is the method or composition for use of embodiment 27, wherein the composition is administered or delivered at least three times.

[0040] Embodiment 29 is the method or composition for use of embodiment 27, wherein the composition is administered or delivered at least four times.

[0041] Embodiment 30 is the method or composition for use of embodiment 27, wherein the composition is administered or delivered up to five, six, seven, eight, nine, or ten times.

[0042] Embodiment 31 is the method or composition for use of any one of embodiments 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

[0043] Embodiment 32 is the method or composition for use of any one of embodiments 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks.

[0044] Embodiment 33 is the method or composition for use of any one of embodiments 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.

[0045] Embodiment 34 is the method or composition of any one of the preceding embodiments, wherein the guide sequence is selected from SEQ ID NOs: 5-82.

[0046] Embodiment 35 is the method or composition of any one of the preceding embodiments, wherein the guide RNA is at least partially complementary to a target sequence present in the human TTR gene.

[0047] Embodiment 36 is the method or composition of embodiment 35, wherein the target sequence is in exon 1, 2, 3, or 4 of the human TTR gene.

[0048] Embodiment 37 is the method or composition of embodiment 35, wherein the target sequence is in exon 1 of the human TTR gene.

[0049] Embodiment 38 is the method or composition of embodiment 35, wherein the target sequence is in exon 2 of the human TTR gene.

[0050] Embodiment 39 is the method or composition of embodiment 35, wherein the target sequence is in exon 3 of the human TTR gene.

[0051] Embodiment 40 is the method or composition of embodiment 35, wherein the target sequence is in exon 4 of the human TTR gene.

[0052] Embodiment 41 is the method or composition of any one of embodiments 1-40, wherein the guide sequence is complementary to a target sequence in the positive strand of TTR.

[0053] Embodiment 42 is the method or composition of any one of embodiments 1-40, wherein the guide sequence is complementary to a target sequence in the negative strand of TTR.

[0054] Embodiment 43 is the method or composition of any one of embodiments 1-40, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the TTR gene, and wherein the composition further comprises a second guide sequence that is complementary to a second target sequence in the negative strand of the TTR gene.

[0055] Embodiment 44 is the method or composition of any one of the preceding embodiments, wherein the guide RNA comprises a crRNA that comprises the guide sequence and further comprises a nucleotide sequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequence at its 3' end.

[0056] Embodiment 45 is the method or composition of any one of the preceding embodiments, wherein the guide RNA is a dual guide (dgRNA).

[0057] Embodiment 46 is the method or composition of embodiment 45, wherein the dual guide RNA comprises a crRNA comprising a nucleotide sequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequence at its 3' end, and a trRNA.

[0058] Embodiment 47 is the method or composition of any one of embodiments 1-43, wherein the guide RNA is a single guide (sgRNA).

[0059] Embodiment 48 is the method or composition of embodiment 47, wherein the sgRNA comprises a guide sequence that has the pattern of SEQ ID NO: 3.

[0060] Embodiment 49 is the method or composition of embodiment 47, wherein the sgRNA comprises the sequence of SEQ ID NO: 3.

[0061] Embodiment 50 is the method or composition of embodiment 48 or 49, wherein each N in SEQ ID NO: 3 is any natural or non-natural nucleotide, wherein the N's form the guide sequence, and the guide sequence targets Cas9 to the TTR gene.

[0062] Embodiment 51 is the method or composition of any one of embodiments 47-50, wherein the sgRNA comprises any one of the guide sequences of SEQ ID NOs: 5-82 and the nucleotides of SEQ ID NO: 126.

[0063] Embodiment 52 is the method or composition of any one of embodiments 47-51, wherein the sgRNA comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID Nos: 87-124.

[0064] Embodiment 53 is the method or composition of embodiment 47, wherein the sgRNA comprises a sequence selected from SEQ ID Nos: 87-124.

[0065] Embodiment 54 is the method or composition of any one of the preceding embodiments, wherein the guide RNA comprises at least one modification.

[0066] Embodiment 55 is the method or composition of embodiment 54, wherein the at least one modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide.

[0067] Embodiment 56 is the method or composition of embodiment 54 or 55, wherein the at least one modification includes a phosphorothioate (PS) bond between nucleotides.

[0068] Embodiment 57 is the method or composition of any one of embodiments 54-56, wherein the at least one modification includes a 2'-fluoro (2'-F) modified nucleotide.

[0069] Embodiment 58 is the method or composition of any one of embodiments 54-57, wherein the at least one modification includes a modification at one or more of the first five nucleotides at the 5' end.

[0070] Embodiment 59 is the method or composition of any one of embodiments 54-58, wherein the at least one modification includes a modification at one or more of the last five nucleotides at the 3' end.

[0071] Embodiment 60 is the method or composition of any one of embodiments 54-59, wherein the at least one modification includes PS bonds between the first four nucleotides.

[0072] Embodiment 61 is the method or composition of any one of embodiments 54-60, wherein the at least one modification includes PS bonds between the last four nucleotides.

[0073] Embodiment 62 is the method or composition of any one of embodiments 54-61, wherein the at least one modification includes 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end.

[0074] Embodiment 63 is the method or composition of any one of embodiments 54-62, wherein the at least one modification includes 2'-O-Me modified nucleotides at the last three nucleotides at the 3' end.

[0075] Embodiment 64 is the method or composition of any one of embodiments 54-63, wherein the guide RNA comprises the modified nucleotides of SEQ ID NO: 3.

[0076] Embodiment 65 is the method or composition of any one of embodiments 1-64, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0077] Embodiment 66 is the method or composition of any one of embodiments 1-65, wherein the guide RNA is associated with a lipid nanoparticle (LNP).

[0078] Embodiment 67 is the method or composition of embodiment 66, wherein the LNP comprises a CCD lipid.

[0079] Embodiment 68 is the method or composition of embodiment 67, wherein the CCD lipid is Lipid a or Lipid B.

[0080] Embodiment 69 is the method or composition of embodiment 66-68, wherein the LNP comprises a neutral lipid.

[0081] Embodiment 70 is the method or composition of embodiment 69, wherein the neutral lipid is DSPC

[0082] Embodiment 71 is the method or composition of any one of embodiments 66-70, wherein the LNP comprises a helper lipid.

[0083] Embodiment 72 is the method or composition of embodiment 71, wherein the helper lipid is cholesterol.

[0084] Embodiment 73 is the method or composition of any one of embodiments 66-72, wherein the LNP comprises a stealth lipid.

[0085] Embodiment 74 is the method or composition of embodiment 73, wherein the stealth lipid is PEG2k-DMG.

[0086] Embodiment 75 is the method or composition of any one of the preceding embodiments, wherein the composition further comprises an RNA-guided DNA binding agent.

[0087] Embodiment 76 is the method or composition of any one of the preceding embodiments, wherein the composition further comprises an mRNA that encodes an RNA-guided DNA binding agent.

[0088] Embodiment 77 is the method or composition of embodiment 75 or 76, wherein the RNA-guided DNA binding agent is a Cas cleavase.

[0089] Embodiment 78 is the method or composition of embodiment 77, wherein the RNA-guided DNA binding agent is Cas9.

[0090] Embodiment 79 is the method or composition of any one of embodiments 75-78, wherein the RNA-guided DNA binding agent is modified.

[0091] Embodiment 80 is the method or composition of any one of embodiments 75-79, wherein the RNA-guided DNA binding agent is a nickase.

[0092] Embodiment 81 is the method or composition of embodiment 79 or 80, wherein the modified RNA-guided DNA binding agent comprises a nuclear localization signal (NLS).

[0093] Embodiment 82 is the method or composition of any one of embodiments 75-81, wherein the RNA-guided DNA binding agent is a Cas from a Type-II CRISPR/Cas system.

[0094] Embodiment 83 is the method or composition of any one of the preceding embodiments, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.

[0095] Embodiment 84 is the method or composition for use of any one of embodiments 1-5 or 8-83, wherein the composition reduces or prevents amyloids or amyloid fibrils comprising TTR.

[0096] Embodiment 85 is the method or composition for use of embodiment 84, wherein the amyloids or amyloid fibrils are in the nerves, heart, or gastrointestinal track.

[0097] Embodiment 86 is the method or composition for use of any one of embodiments 1-5 or 8-83, wherein non-homologous ending joining (NHEJ) leads to a mutation during repair of a DSB in the TTR gene.

[0098] Embodiment 87 is the method or composition for use of embodiment 86, wherein NHEJ leads to a deletion or insertion of a nucleotide(s) during repair of a DSB in the TTR gene.

[0099] Embodiment 88 is the method or composition for use of embodiment 87, wherein the deletion or insertion of a nucleotide(s) induces a frame shift or nonsense mutation in the TTR gene.

[0100] Embodiment 89 is the method or composition for use of embodiment 87, wherein a frame shift or nonsense mutation is induced in the TTR gene of at least 50% of liver cells.

[0101] Embodiment 90 is the method or composition for use of embodiment 89, wherein a frame shift or nonsense mutation is induced in the TTR gene of 50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or 99%-100% of liver cells.

[0102] Embodiment 91 is the method or composition for use of any one of embodiments 87-90, wherein a deletion or insertion of a nucleotide(s) occurs in the TTR gene at least 50-fold or more than in off-target sites.

[0103] Embodiment 92 is the method or composition for use of embodiment 91, wherein the deletion or insertion of a nucleotide(s) occurs in the TTR gene 50-fold to 150-fold, 150-fold to 500-fold, 500-fold to 1500-fold, 1500-fold to 5000-fold, 5000-fold to 15000-fold, 15000-fold to 30000-fold, or 30000-fold to 60000-fold more than in off-target sites.

[0104] Embodiment 93 is the method or composition for use of any one of embodiments 87-92, wherein the deletion or insertion of a nucleotide(s) occurs at less than or equal to 3, 2, 1, or 0 off-target site(s) in primary human hepatocytes, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

[0105] Embodiment 94 is the method or composition for use of embodiment 93, wherein the deletion or insertion of a nucleotide(s) occurs at a number of off-target sites in primary human hepatocytes that is less than the number of off-target sites at which a deletion or insertion of a nucleotide(s) occurs in Cas9-overexpressing cells, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

[0106] Embodiment 95 is the method or composition for use of embodiment 94, wherein the Cas9-overexpressing cells are HEK293 cells stably expressing Cas9.

[0107] Embodiment 96 is the method or composition for use of any one of embodiments 93-95, wherein the number of off-target sites in primary human hepatocytes is determined by analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

[0108] Embodiment 97 is the method or composition for use of any one of embodiments 93-95, wherein the number of off-target sites in primary human hepatocytes is determined by an oligonucleotide insertion assay comprising analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

[0109] Embodiment 98 is the method or composition of any one of embodiments 1-43 or 47-97, wherein the sequence of the guide RNA is:

[0110] a) SEQ ID NO: 92 or 104;

[0111] b) SEQ ID NO: 87, 89, 96, or 113;

[0112] c) SEQ ID NO: 100, 102, 106, 111, or 112; or

[0113] d) SEQ ID NO: 88, 90, 91, 93, 94, 95, 97, 101, 103, 108, or 109,

optionally wherein the guide RNA does not produce indels at off-target site(s) that occur in a protein coding region in the genome of primary human hepatocytes.

[0114] Embodiment 99 is the method or composition for use of any one of embodiments 1-5 or 8-98, wherein administering the composition reduces levels of TTR in the subject.

[0115] Embodiment 100 is the method or composition for use of embodiment 99, wherein the levels of TTR are reduced by at least 50%.

[0116] Embodiment 101 is the method or composition for use of embodiment 100, wherein the levels of TTR are reduced by 50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or 99%-100%.

[0117] Embodiment 102 is the method or composition for use of embodiment 100 or 101, wherein the levels of TTR are measured in serum, plasma, blood, cerebral spinal fluid, or sputum.

[0118] Embodiment 103 is the method or composition for use of embodiment 100 or 101, wherein the levels of TTR are measured in liver, choroid plexus, and/or retina.

[0119] Embodiment 104 is the method or composition for use of any one of embodiments 99-103, wherein the levels of TTR are measured via enzyme-linked immunosorbent assay (ELISA).

[0120] Embodiment 105 is the method or composition for use of any one of embodiments 1-5 or 8-104, wherein the subject has ATTR.

[0121] Embodiment 106 is the method or composition for use of any one of embodiments 1-5 or 8-105, wherein the subject is human.

[0122] Embodiment 107 is the method or composition for use of embodiment 105 or 106, wherein the subject has ATTRwt.

[0123] Embodiment 108 is the method or composition for use of embodiment 105 or 106, wherein the subject has hereditary ATTR.

[0124] Embodiment 109 is the method or composition for use of any one of embodiments 1-5, 8-106, or 108, wherein the subject has a family history of ATTR.

[0125] Embodiment 110 is the method or composition for use of any one of embodiments 1-5, 8-106, or 108-109, wherein the subject has familial amyloid polyneuropathy.

[0126] Embodiment 111 is the method or composition for use of any one of embodiments 1-5 or 8-110, wherein the subject has only or predominantly nerve symptoms of ATTR.

[0127] Embodiment 112 is the method or composition for use of any one of embodiments 1-5 or 8-110, wherein the subject has familial amyloid cardiomyopathy.

[0128] Embodiment 113 is the method or composition for use of any one of embodiments 1-5, 8-109, or 112, wherein the subject has only or predominantly cardiac symptoms of ATTR.

[0129] Embodiment 114 is the method or composition for use of any one of embodiments 1-5 or 8-113, wherein the subject expresses TTR having a V30 mutation.

[0130] Embodiment 115 is the method or composition for use of embodiment 114, wherein the V30 mutation is V30A, V30G, V30L, or V30M.

[0131] Embodiment 116 is the method or composition for use of embodiment any one of embodiments 1-5 or 8-113, wherein the subject expresses TTR having a T60 mutation.

[0132] Embodiment 117 is the method or composition for use of embodiment 116, wherein the T60 mutation is T60A.

[0133] Embodiment 118 is the method or composition for use of embodiment any one of embodiments 1-5 or 8-113, wherein the subject expresses TTR having a V122 mutation.

[0134] Embodiment 119 is the method or composition for use of embodiment 118, wherein the V122 mutation is V122A, V122I, or V122(-).

[0135] Embodiment 120 is the method or composition for use of any one of embodiments 1-5 or 8-119, wherein the subject expresses wild-type TTR.

[0136] Embodiment 121 is the method or composition for use of any one of embodiments 1-5, 8-107, or 120, wherein the subject does not express TTR having a V30, T60, or V122 mutation.

[0137] Embodiment 122 is the method or composition for use of any one of embodiments 1-5, 8-107, or 120-121, wherein the subject does not express TTR having a pathological mutation.

[0138] Embodiment 123 is the method or composition for use of embodiment 121, wherein the subject is homozygous for wild-type TTR.

[0139] Embodiment 124 is the method or composition for use of any one of embodiments 1-5 or 8-123, wherein after administration the subject has an improvement, stabilization, or slowing of change in symptoms of sensorimotor neuropathy.

[0140] Embodiment 125 is the method or composition for use of embodiment 124, wherein the improvement, stabilization, or slowing of change in sensory neuropathy is measured using electromyogram, nerve conduction tests, or patient-reported outcomes.

[0141] Embodiment 126 is the method or composition for use of any one of embodiments 1-5 or 8-125, wherein the subject has an improvement, stabilization, or slowing of change in symptoms of congestive heart failure.

[0142] Embodiment 127 is the method or composition for use of embodiment 126, wherein the improvement, stabilization, or slowing of change in congestive heart failure is measured using cardiac biomarker tests, lung function tests, chest x-rays, or electrocardiography.

[0143] Embodiment 128 is the method or composition for use of any one of embodiments 1-5 or 8-127, wherein the composition or pharmaceutical formulation is administered via a viral vector.

[0144] Embodiment 129 is the method or composition for use of any one of embodiments 1-5 or 8-127, wherein the composition or pharmaceutical formulation is administered via lipid nanoparticles.

[0145] Embodiment 130 is the method or composition for use of any one of embodiments 1-5 or 8-129, wherein the subject is tested for specific mutations in the TTR gene before administering the composition or formulation.

[0146] Embodiment 131 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 5.

[0147] Embodiment 132 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 6.

[0148] Embodiment 133 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 7.

[0149] Embodiment 134 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 8.

[0150] Embodiment 135 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 9.

[0151] Embodiment 136 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 10.

[0152] Embodiment 137 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 11.

[0153] Embodiment 138 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 12.

[0154] Embodiment 139 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 13.

[0155] Embodiment 140 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 14.

[0156] Embodiment 141 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 15.

[0157] Embodiment 142 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 16.

[0158] Embodiment 143 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 17.

[0159] Embodiment 144 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 18.

[0160] Embodiment 145 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 19.

[0161] Embodiment 146 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 20.

[0162] Embodiment 147 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 21.

[0163] Embodiment 148 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 22.

[0164] Embodiment 149 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 23.

[0165] Embodiment 150 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 24.

[0166] Embodiment 151 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 25.

[0167] Embodiment 152 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 26.

[0168] Embodiment 153 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 27.

[0169] Embodiment 154 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 28.

[0170] Embodiment 155 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 29.

[0171] Embodiment 156 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 30.

[0172] Embodiment 157 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 31.

[0173] Embodiment 158 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 32.

[0174] Embodiment 159 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 33.

[0175] Embodiment 160 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 34.

[0176] Embodiment 161 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 35.

[0177] Embodiment 162 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 36.

[0178] Embodiment 163 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 37.

[0179] Embodiment 164 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 38.

[0180] Embodiment 165 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 39.

[0181] Embodiment 166 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 40.

[0182] Embodiment 167 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 41.

[0183] Embodiment 168 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 42.

[0184] Embodiment 169 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 43.

[0185] Embodiment 170 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 44.

[0186] Embodiment 171 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 45.

[0187] Embodiment 172 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 46.

[0188] Embodiment 173 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 47.

[0189] Embodiment 174 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 48.

[0190] Embodiment 175 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 49.

[0191] Embodiment 176 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 50.

[0192] Embodiment 177 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 51.

[0193] Embodiment 178 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 52.

[0194] Embodiment 179 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 53.

[0195] Embodiment 180 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 54.

[0196] Embodiment 181 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 55.

[0197] Embodiment 182 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 56.

[0198] Embodiment 183 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 57.

[0199] Embodiment 184 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 58.

[0200] Embodiment 185 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 59.

[0201] Embodiment 186 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 60.

[0202] Embodiment 187 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 61.

[0203] Embodiment 188 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 62.

[0204] Embodiment 189 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 63.

[0205] Embodiment 190 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 64.

[0206] Embodiment 191 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 65.

[0207] Embodiment 192 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 66.

[0208] Embodiment 193 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 67.

[0209] Embodiment 194 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 68.

[0210] Embodiment 195 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 69.

[0211] Embodiment 196 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 70.

[0212] Embodiment 197 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 71.

[0213] Embodiment 198 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 72.

[0214] Embodiment 199 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 73.

[0215] Embodiment 200 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 74.

[0216] Embodiment 201 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 75.

[0217] Embodiment 202 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 76.

[0218] Embodiment 203 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 77.

[0219] Embodiment 204 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 78.

[0220] Embodiment 205 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 79.

[0221] Embodiment 206 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 80.

[0222] Embodiment 207 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 81.

[0223] Embodiment 208 is the method or composition of any one of embodiments 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 82.

[0224] Embodiment 209 is a use of a composition or formulation of any of embodiments 6-208 for the preparation of a medicament for treating a human subject having ATTR.

[0225] Also disclosed is the use of a composition or formulation of any of the foregoing embodiments for the preparation of a medicament for treating a human subject having ATTR. Also disclosed are any of the foregoing compositions or formulations for use in treating ATTR or for use in modifying (e.g., forming an indel in, or forming a frameshift or nonsense mutation in) a TTR gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0226] FIG. 1 shows a schematic of chromosome 18 with the regions of the TTR gene that are targeted by the guide sequences provided in Table 1.

[0227] FIG. 2 shows off-target analysis in HEK293_Cas9 cells of certain dual guide RNAs targeting TTR. The on-target site is designated by a filled square for each dual guide RNA tested, whereas closed circles represent a potential off-target site.

[0228] FIG. 3 shows off-target analysis in HEK_Cas9 cells of certain single guide RNAs targeting TTR. The on-target site is designated by a filled square for each single guide RNA tested, whereas open circles represent a potential off-target site.

[0229] FIG. 4 shows dose response curves of lipid nanoparticle formulated human TTR specific sgRNAs on primary human hepatocytes.

[0230] FIG. 5 shows dose response curves of lipid nanoparticle formulated human TTR specific sgRNAs on primary cyno hepatocytes.

[0231] FIG. 6 shows dose response curves of lipid nanoparticle formulated cyno TTR specific sgRNAs on primary cyno hepatocytes.

[0232] FIG. 7 shows percent editing (% edit) of TTR and reduction of secreted TTR following administration of the guide in HUH7 cells sequences provided on the x-axis. The values are normalized to the amount of alpha-1-antitrypsin (AAT) protein.

[0233] FIG. 8 shows western blot analysis of intracellular TTR following administration of targeted guides (listed in Table 1) in HUH7 cells.

[0234] FIG. 9 shows percentage liver editing of TTR observed following administration of LNP formulations to mice with humanized (G481-G499) or murine (G282) TTR. Note: the first three `0`s in each Guide ID is omitted from the Figure, for example "G481" is "G000481" in Tables 2 and 3.

[0235] FIGS. 10A-B show serum TTR levels observed following the dosing regimens indicated on the horizontal axis as .mu.g/ml (FIG. 10A) or percentage of TSS control (FIG. 10B). MPK=mg/kg throughout.

[0236] FIGS. 11A-B show serum TTR levels observed following the dosing regimens indicated on the horizontal axis for 1 mg/kg (FIG. 11A) or 0.5 mg/kg dosages (FIG. 11B). Data for a single 2 mg/kg dose is included as the right column in both panels.

[0237] FIGS. 12A-B show percentage liver editing observed following the dosing regimens indicated on the horizontal axis for 1 mg/kg (FIG. 12A) or 0.5 mg/kg dosages (FIG. 12B). FIG. 12C shows percentage liver editing observed following a single dose at 0.5, 1, or 2 mg/kg.

[0238] FIG. 13 shows percent liver editing observed following administration of LNP formulations to mice humanized with respect to the TTR gene. Note: the first three `0`s in each Guide ID is omitted from the Figure, for example "G481" is "G000481" in Tables 2 and

[0239] FIGS. 14A-B show that there is correlation between liver editing (FIG. 14A) and serum human TTR levels (FIG. 14B) following administration of LNP formulations to mice humanized with respect to the TTR gene. Note: the first three `0`s in each Guide ID is omitted from the Figure, for example "G481" is "G000481" in Tables 2 and 3.

[0240] FIGS. 15A-B show that there is a dose response with respect to percent editing (FIG. 15A) and serum TTR levels (FIG. 15B) in wild type mice following administration of LNP formulations comprising guide G502, which is cross homologous between mouse and cyno.

[0241] FIG. 16 shows dose response curves of lipid nanoparticle formulated human TTR specific sgRNAs on primary cyno hepatocytes.

[0242] FIG. 17 shows dose response curves of lipid nanoparticle formulated cyno TTR specific sgRNAs on primary human hepatocytes.

[0243] FIG. 18 shows dose response curves of lipid nanoparticle formulated cyno TTR specific sgRNAs on primary cyno hepatocytes.

[0244] FIGS. 19A-D show serum TTR (% TSS; FIGS. 19A and 19C) and editing results following dosing of LNP formulations at the indicated ratios and amounts (FIGS. 19B and 19D).

[0245] FIG. 20 shows off-target analysis of certain single guide RNAs in Primary Human Hepatocytes (PHH) targeting TTR. In the graph, filled squares represent the identification of the on-target cut site, while open circles represent the identification of potential off-target sites.

[0246] FIGS. 21A-B show percent editing on-target (ONT, FIG. 21A) and at two off-target sites (OT2 and OT4) in primary human hepatocytes following administration of lipid nanoparticle formulated G000480. FIG. 21B is a re-scaled version of the OT2, OT4, and negative control (Neg Cont) data in FIG. 21A.

[0247] FIGS. 22A-B show percent editing on-target (ONT, FIG. 22A) and at an off-target site (OT4) in primary human hepatocytes following administration of lipid nanoparticle formulated G000486. FIG. 22B is a re-scaled version of the OT4 and negative control (Neg Cont) data in FIG. 22A.

[0248] FIGS. 23A-B show percent editing (FIG. 23A) and number of insertion and deletion events at the TTR locus (FIG. 23B). FIG. 23A shows percent editing at the TTR locus in control and treatment (dosed with lipid nanoparticle formulated TTR specific sgRNA) groups. FIG. 23B shows the number of insertion and deletion events at the TTR locus when editing was observed in the treatment group of FIG. 23A.

[0249] FIGS. 24A-B show TTR levels in circulating serum (FIG. 24A) and cerebrospinal fluid (CSF) (FIG. 24B), respectively, in .mu.g/mL for control and treatment (dosed with lipid nanoparticle formulated TTR specific sgRNA) groups. Treatment resulted in >99% knockdown of TTR levels in serum.

[0250] FIGS. 25A-D show immunohistochemistry images with staining for TTR in stomach (FIG. 25A), colon (FIG. 25B), sciatic nerve (FIG. 25C), and dorsal root ganglion (DRG) (FIG. 25D) from control and treatment (dosed with lipid nanoparticle formulated TTR specific sgRNA) mice. At right, bar graphs show reduction in TTR staining 8 weeks after treatment in treated mice as measured by percent occupied area for each tissue type.

[0251] FIGS. 26A-C show liver TTR editing (FIG. 26A) and serum TTR results (in .mu.g/mL (FIG. 26B) and as percentage of TSS-treated control (FIG. 26C)), respectively, from humanized TTR mice dosed with LNP formulations across a range of doses with guides G000480, G000488, G000489 and G000502 and containing Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide.

[0252] FIGS. 27A-C show liver TTR editing (FIG. 27A) and serum TTR results (in .mu.g/mL (FIG. 27B) and as percentage of TSS-treated control (FIG. 27C)), respectively, from humanized TTR mice dosed with LNP formulations across a range of doses with guides G000481, G000482, G000486 and G000499 and containing Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide.

[0253] FIGS. 28A-C show liver TTR editing (FIG. 28A) and serum TTR results (in .mu.g/mL (FIG. 28B) and as percentage of TSS-treated control (FIG. 28C)), respectively, from humanized TTR mice dosed with LNP formulations across a range of doses with guides G000480, G000481, G000486, G000499 and G000502 and containing Cas9 mRNA (SEQ ID NO: 1) in a 1:2 ratio by weight to the guide.

[0254] FIG. 29 shows relative expression of TTR mRNA in primary human hepatocytes (PHH) after treatment with LNPs comprising Cas9 mRNA and a gRNA as indicated, as compared to negative (untreated) controls.

[0255] FIG. 30 shows relative expression of TTR mRNA in primary human hepatocytes (PHH) after treatment with LNPs comprising Cas9 mRNA and a gRNA as indicated, as compared to negative (untreated) controls.

DETAILED DESCRIPTION

[0256] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0257] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells and the like.

[0258] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0259] Unless specifically noted in the above specification, embodiments in the specification that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments in the specification that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments in the specification that recite "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in an inclusive sense, i.e., equivalent to "and/or," unless the context clearly indicates otherwise.

[0260] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

I. Definitions

[0261] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0262] "Polynucleotide" and "nucleic acid" are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid "backbone" can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds ("peptide nucleic acids" or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2' methoxy or 2' halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N.sup.4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O.sup.6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O.sup.4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11.sup.th ed., 1992). Nucleic acids can include one or more "abasic" residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes "locked nucleic acid" (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0263] "Guide RNA", "gRNA", and "guide" are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.

[0264] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. A "guide sequence" may also be referred to as a "targeting sequence," or a "spacer sequence." A guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 88%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.

[0265] Target sequences for Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be "complementary to a target sequence", it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0266] As used herein, an "RNA-guided DNA binding agent" means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof ("dCas DNA binding agents"). "Cas nuclease", also called "Cas protein", as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas cleavases/nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a "Class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA binding activity, such as a Cas9 nuclease or a Cpf1 nuclease. Class 2 Cas nucleases include Class 2 Cas cleavases and Class 2 Cas nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g, K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. "Cas9" encompasses Spy Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0267] "Modified uridine" is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton. In some embodiments, a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine.

[0268] "Uridine position" as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide in which "100% of the uridine positions are modified uridines" contains a modified uridine at every position that would be a uridine in a conventional RNA (where all bases are standard A, U, C, or G bases) of the same sequence. Unless otherwise indicated, a U in a polynucleotide sequence of a sequence table or sequence listing in, or accompanying, this disclosure can be a uridine or a modified uridine.

[0269] As used herein, a first sequence is considered to "comprise a sequence with at least X % identity to" a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.

[0270] "mRNA" is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2'-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxy ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.

[0271] As used herein, the "minimum uridine content" of a given open reading frame (ORF) is the uridine content of an ORF that (a) uses a minimal uridine codon at every position and (b) encodes the same amino acid sequence as the given ORF. The minimal uridine codon(s) for a given amino acid is the codon(s) with the fewest uridines (usually 0 or 1 except for a codon for phenylalanine, where the minimal uridine codon has 2 uridines). Modified uridine residues are considered equivalent to uridines for the purpose of evaluating minimum uridine content.

[0272] As used herein, the "minimum uridine dinucleotide content" of a given open reading frame (ORF) is the lowest possible uridine dinucleotide (UU) content of an ORF that (a) uses a minimal uridine codon (as discussed above) at every position and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed in absolute terms as the enumeration of UU dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the uridines of uridine dinucleotides (for example, AUUAU would have a uridine dinucleotide content of 40% because 2 of 5 positions are occupied by the uridines of a uridine dinucleotide). Modified uridine residues are considered equivalent to uridines for the purpose of evaluating minimum uridine dinucleotide content.

[0273] As used herein, "TTR" refers to transthyretin, which is the gene product of a TTR gene.

[0274] As used herein, "amyloid" refers to abnormal aggregates of proteins or peptides that are normally soluble. Amyloids are insoluble, and amyloids can create proteinaceous deposits in organs and tissues. Proteins or peptides in amyloids may be misfolded into a form that allows many copies of the protein to stick together to form fibrils. While some forms of amyloid may have normal functions in the human body, "amyloids" as used herein refers to abnormal or pathologic aggregates of protein. Amyloids may comprise a single protein or peptide, such as TTR, or they may comprise multiple proteins or peptides, such as TTR and additional proteins.

[0275] As used herein, "amyloid fibrils" refers to insoluble fibers of amyloid that are resistant to degradation. Amyloid fibrils can produce symptoms based on the specific protein or peptide and the tissue and cell type in which it has aggregated.

[0276] As used herein, "amyloidosis" refers to a disease characterized by symptoms caused by deposition of amyloid or amyloid fibrils. Amyloidosis can affect numerous organs including the heart, kidney, liver, spleen, nervous system, and digestive track.

[0277] As used herein, "ATTR," "TTR-related amyloidosis," "TTR amyloidosis," "ATTR amyloidosis," or "amyloidosis associated with TTR" refers to amyloidosis associated with deposition of TTR.

[0278] As used herein, "familial amyloid cardiomyopathy" or "FAC" refers to a hereditary transthyretin amyloidosis (ATTR) characterized primarily by restrictive cardiomyopathy. Congestive heart failure is common in FAC. Average age of onset is approximately 60-70 years of age, with an estimated life expectancy of 4-5 years after diagnosis.

[0279] As used herein, "familial amyloid polyneuropathy" or "FAP" refers to a hereditary transthyretin amyloidosis (ATTR) characterized primarily by sensorimotor neuropathy. Autonomic neuropathy is common in FAP. While neuropathy is a primary feature, symptoms of FAP may also include cachexia, renal failure, and cardiac disease. Average age of onset of FAP is approximately 30-50 years of age, with an estimated life expectancy of 5-15 after diagnosis.

[0280] As used herein, "wild-type ATTR" and "ATTRwt" refer to ATTR not associated with a pathological TTR mutation such as T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(-). ATTRwt has also been referred to as senile systemic amyloidosis. Onset typically occurs in men aged 60 or higher with the most common symptoms being congestive heart failure and abnormal heart rhythm such as atrial fibrillation. Additional symptoms include consequences of poor heart function such as shortness of breath, fatigue, dizziness, swelling (especially in the legs), nausea, angina, disrupted sleep, and weight loss. A history of carpal tunnel syndrome indicates increased risk for ATTRwt and may in some cases be indicative of early-stage disease. ATTRwt generally leads to decreasing heart function over time but can have a better prognosis than hereditary ATTR because wild-type TTR deposits accumulate more slowly. Existing treatments are similar to other forms of ATTR (other than liver transplantation) and are generally directed to supporting or improving heart function, ranging from diuretics and limited fluid and salt intake to anticoagulants, and in severe cases, heart transplants. Nonetheless, like FAC, ATTRwt can result in death from heart failure, sometimes within 3-5 years of diagnosis.

[0281] Guide sequences useful in the guide RNA compositions and methods described herein are shown in Table 1 and throughout the application.

[0282] As used herein, "hereditary ATTR" refers to ATTR that is associated with a mutation in the sequence of the TTR gene. Known mutations in the TTR gene associated with ATTR include those resulting in TTR with substitutions of T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(-).

[0283] As used herein, "indels" refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in a target nucleic acid.

[0284] As used herein, "knockdown" refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both). Knockdown of a protein can be measured either by detecting protein secreted by tissue or population of cells (e.g., in serum or cell media) or by detecting total cellular amount of the protein from a tissue or cell population of interest. Methods for measuring knockdown of mRNA are known, and include sequencing of mRNA isolated from a tissue or cell population of interest. In some embodiments, "knockdown" may refer to some loss of expression of a particular gene product, for example a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed or secreted by a population of cells (including in vivo populations such as those found in tissues).

[0285] As used herein, "knockout" refers to a loss of expression of a particular protein in a cell. Knockout can be measured either by detecting the amount of protein secretion from a tissue or population of cells (e.g., in serum or cell media) or by detecting total cellular amount of a protein a tissue or a population of cells. In some embodiments, the methods of the disclosure "knockout" TTR in one or more cells (e.g., in a population of cells including in vivo populations such as those found in tissues). In some embodiments, a knockout is not the formation of mutant TTR protein, for example, created by indels, but rather the complete loss of expression of TTR protein in a cell.

[0286] As used herein, "mutant TTR" refers to a gene product of TTR (i.e., the TTR protein) having a change in the amino acid sequence of TTR compared to the wildtype amino acid sequence of TTR. The human wild-type TTR sequence is available at NCBI Gene ID: 7276; Ensembl: Ensembl: ENSG00000118271. Mutants forms of TTR associated with ATTR, e.g., in humans, include T60A, V30M, V30A, V30G, V30L, V122I, V122A, or V122(-).

[0287] As used herein, "mutant TTR" or "mutant TTR allele" refers to a TTR sequence having a change in the nucleotide sequence of TTR compared to the wildtype sequence (NCBI Gene ID: 7276; Ensembl: ENSG00000118271).

[0288] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.

[0289] As used herein, a "target sequence" refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.

[0290] As used herein, "treatment" refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of ATTR may comprise alleviating symptoms of ATTR.

[0291] "Modified uridine" is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton, e.g., N1-methyl pseudouridine. In some embodiments, a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine.

[0292] As used herein, a first sequence is considered to "comprise a sequence with at least X % identity to" a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine as a complement). Thus, for example, the sequence 5'-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server are generally appropriate.

[0293] The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

II. Compositions

[0294] A. Compositions Comprising Guide RNA (gRNAs)

[0295] Provided herein are compositions useful for editing the TTR gene, e.g., using a guide RNA with an RNA-guided DNA binding agent (e.g., a CRISPR/Cas system). The compositions may be administered to subjects having wild-type or non-wild type TTR gene sequences, such as, for example, subjects with ATTR, which may be ATTR wt or a hereditary or familial form of ATTR. Guide sequences targeting the TTR gene are shown in Table 1 at SEQ ID Nos: 5-82.

TABLE-US-00001 TABLE 1 TTR targeted guide sequences, nomenclature, chromosomal coordinates, and sequence. SEQ ID Guide De- Chromosomal No. ID scription Species Location Guide Sequences* 5 CR003335 TTR Human chr18:315919 CUGCUCCUCCUCUGCCUUGC (Exon 1) 17-31591937 6 CR003336 TTR Human chr18:315919 CCUCCUCUGCCUUGCUGGAC (Exon 1) 22-31591942 7 CR003337 TTR Human chr18:315919 CCAGUCCAGCAAGGCAGAGG (Exon 1) 25-31591945 8 CR003338 TTR Human chr18:315919 AUACCAGUCCAGCAAGGCAG (Exon 1) 28-31591948 9 CR003339 TTR Human chr18:315919 ACACAAAUACCAGUCCAGCA (Exon 1) 34-31591954 10 CR003340 TTR Human chr18:315919 UGGACUGGUAUUUGUGUCUG (Exon 1) 37-31591957 11 CR003341 TTR Human chr18:315919 CUGGUAUUUGUGUCUGAGGC (Exon 1) 41-31591961 12 CR003342 TTR Human chr18:315928 CUUCUCUACACCCAGGGCAC (Exon 2) 80-31592900 13 CR003343 TTR Human chr18:315929 CAGAGGACACUUGGAUUCAC (Exon 2) 02-31592922 14 CR003344 TTR Human chr18:315929 UUUGACCAUCAGAGGACACU (Exon 2) 11-31592931 15 CR003345 TTR Human chr18:315929 UCUAGAACUUUGACCAUCAG (Exon 2) 19-31592939 16 CR003346 TTR Human chr18:315929 AAAGUUCUAGAUGCUGUCCG (Exon 2) 28-31592948 17 CR003347 TTR Human chr18:315929 CAUUGAUGGCAGGACUGCCU (Exon 2) 48-31592968 18 CR003348 TTR Human chr18:315929 AGGCAGUCCUGCCAUCAAUG (Exon 2) 48-31592968 19 CR003349 TTR Human chr18:315929 UGCACGGCCACAUUGAUGGC (Exon 2) 58-31592978 20 CR003350 TTR Human chr18:315929 CACAUGCACGGCCACAUUGA (Exon 2) 62-31592982 21 CR003351 TTR Human chr18:315929 AGCCUUUCUGAACACAUGCA (Exon 2) 74-31592994 22 CR003352 TTR Human chr18:315929 GAAAGGCUGCUGAUGACACC (Exon 2) 86-31593006 23 CR003353 TTR Human chr18:315929 AAAGGCUGCUGAUGACACCU (Exon 2) 87-31593007 24 CR003354 TTR Human chr18:315930 ACCUGGGAGCCAUUUGCCUC (Exon 2) 03-31593023 25 CR003355 TTR Human chr18:315930 CCCAGAGGCAAAUGGCUCCC (Exon 2) 07-31593027 26 CR003356 TTR Human chr18:315930 GCAACUUACCCAGAGGCAAA (Exon 2) 15-31593035 27 CR003357 TTR Human chr18:315930 UUCUUUGGCAACUUACCCAG (Exon 2) 22-31593042 28 CR003358 TTR Human chr18:315951 AUGCAGCUCUCCAGACUCAC (Exon 3) 27-31595147 29 CR003359 TTR Human chr18:315951 AGUGAGUCUGGAGAGCUGCA (Exon 3) 26-31595146 30 CR003360 TTR Human chr18:315951 GUGAGUCUGGAGAGCUGCAU (Exon 3) 27-31595147 31 CR003361 TTR Human chr18:315951 GCUGCAUGGGCUCACAACUG (Exon 3) 40-31595160 32 CR003362 TTR Human chr18:315951 GCAUGGGCUCACAACUGAGG (Exon 3) 43-31595163 33 CR003363 TTR Human chr18:315951 ACUGAGGAGGAAUUUGUAGA (Exon 3) 56-31595176 34 CR003364 TTR Human chr18:315951 CUGAGGAGGAAUUUGUAGAA (Exon 3) 57-31595177 35 CR003365 TTR Human chr18:315951 UGUAGAAGGGAUAUACAAAG (Exon 3) 70-31595190 36 CR003366 TTR Human chr18:315951 AAAUAGACACCAAAUCUUAC (Exon 3) 93-31595213 37 CR003367 TTR Human chr18:315951 AGACACCAAAUCUUACUGGA (Exon 3) 97-31595217 38 CR003368 TTR Human chr18:315952 AAGUGCCUUCCAGUAAGAUU (Exon 3) 05-31595225 39 CR003369 TTR Human chr18:315952 CUCUGCAUGCUCAUGGAAUG (Exon 3) 35-31595255 40 CR003370 TTR Human chr18:315952 CCUCUGCAUGCUCAUGGAAU (Exon 3) 36-31595256 41 CR003371 TTR Human chr18:315952 ACCUCUGCAUGCUCAUGGAA (Exon 3) 37-31595257 42 CR003372 TTR Human chr18:315952 UACUCACCUCUGCAUGCUCA (Exon 3) 42-31595262 43 CR003373 TTR Human chr18:315985 GUAUUCACAGCCAACGACUC (Exon 4) 70-31598590 44 CR003374 TTR Human chr18:315985 GCGGCGGGGGCCGGAGUCGU (Exon 4) 83-31598603 45 CR003375 TTR Human chr18:315985 AAUGGUGUAGCGGCGGGGGC (Exon 4) 92-31598612 46 CR003376 TTR Human chr18:315985 CGGCAAUGGUGUAGCGGCGG (Exon 4) 96-31598616 47 CR003377 TTR Human chr18:315985 GCGGCAAUGGUGUAGCGGCG (Exon 4) 97-31598617 48 CR003378 TTR Human chr18:315985 GGCGGCAAUGGUGUAGCGGC (Exon 4) 98-31598618 49 CR003379 TTR Human chr18:315985 GGGCGGCAAUGGUGUAGCGG (Exon 4) 99-31598619 50 CR003380 TTR Human chr18:315986 GCAGGGCGGCAAUGGUGUAG (Exon 4) 02-31598622 51 CR003381 TTR Human chr18:315986 GGGGCUCAGCAGGGCGGCAA (Exon 4) 10-31598630 52 CR003382 TTR Human chr18:315986 GGAGUAGGGGCUCAGCAGGG (Exon 4) 16-31598636 53 CR003383 TTR Human chr18:315986 AUAGGAGUAGGGGCUCAGCA (Exon 4) 19-31598639 54 CR003384 TTR Human chr18:315986 AAUAGGAGUAGGGGCUCAGC (Exon 4) 20-31598640 55 CR003385 TTR Human chr18:315986 CCCCUACUCCUAUUCCACCA (Exon 4) 26-31598646 56 CR003386 TTR Human chr18:315986 CCGUGGUGGAAUAGGAGUAG (Exon 4) 29-31598649 57 CR003387 TTR Human chr18:315986 GCCGUGGUGGAAUAGGAGUA (Exon 4) 30-31598650 58 CR003388 TTR Human chr18:315986 GACGACAGCCGUGGUGGAAU (Exon 4) 37-31598657 59 CR003389 TTR Human chr18:315986 AUUGGUGACGACAGCCGUGG (Exon 4) 43-31598663 60 CR003390 TTR Human chr18:315986 GGGAUUGGUGACGACAGCCG (Exon 4) 46-31598666 61 CR003391 TTR Human chr18:315986 GGCUGUCGUCACCAAUCCCA (Exon 4) 47-31598667 62 CR003392 TTR Human chr18:315986 AGUCCCUCAUUCCUUGGGAU (Exon 4) 61-31598681 63 CR005298 TTR Human chr18:315918 UCCACUCAUUCUUGGCAGGA (Exon 1) 83-31591903 64 CR005299 TTR Human chr18:315986 AGCCGUGGUGGAAUAGGAGU (Exon 4) 31-31598651 65 CR005300 TTR Human chr18:315919 UCACAGAAACACUCACCGUA (Exon 1) 67-31591987 66 CR005301 TTR Human chr18:315919 GUCACAGAAACACUCACCGU (Exon 1) 68-31591988 67 CR005302 TTR Human chr18:315928 ACGUGUCUUCUCUACACCCA (Exon 2) 74-31592894 68 CR005303 TTR Human chr18:315929 UGAAUCCAAGUGUCCUCUGA (Exon 2) 03-31592923 69 CR005304 TTR Human chr18:315929 GGCCGUGCAUGUGUUCAGAA (Exon 2) 69-31592989 70 CR005305 TTR Human chr18:315951 UAUAGGAAAACCAGUGAGUC (Exon 3) 14-31595134 71 CR005306 TTR Human chr18:315952 AAAUCUUACUGGAAGGCACU (Exon 3) 04-31595224 72 CR005307 TTR Human chr18:315985 UGUCUGUCUUCUCUCAUAGG (Exon 4) 48-31598568 73 CR000689 TTR Cyno chr18:506815 ACACAAAUACCAGUCCAGCG 33-50681553 74 CR005364 TTR Cyno chr18:506804 AAAGGCUGCUGAUGAGACCU 81-50680501 75 CR005365 TTR Cyno chr18:506805 CAUUGACAGCAGGACUGCCU 20-50680540 76 CR005366 TTR Cyno chr18:506815 AUACCAGUCCAGCGAGGCAG 39-50681559 77 CR005367 TTR Cyno chr18:506815 CCAGUCCAGCGAGGCAGAGG 42-50681562 78 CR005368 TTR Cyno chr18:506815 CCUCCUCUGCCUCGCUGGAC 45-50681565 79 CR005369 TTR Cyno chr18:506805 AAAGUUCUAGAUGCCGUCCG 40-50680560 80 CR005370 TTR Cyno chr18:506805 ACUUGUCUUCUCUAUACCCA 94-50680614 81 CR005371 TTR Cyno chr18:506782 AAGUGACUUCCAGUAAGAUU 16-50678236 82 CR005372 TTR Cyno chr18:506804 AAAAGGCUGCUGAUGAGACC 82-50680502

[0296] Each of the Guide Sequences above may further comprise additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the Guide Sequence at its 3' end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 126). In the case of a sgRNA, the above Guide Sequences may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3' end of the Guide Sequence: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 125) in 5' to 3' orientation.

[0297] In some embodiments, the sgRNA is modified. In some embodiments, the sgRNA comprises the modification pattern shown below in SEQ ID NO: 3, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence as described herein and the modified sgRNA comprises the following sequence: mN*mN*mN*GUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 3), where "N" may be any natural or non-natural nucleotide. For example, encompassed herein is SEQ ID NO: 3, where the N's are replaced with any of the guide sequences disclosed herein. The modifications remain as shown in SEQ ID NO: 3 despite the substitution of N's for the nucleotides of a guide. That is, although the nucleotides of the guide replace the "N's", the first three nucleotides are 2'OMe modified and there are phosphorothioate linkages between the first and second nucleotides, the second and third nucleotides and the third and fourth nucleotides.

[0298] In some embodiments, any one of the sequences recited in Table 2 is encompassed.

TABLE-US-00002 TABLE 2 TTR targeted sgRNA sequences SEQ ID Guide Target and No. ID Description Species Sequence 87 G000480 TTR Human mA*mA*mA*GGCUGCUGAUGACACCUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 88 G000481 TTR Human mU*mC*mU*AGAACUUUGACCAUCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 89 G000482 TTR Human mU*mG*mU*AGAAGGGAUAUACAAAGG sgRNA UUUUAGAmGmCmUmAmGmAmAmAmUm modified AmGmCAAGUUAAAAUAAGGCUAGUCCG sequence UUAUCAmAmCmUmUmGmAmAmAmAmA mGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU 90 G000483 TTR Human mU*mC*mC*ACUCAUUCUUGGCAGGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 91 G000484 TTR Human mA*mG*mA*CACCAAAUCUUACUGGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 92 G000485 TTR Human mC*mC*mU*CCUCUGCCUUGCUGGACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 93 G000486 TTR Human mA*mC*mA*CAAAUACCAGUCCAGCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 94 G000487 TTR Human mU*mU*mC*UUUGGCAACUUACCCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 95 G000488 TTR Human mA*mA*mA*GUUCUAGAUGCUGUCCGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 96 G000489 TTR Human mU*mU*mU*GACCAUCAGAGGACACUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 97 G000490 TTR Human mA*mA*mA*UAGACACCAAAUCUUACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 98 G000491 TTR Human mA*mU*mA*CCAGUCCAGCAAGGCAGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 99 G000492 TTR Human mC*mU*mU*CUCUACACCCAGGGCACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 100 G000493 TTR Human mA*mA*mG*UGCCUUCCAGUAAGAUUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 101 G000494 TTR Human mG*mU*mG*AGUCUGGAGAGCUGCAUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 102 G000495 TTR Human mC*mA*mG*AGGACACUUGGAUUCACGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 103 G000496 TTR Human mG*mG*mC*CGUGCAUGUGUUCAGAAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 104 G000497 TTR Human mC*mU*mG*CUCCUCCUCUGCCUUGCGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 105 G000498 TTR Human mA*mG*mU*GAGUCUGGAGAGCUGCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 106 G000499 TTR Human mU*mG*mA*AUCCAAGUGUCCUCUGAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 107 G000500 TTR Human mC*mC*mA*GUCCAGCAAGGCAGAGGGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 108 G000501 TTR Human mU*mC*mA*CAGAAACACUCACCGUAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 109 G000567 TTR Human mG*mA*mA*AGGCUGCUGAUGACACCGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 110 G000568 TTR Human mG*mG*mC*UGUCGUCACCAAUCCCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 111 G000570 TTR Human mC*mA*mU*UGAUGGCAGGACUGCCUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 112 G000571 TTR Human mG*mU*mC*ACAGAAACACUCACCGUGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 113 G000572 TTR Human mC*mC*mC*CUACUCCUAUUCCACCAGU sgRNA UUUAGAmGmCmUmAmGmAmAmAmUmA modified mGmCAAGUUAAAAUAAGGCUAGUCCGU sequence UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 114 G000502 TTR Cyno Cyno mA*mC*mA*CAAAUACCAGUCCAGCGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 115 G000503 TTR Cyno Cyno mA*mA*mA*AGGCUGCUGAUGAGACCGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 116 G000504 TTR Cyno Cyno mA*mA*mA*GGCUGCUGAUGAGACCUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 117 G000505 TTR Cyno Cyno mC*mA*mU*UGACAGCAGGACUGCCUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 118 G000506 TTR Cyno Cyno mA*mU*mA*CCAGUCCAGCGAGGCAGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 119 G000507 TTR Cyno Cyno mC*mC*mA*GUCCAGCGAGGCAGAGGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 120 G000508 TTR Cyno Cyno mC*mC*mU*CCUCUGCCUCGCUGGACGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 121 G000509 TTR Cyno Cyno mA*mA*mA*GUUCUAGAUGCCGUCCGGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm

GmGmUmGmCmU*mU*mU*mU 122 G000510 TTR Cyno Cyno mA*mC*mU*UGUCUUCUCUAUACCCAGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 123 G000511 TTR Cyno Cyno mA*mA*mG*UGACUUCCAGUAAGAUUGU specific UUUAGAmGmCmUmAmGmAmAmAmUmA sgRNA mGmCAAGUUAAAAUAAGGCUAGUCCGU modified UAUCAmAmCmUmUmGmAmAmAmAmAm sequence GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU 124 G000282 TTR Mouse mU*mU*mA*CAGCCACGUCUACAGCAGU UUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGU UAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmU*mU*mU*mU * = PS linkage; 'm' = 2'-O-Me nucleotide

[0299] An alignment mapping of the Guide IDs with the corresponding sgRNA IDs as well as homology to the cyno genome and cyno matched guide IDs are provided in Table 3.

TABLE-US-00003 TABLE 3 TTR targeted guide sequence ID mapping and Cyno Homology De- Human Human Number Cyno Cyno scrip- Dual Single Mismatches to Matched Matched tion Guide ID Guide ID Cyno Genome dgRNA ID sgRNA ID TTR CR003335 G000497 1 TTR CR003336 G000485 1 CR005368 G000508 TTR CR003337 G000500 1 CR005367 G000507 TTR CR003338 G000491 1 CR005366 G000506 TTR CR003339 G000486 1 CR000689 G000502 TTR CR003340 0 TTR CR003341 0 TTR CR003342 G000492 no PAM in cyno TTR CR003343 G000495 no PAM in cyno TTR CR003344 G000489 0 TTR CR003345 G000481 0 TTR CR003346 G000488 1 CR005369 G000509 TTR CR003347 G000570 2 CR005365 G000505 TTR CR003348 2 TTR CR003349 >3 TTR CR003350 no PAM in cyno TTR CR003351 no PAM in cyno TTR CR003352 G000567 2 CR005372 G000503 TTR CR003353 G000480 1 CR005364 G000504 TTR CR003354 1 TTR CR003355 1 TTR CR003356 3 TTR CR003357 G000487 >3 TTR CR003358 0 TTR CR003359 G000498 0 TTR CR003360 G000494 0 TTR CR003361 0 TTR CR003362 0 TTR CR003363 0 TTR CR003364 0 TTR CR003365 G000482 0 TTR CR003366 G000490 0 TTR CR003367 G000484 no PAM in cyno TTR CR003368 G000493 1 CR005371 G000511 TTR CR003369 0 TTR CR003370 0 TTR CR003371 0 TTR CR003372 0 TTR CR003373 1 TTR CR003374 2 TTR CR003375 2 TTR CR003376 2 TTR CR003377 2 TTR CR003378 2 TTR CR003379 2 TTR CR003380 1 TTR CR003381 1 TTR CR003382 0 TTR CR003383 0 TTR CR003384 0 TTR CR003385 G000572 0 TTR CR003386 0 TTR CR003387 0 TTR CR003388 0 TTR CR003389 G000569 0 TTR CR003390 0 TTR CR003391 G000568 0 TTR CR003392 0 TTR CR005298 G000483 1 TTR CR005299 0 TTR CR005300 G000501 no PAM in cyno TTR CR005301 G000571 0 TTR CR005302 2 CR005370 G000510 TTR CR005303 G000499 0 TTR CR005304 G000496 >3 TTR CR005305 0 TTR CR005306 1 TTR CR005307 0

[0300] In some embodiments, the invention provides a composition comprising one or more guide RNA (gRNA) comprising guide sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in TTR. The gRNA may comprise a crRNA comprising a guide sequence shown in Table 1. The gRNA may comprise a crRNA comprising 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1. In some embodiments, the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1. In some embodiments, the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 1. The gRNA may further comprise a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA), or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.

[0301] In each of the composition, use, and method embodiments described herein, the guide RNA may comprise two RNA molecules as a "dual guide RNA" or "dgRNA". The dgRNA comprises a first RNA molecule comprising a crRNA comprising, e.g., a guide sequence shown in Table 1, and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form a RNA duplex via the base pairing between portions of the crRNA and the trRNA.

[0302] In each of the composition, use, and method embodiments described herein, the guide RNA may comprise a single RNA molecule as a "single guide RNA" or "sgRNA". The sgRNA may comprise a crRNA (or a portion thereof) comprising a guide sequence shown in Table 1 covalently linked to a trRNA. The sgRNA may comprise 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via one or more bonds that are not a phosphodiester bond.

[0303] In some embodiments, the trRNA may comprise all or a portion of a trRNA sequence derived from a naturally-occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild type trRNA. The length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.

[0304] In some embodiments, the invention provides a composition comprising one or more guide RNAs comprising a guide sequence of any one of SEQ ID NOs: 5-82.

[0305] In one aspect, the invention provides a composition comprising a gRNA that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 5-82.

[0306] In other embodiments, the composition comprises at least one, e.g., at least two gRNA's comprising guide sequences selected from any two or more of the guide sequences of SEQ ID NOs: 5-82. In some embodiments, the composition comprises at least two gRNA's that each comprise a guide sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 5-82.

[0307] In some embodiments, the gRNA is a sgRNA comprising any one of the sequences shown in Table 2 (SEQ ID Nos. 87-124). In some embodiments, the gRNA is a sgRNA comprising any one of the sequences shown in Table 2 (SEQ ID Nos. 87-124, but without the modifications as shown (i.e., unmodified SEQ ID Nos. 87-124). In some embodiments, the sgRNA comprises a sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID Nos. 87-124. In some embodiments, the sgRNA comprises a sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID Nos. 87-124, but without the modifications as shown (i.e., unmodified SEQ ID Nos. 87-124). In some embodiments, the sgRNA comprises any one of the guide sequences shown in Table 1 in place of the guide sequences shown in the sgRNA sequences of Table 2 at SEQ ID Nos: 87-124, with or without the modifications.

[0308] The guide RNA compositions of the present invention are designed to recognize (e.g., hybridize to) a target sequence in the TTR gene. For example, the TTR target sequence may be recognized and cleaved by a provided Cas cleavase comprising a guide RNA. In some embodiments, an RNA-guided DNA binding agent, such as a Cas cleavase, may be directed by a guide RNA to a target sequence of the TTR gene, where the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-guided DNA binding agent, such as a Cas cleavase, cleaves the target sequence.

[0309] In some embodiments, the selection of the one or more guide RNAs is determined based on target sequences within the TTR gene.

[0310] Without being bound by any particular theory, mutations (e.g., frameshift mutations resulting from indels occurring as a result of a nuclease-mediated DSB) in certain regions of the gene may be less tolerable than mutations in other regions of the gene, thus the location of a DSB is an important factor in the amount or type of protein knockdown that may result. In some embodiments, a gRNA complementary or having complementarity to a target sequence within TTR is used to direct the RNA-guided DNA binding agent to a particular location in the TTR gene. In some embodiments, gRNAs are designed to have guide sequences that are complementary or have complementarity to target sequences in exon 1, exon 2, exon 3, or exon 4 of TTR.

[0311] In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence present in the human TTR gene. In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides.

[0312] In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered.

[0313] In some embodiments, the RNA-guided DNA-binding agent is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or VI). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modifications thereof. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. For discussion of various CRISPR systems and Cas nucleases see, e.g., Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015).

[0314] Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.

[0315] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.

[0316] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments, the Cas9 is capable of inducing a double strand break in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 203. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 204. An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 210.

[0317] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as FokI. In some embodiments, a Cas nuclease may be a modified nuclease.

[0318] In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.

[0319] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 206. An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 207. An exemplary Cas9 nickase mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 211.

[0320] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.

[0321] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell October 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB--A0Q7Q2 (CPF1_FRATN)).

[0322] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[0323] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980 A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 208. An exemplary dCas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 209. An exemplary dCas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 212.

[0324] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0325] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-10 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. It may also be inserted within the RNA-guided DNA binding agent sequence. In other embodiments, the RNA-guided DNA-binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 274) or PKKKRRV (SEQ ID NO: 275). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 276). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 274) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site.

[0326] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).

[0327] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6.times.His, 8.times.His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0328] In additional embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria.

[0329] In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression," Cell 152:1173-83 (2013); Perez-Pinera et al., "RNA-guided gene activation by CRISPR-Cas9-based transcription factors," Nat. Methods 10:973-6 (2013); Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes," Cell 154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.

[0330] B. Modified gRNAs and mRNAs

[0331] In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a "modified" gRNA or "chemically modified" gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called "modified." Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with "dephospho" linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).

[0332] As noted above, in some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered. In some embodiments, the ORF encoding an RNA-guided DNA nuclease is a "modified RNA-guided DNA binding agent ORF" or simply a "modified ORF," which is used as shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity to any one of SEQ ID NOs: 201, 204, 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266; (4) the modified ORF consists of a set of codons of which at least 75% of the codons are codons listed in the Table 3A of Minimal Uridine Codons; or (5) the modified ORF comprises at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the foregoing ways. In some embodiments, the modified ORF comprises at least one modified uridine and is modified in at least one, two, three, or all of (1)-(4) above.

TABLE-US-00004 TABLE 3A of Minimal Uridine Codons Amino Acid Minimal uridine codon A Alanine GCA or GCC or GCG G Glycine GGA or GGC or GGG V Valine GUC or GUA or GUG D Aspartic acid GAC E Glutamic acid GAA or GAG I Isoleucine AUC or AUA T Threonine ACA or ACC or ACG N Asparagine AAC K Lysine AAG or AAA S Serine AGC R Arginine AGA or AGG L Leucine CUG or CUA or CUC P Proline CCG or CCA or CCC H Histidine CAC Q Glutamine CAG or CAA F Phenylalanine UUC Y Tyrosine UAC C Cysteine UGC W Tryptophan UGG M Methionine AUG

[0333] In any of the foregoing embodiments, the modified ORF may consist of a set of codons of which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in Table 3A showing Minimal Uridine Codons.

[0334] In any of the foregoing embodiments, the modified ORF may comprise a sequence with at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 201, 204, 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0335] In any of the foregoing embodiments, the modified ORF may have a uridine content ranging from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine content.

[0336] In any of the foregoing embodiments, the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine dinucleotide content.

[0337] In any of the foregoing embodiments, the modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0338] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in an mRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine.

[0339] In some embodiments, the mRNA comprises at least one UTR from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered constitutively expressed in a mammal if it is continually transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5' UTR, 3' UTR, or 5' and 3' UTRs from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

[0340] In some embodiments, the mRNA comprises at least one UTR from Hydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD), e.g., a 5' UTR from HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, for example, human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5' UTR, 3' UTR, or 5' and 3' UTRs from a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5' UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3' UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises 5' and 3' UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0341] In some embodiments, the mRNA comprises 5' and 3' UTRs that are from the same source, e.g., a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.

[0342] In some embodiments, the mRNA does not comprise a 5' UTR, e.g., there are no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5' cap and the start codon, but does not have any additional 5' UTR. In some embodiments, the mRNA does not comprise a 3' UTR, e.g., there are no additional nucleotides between the stop codon and the poly-A tail.

[0343] In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect translation initiation and the overall yield of a polypeptide translated from an mRNA. A Kozak sequence includes a methionine codon that can function as the start codon. A minimal Kozak sequence is NNNRUGN wherein at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R means a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccRccAUGG (SEQ ID NO: 277) with zero mismatches or with up to one, two, or three mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQ ID NO: 278) with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase.

[0344] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 1, optionally wherein the ORF of SEQ ID NO: 1 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0345] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 244, optionally wherein the ORF of SEQ ID NO: 244 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0346] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 256, optionally wherein the ORF of SEQ ID NO: 256 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0347] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 257, optionally wherein the ORF of SEQ ID NO: 257 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0348] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 257, optionally wherein the ORF of SEQ ID NO: 258 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0349] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 259, optionally wherein the ORF of SEQ ID NO: 259 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0350] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 260, optionally wherein the ORF of SEQ ID NO: 260 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0351] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 261, optionally wherein the ORF of SEQ ID NO: 261 (i.e., SEQ ID NO: 204) is substituted with an alternative ORF of any one of SEQ ID NO: 210, 214, 215, 223, 224, 250, 252, 254, 265, or 266.

[0352] In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 243, 244, or 256-261 is 95%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 243, 244, or 256-261 is 98%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 243, 244, or 256-261 is 99%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 243, 244, or 256-261 is 100%.

[0353] In some embodiments, an mRNA disclosed herein comprises a 5' cap, such as a Cap0, Cap1, or Cap2. A 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5'-triphosphate to the 5' position of the first nucleotide of the 5'-to-3' chain of the mRNA, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2'-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as "non-self" by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA.

[0354] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap in which the 2' position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) "Synthesis and properties of mRNAs containing the novel `anti-reverse` cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG," RNA 7: 1486-1495. The ARCA structure is shown below.

##STR00001##

[0355] CleanCap.TM. AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap.TM. GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally. 3'-O-methylated versions of CleanCap.TM. AG and CleanCap.TM. GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively. The CleanCap.TM. AG structure is shown below.

##STR00002##

[0356] Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479. For additional discussion of caps and capping approaches, see, e.g., WO2017/053297 and Ishikawa et al., Nucl. Acids. Symp. Ser. (2009) No. 53, 129-130.

[0357] In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some instances, the poly-A tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more locations within the poly-A tail. The poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide. As used herein, "non-adenine nucleotides" refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or a sequence of interest. In some instances, the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals.

[0358] In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is after one, two, three, four, five, six, or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.

[0359] The poly-A tail may comprise one sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

[0360] In some embodiments, the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some instances, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0361] In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some instances, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some instances, where more than one non-adenine nucleotide is present, the non-adenine nucleotide may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 4.

[0362] In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some instances, the poly-A tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more locations within the poly-A tail. The poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide. As used herein, "non-adenine nucleotides" refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest. In some instances, the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals.

[0363] In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is after one, two, three, four, five, six, or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.

[0364] The poly-A tail of the present invention may comprise one sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

[0365] In some embodiments, the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some instances, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0366] In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some instances, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some instances, where more than one non-adenine nucleotide is present, the non-adenine nucleotide may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 4:

TABLE-US-00005 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAAAAAAAAAAAA AAAAAAAAAAAAACCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAA.

[0367] Chemical modifications such as those listed above can be combined to provide modified gRNAs and/or mRNAs comprising nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA.

[0368] In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified gRNA are modified nucleosides or nucleotides.

[0369] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0370] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0371] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[0372] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0373] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0374] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[0375] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include "locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C.sub.1-6 alkylene or C.sub.1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH.sub.2).sub.n-amino, (wherein amino can be, e.g., NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can included "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, e.g., a PEG derivative).

[0376] "Deoxy" 2' modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-- amino (wherein amino can be, e.g., as described herein), --NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[0377] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.

[0378] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

[0379] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification. In certain embodiments, one or more or all of the nucleotides in single stranded overhang of a guide RNA molecule are deoxynucleotides.

[0380] In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in U.S. 62/431,756, filed Dec. 8, 2016, titled "Chemically Modified Guide RNAs," the contents of which are hereby incorporated by reference in their entirety.

[0381] In some embodiments, the invention comprises a gRNA comprising one or more modifications. In some embodiments, the modification comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides.

[0382] The terms "mA," "mC," "mU," or "mG" may be used to denote a nucleotide that has been modified with 2'-O-Me.

[0383] Modification of 2'-O-methyl can be depicted as follows:

##STR00003##

[0384] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2'-fluoro (2'-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

[0385] In this application, the terms "fA," "fC," "fU," or "fG" may be used to denote a nucleotide that has been substituted with 2'-F.

[0386] Substitution of 2'-F can be depicted as follows:

##STR00004##

[0387] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[0388] A "*" may be used to depict a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS bond.

[0389] In this application, the terms "mA*," "mC*," "mU*," or "mG*" may be used to denote a nucleotide that has been substituted with 2'-O-Me and that is linked to the next (e.g., 3') nucleotide with a PS bond.

[0390] The diagram below shows the substitution of S-- into a nonbridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

##STR00005##

[0391] Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base:

##STR00006##

[0392] Inverted bases refer to those with linkages that are inverted from the normal 5' to 3' linkage (i.e., either a 5' to 5' linkage or a 3' to 3' linkage). For example:

##STR00007##

[0393] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5' nucleotide via a 5' to 5' linkage, or an abasic nucleotide may be attached to the terminal 3' nucleotide via a 3' to 3' linkage. An inverted abasic nucleotide at either the terminal 5' or 3' nucleotide may also be called an inverted abasic end cap.

[0394] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2'-O-Me, 2'-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.

[0395] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[0396] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise an inverted abasic nucleotide.

[0397] In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID No: 3, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence that directs a nuclease to a target sequence.

[0398] In some embodiments, the guide RNA comprises a sgRNA shown in any one of SEQ ID No: 87-124. In some embodiments, the guide RNA comprises a sgRNA comprising any one of the guide sequences of SEQ ID No: 5-82 and the nucleotides of SEQ ID No: 125, wherein the nucleotides of SEQ ID No: 125 are on the 3' end of the guide sequence, and wherein the guide sequence may be modified as shown in SEQ ID No: 3.

[0399] C. Ribonucleoprotein Complex

[0400] In some embodiments, a composition is encompassed comprising one or more gRNAs comprising one or more guide sequences from Table 1 or one or more sgRNAs from Table 2 and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9. In some embodiments, the encoded RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. For discussion of various CRISPR systems and Cas nucleases see, e.g., Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015).

[0401] Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.

[0402] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.

[0403] In some embodiments, the gRNA together with an RNA-guided DNA binding agent is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the gRNA together with a Cas nuclease is called a Cas RNP. In some embodiments, the RNP comprises Type-I, Type-II, or Type-III components. In some embodiments, the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP.

[0404] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA.

[0405] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments, the Cas9 is capable of inducing a double strand break in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 203. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 204. An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 210.

[0406] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as FokI. In some embodiments, a Cas nuclease may be a modified nuclease.

[0407] In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.

[0408] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 206. An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 207. An exemplary Cas9 nickase mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 211.

[0409] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.

[0410] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell October 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB--A0Q7Q2 (CPF1_FRATN)).

[0411] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[0412] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980 A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 208. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 209. An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 212.

[0413] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0414] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-10 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. In some embodiments, the RNA-guided DNA-binding agent may be fused C-terminally to at least one NLS. An NLS may also be inserted within the RNA-guided DNA binding agent sequence. In other embodiments, the RNA-guided DNA-binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 274) or PKKKRRV (SEQ ID NO: 275). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 276). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 274) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site. In some embodiments, one or more NLS(s) according to any of the foregoing embodiments are present in the RNA-guided DNA-binding agent in combination with one or more additional heterologous functional domains, such as any of the heterologous functional domains described below.

[0415] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub 1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).

[0416] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6.times.His, 8.times.His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0417] In additional embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria.

[0418] In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression," Cell 152:1173-83 (2013); Perez-Pinera et al., "RNA-guided gene activation by CRISPR-Cas9-based transcription factors," Nat. Methods 10:973-6 (2013); Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes," Cell 154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.

[0419] D. Determination of Efficacy of gRNAs

[0420] In some embodiments, the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the gRNA is expressed together with an RNA-guided DNA nuclease, such as a Cas protein. In some embodiments, the gRNA is delivered to or expressed in a cell line that already stably expresses an RNA-guided DNA nuclease, such as a Cas protein. In some embodiments the gRNA is delivered to a cell as part of a RNP. In some embodiments, the gRNA is delivered to a cell along with a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease.

[0421] As described herein, use of an RNA-guided DNA nuclease and a guide RNA disclosed herein can lead to double-stranded breaks in the DNA which can produce errors in the form of insertion/deletion (indel) mutations upon repair by cellular machinery. Many mutations due to indels alter the reading frame or introduce premature stop codons and, therefore, produce a non-functional protein.

[0422] In some embodiments, the efficacy of particular gRNAs is determined based on in vitro models. In some embodiments, the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments, the in vitro model is HUH7 human hepatocarcinoma cells. In some embodiments, the in vitro model is HepG2 cells. In some embodiments, the in vitro model is primary human hepatocytes. In some embodiments, the in vitro model is primary cynomolgus hepatocytes. With respect to using primary human hepatocytes, commercially available primary human hepatocytes can be used to provide greater consistency between experiments. In some embodiments, the number of off-target sites at which a deletion or insertion occurs in an in vitro model (e.g., in primary human hepatocytes) is determined, e.g., by analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA. In some embodiments, such a determination comprises analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide. Exemplary procedures for such determinations are provided in the working examples below.

[0423] In some embodiments, the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process. In some embodiments, a cell line comparison of data with selected gRNAs is performed. In some embodiments, cross screening in multiple cell models is performed.

[0424] In some embodiments, the efficacy of particular gRNAs is determined based on in vivo models. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse which expresses a human TTR gene, which may be a mutant human TTR gene. In some embodiments, the in vivo model is a non-human primate, for example cynomolgus monkey.

[0425] In some embodiments, the efficacy of a guide RNA is measured by percent editing of TTR. In some embodiments, the percent editing of TTR is compared to the percent editing necessary to achieve knockdown of TTR protein, e.g., in the cell culture media in the case of an in vitro model or in serum or tissue in the case of an in vivo model.

[0426] In some embodiments, the efficacy of a guide RNA is measured by the number and/or frequency of indels at off-target sequences within the genome of the target cell type. In some embodiments, efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g., <5%) in a cell population and/or relative to the frequency of indel creation at the target site. Thus, the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., a hepatocyte), or which produce a frequency of off-target indel formation of <5% in a cell population and/or relative to the frequency of indel creation at the target site. In some embodiments, the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., hepatocyte). In some embodiments, guide RNAs are provided which produce indels at less than 5 off-target sites, e.g., as evaluated by one or more methods described herein. In some embodiments, guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 off-target site(s) e.g., as evaluated by one or more methods described herein. In some embodiments, the off-target site(s) does not occur in a protein coding region in the target cell (e.g., hepatocyte) genome.

[0427] In some embodiments, detecting gene editing events, such as the formation of insertion/deletion ("indel") mutations and homology directed repair (HDR) events in target DNA utilize linear amplification with a tagged primer and isolating the tagged amplification products (herein after referred to as "LAM-PCR," or "Linear Amplification (LA)" method).

[0428] In some embodiments, the method comprises isolating cellular DNA from a cell that has been induced to have a double strand break (DSB) and optionally that has been provided with an HDR template to repair the DSB; performing at least one cycle of linear amplification of the DNA with a tagged primer; isolating the linear amplification products that comprise tag, thereby discarding any amplification product that was amplified with a non-tagged primer; optionally further amplifying the isolated products; and analyzing the linear amplification products, or the further amplified products, to determine the presence or absence of an editing event such as, for example, a double strand break, an insertion, deletion, or HDR template sequence in the target DNA. In some instances, the editing event can be quantified. Quantification and the like as used herein (including in the context of HDR and non-HDR editing events such as indels) includes detecting the frequency and/or type(s) of editing events in a population.

[0429] In some embodiments, only one cycle of linear amplification is conducted.

[0430] In some instances, the tagged primer comprises a molecular barcode. In some embodiments, the tagged primer comprises a molecular barcode, and only one cycle of linear amplification is conducted.

[0431] In some embodiments, the analyzing step comprises sequencing the linear amplified products or the further amplified products. Sequencing may comprise any method known to those of skill in the art, including, next generation sequencing, and cloning the linear amplification products or further amplified products into a plasmid and sequencing the plasmid or a portion of the plasmid. In other aspects, the analyzing step comprises performing digital PCR (dPCR) or droplet digital PCR (ddPCR) on the linear amplified products or the further amplified products. In other instances, the analyzing step comprises contacting the linear amplified products or the further amplified products with a nucleic acid probe designed to identify DNA comprising HDR template sequence and detecting the probes that have bound to the linear amplified product(s) or further amplified product(s). In some embodiments, the method further comprises determining the location of the HDR template in the target DNA.

[0432] In certain embodiments, the method further comprises determining the sequence of an insertion site in the target DNA, wherein the insertion site is the location where the HDR template incorporates into the target DNA, and wherein the insertion site may include some target DNA sequence and some HDR template sequence.

[0433] In some embodiments, the linear amplification of the target DNA with a tagged primer is performed for 1-50 cycles, 1-60 cycles, 1-70 cycles, 1-80 cycles, 1-90 cycles, or 1-100 cycles.

[0434] In some embodiments, the linear amplification of the target DNA with a tagged primer comprises a denaturation step to separate DNA duplexes, an annealing step to allow primer binding, and an elongation step. In some embodiments, the linear amplification is isothermal (does not require a change in temperature). In some embodiments, the isothermal linear amplification is a loop-mediated isothermal amplification (LAMP), a strand displacement amplification (SDA), a helicase-dependent amplification, or a nicking enzyme amplification reaction.

[0435] In some embodiments, the tagged primer anneals to the target DNA at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 1,000, at least 5,000, or at least 10,000 nucleotides away from of the expected editing event location, e.g., the insertion, deletion, or template insertion site.

[0436] In some embodiments, the tagged primer comprises a molecular barcode. In some embodiments, the molecular barcode comprises a sequence that is not complementary to the target DNA. In some embodiments, the molecular barcode comprises 6, 8, 10, or 12 nucleotides.

[0437] In some embodiments, the tag on the primer is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescein isothiocyanate (FITC).

[0438] In some embodiments, the linear amplification product(s) are isolated using a capture reagent specific for the tag on the primer. In some embodiments, the capture reagent is on a bead, solid support, matrix, or column. In some embodiments, the isolation step comprises contacting the linear amplification product(s) with a capture reagent specific for the tag on the primer. In some embodiments, the capture reagent is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescein isothiocyanate (FITC).

[0439] In some embodiments, the tag is biotin and capture reagent is streptavidin. In some embodiments, the tag is streptavidin and the capture reagent is biotin. In some embodiments, the tag is on the 5' terminus of the primer, the 3' terminus of the primer, or internal to the primer. In some embodiments, the tag and/or the capture reagent is removed after the isolation step. In some embodiments, the tag and/or the capture reagent is not removed, and the further amplifying and analyzing steps are performed in the presence of tag and/or capture.

[0440] In some embodiments, the further amplification is non-linear. In some embodiments, the further amplification is digital PCR, qPCR, or RT-PCR. In some embodiments, the sequencing is next generation sequencing (NGS).

[0441] In some embodiments, the target DNA is genomic or mitochondrial. In some embodiments, the target DNA is genomic DNA of a prokaryotic or eukaryotic cell. In some embodiments, the target DNA is mammalian. The target DNA may be from a non-dividing cell or a dividing cell. In some embodiments, the target DNA may be from a primary cell. In some embodiments, the target DNA is from a replicating cell.

[0442] In some instances, the cellular DNA is sheared prior to linear amplification. In some embodiments, the sheared DNA has an average size between 0.5 kb and 20 kb. In some instances, the cellular DNA is sheared to an average size of 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12.0, 12.25, 12.5, 12.75, 13.0, 13.25, 13.5, 13.75, 14.0, 14.25, 14.5, 14.75, 15.0, 15.25, 15.5, 15.75, 16.0, 16.25, 16.5, 16.75, 17.0, 17.25, 17.5, 17.75, 18.0, 18.25, 18.5, 18.75, 19.0, 19.25, 19.5, 19.75, or 20.0 kb. In some instances, the cellular DNA is sheared to an average size of about 1.5 kb.

[0443] In some embodiments, the efficacy of a guide RNA is measured by secretion of TTR. In some embodiments, secretion of TTR is measured using an enzyme-linked immunosorbent assay (ELISA) assay with cell culture media or serum. In some embodiments, secretion of TTR is measured in the same in vitro or in vivo systems or models used to measure editing. In some embodiments, secretion of TTR is measured in primary human hepatocytes. In some embodiments, secretion of TTR is measured in HUH7 cells. In some embodiments, secretion of TTR is measured in HepG2 cells.

[0444] ELISA assays are generally known to the skilled artisan and can be designed to determine serum TTR levels. In one exemplary embodiment, blood is collected and the serum is isolated. The total TTR serum levels may be determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111) or similar kit for measuring human TTR. If no kit is available, an ELISA can be developed using plates that are pre-coated with capture antibody specific for the TTR one is measuring. The plate is next incubated at room temperature for a period of time before washing. Enzyme-anti-TTR antibody conjugate is added and inncubated. Unbound antibody conjugate is removed and the plate washed before the addition of the chromogenic substrate solution that reactes with the enzyme. The plate is read on an appropriate plate reader at an absorbance specific for the enzyme and substrate used.

[0445] In some embodiments, the amount of TTR in cells (including those from tissue) measures efficacy of a gRNA. In some embodiments, the amount of TTR in cells is measured using western blot. In some embodiments, the cell used is HUH7 cells. In some embodiments, the cell used is a primary human hepatocyte. In some embodiments, the cell used is a primar cell obtained from an animal. In some embodiments, the amount of TTR is compared to the amount of glyceraldehyde 3-phosphate dehydrogenase GAPDH (a housekeeping gene) to control for changes in cell number.

III. LNP Formulations and Treatment of ATTR

[0446] In some embodiments, a method of inducing a double-stranded break (DSB) within the TTR gene is provided comprising administering a composition comprising a guide RNA comprising any one or more guide sequences of SEQ ID Nos: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID Nos: 5-82 are administered to induce a DSB in the TTR gene. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

[0447] In some embodiments, a method of modifying the TTR gene is provided comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID Nos: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID Nos: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124, are administered to modify the TTR gene. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

[0448] In some embodiments, a method of treating ATTR is provided comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124 are administered to treat ATTR. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

[0449] In some embodiments, a method of reducing TTR serum concentration is provided comprising administering a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 5-82 or any one or more of the sgRNAs of SEQ ID Nos: 87-124 are administered to reduce or prevent the accumulation of TTR in amyloids or amyloid fibrils. The gRNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

[0450] In some embodiments, a method of reducing or preventing the accumulation of TTR in amyloids or amyloid fibrils of a subject is provided comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs: 5-82, or any one or more of the sgRNAs of SEQ ID Nos: 87-124. In some embodiments, a method of reducing or preventing the accumulation of TTR in amyloids or amyloid fibrils of a subject is provided comprising administering a composition comprising any one or more of the sgRNAs of SEQ ID Nos: 87-113. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 5-82 or any one or more of the sgRNAs of SEQ ID Nos: 87-124 are administered to reduce or prevent the accumulation of TTR in amyloids or amyloid fibrils. The gRNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9).

[0451] In some embodiments, the gRNAs comprising the guide sequences of Table 1 or one or more sgRNAs from Table 2 together with an RNA-guided DNA nuclease such as a Cas nuclease induce DSBs, and non-homologous ending joining (NHEJ) during repair leads to a mutation in the TTR gene. In some embodiments, NHEJ leads to a deletion or insertion of a nucleotide(s), which induces a frame shift or nonsense mutation in the TTR gene.

[0452] In some embodiments, administering the guide RNAs of the invention (e.g., in a composition provided herein) reduces levels (e.g., serum levels) of TTR in the subject, and therefore prevents accumulation and aggregation of TTR in amyloids or amyloid fibrils.

[0453] In some embodiments, reducing or preventing the accumulation of TTR in amyloids or amyloid fibrils of a subject comprises reducing or preventing TTR deposition in one or more tissues of the subject, such as stomach, colon, or nervous tissue. In some embodiments, the nervous tissue comprises sciatic nerve or dorsal root ganglion. In some embodiments, TTR deposition is reduced in two, three, or four of the stomach, colon, dorsal root ganglion, and sciatic nerve. The level of deposition in a given tissue can be determined using a biopsy sample, e.g., using immunostaining. In some embodiments, reducing or preventing the accumulation of TTR in amyloids or amyloid fibrils of a subject and/or reducing or preventing TTR deposition is inferred based on reducing serum TTR levels for a period of time. As discussed in the examples, it has been found that reducing serum TTR levels in accordance with methods and uses provided herein can result in clearance of deposited TTR from tissues such as those discussed above and in the examples, e.g., as measured 8 weeks after administration of the composition.

[0454] In some embodiments, the subject is mammalian. In some embodiments, the subject is human. In some embodiments, the subject is cow, pig, monkey, sheep, dog, cat, fish, or poultry.

[0455] In some embodiments, the use of a guide RNAs comprising any one or more of the guide sequences in Table 1 or one or more sgRNAs from Table 2 (e.g., in a composition provided herein) is provided for the preparation of a medicament for treating a human subject having ATTR.

[0456] In some embodiments, the guide RNAs, compositions, and formulations are administered intravenously. In some embodiments, the guide RNAs, compositions, and formulations are administered into the hepatic circulation.

[0457] In some embodiments, a single administration of a composition comprising a guide RNA provided herein is sufficient to knock down expression of the mutant protein. In some embodiments, a single administration of a composition comprising a guide RNA provided herein is sufficient to knock out expression of the mutant protein in a population of cells. In other embodiments, more than one administration of a composition comprising a guide RNA provided herein may be beneficial to maximize editing via cumulative effects.

[0458] For example, a composition provided herein can be administered 2, 3, 4, 5, or more times, such as 2 times. Administrations can be separated by a period of time ranging from, e.g., 1 day to 2 years, such as 1 to 7 days, 7 to 14 days, 14 days to 30 days, 30 days to 60 days, 60 days to 120 days, 120 days to 183 days, 183 days to 274 days, 274 days to 366 days, or 366 days to 2 years.

[0459] In some embodiments, a composition is administered in an effective amount in the range of 0.01 to 10 mg/kg (mpk), e.g., 0.01 to 0.1 mpk, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, or 10 mpk.

[0460] In some embodiments, the efficacy of treatment with the compositions of the invention is seen at 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years after delivery. In some embodiments, efficacy of treatment with the compositions of the invention is assessed by measuring serum levels of TTR before and after treatment. In some embodiments, efficacy of treatment with the compositions assessed via a reduction of serum levels of TTR is seen at 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or at 11 months.

[0461] In some embodiments, treatment slows or halts disease progression.

[0462] In some embodiments, treatment slows or halts progression of FAP. In some embodiments, treatment results in improvement, stabilization, or slowing of change in symptoms of sensorimotor neuropathy or autonomic neuropathy.

[0463] In some embodiments, treatment results in improvement, stabilization, or slowing of change in symptoms of FAC. In some embodiments, treatment results in improvement, stabilization, or slowing of change symptoms of restrictive cardiomyopathy or congestive heart failure.

[0464] In some embodiments, efficacy of treatment is measured by increased survival time of the subject.

[0465] In some embodiments, efficacy of treatment is measured by improvement or slowing of progression in symptoms of sensorimotor or autonomic neuropathy. In some embodiments, efficacy of treatment is measured by an increase or a slowing of decrease in ability to move an area of the body or to feel in any area of the body. In some embodiments, efficacy of treatment is measured by improvement or a slowing of decrease in the ability to swallow; breath; use arms, hands, legs, or feet; or walk. In some embodiments, efficacy of treatment is measured by improvement or a slowing of progression of neuralgia. In some embodiments, the neuralgia is characterized by pain, burning, tingling, or abnormal feeling.

[0466] In some embodiments, efficacy of treatment is measured by improvement or a slowing of increase in postural hypotension, dizziness, gastrointestinal dysmotility, bladder dysfunction, or sexual dysfunction. In some embodiments, efficacy of treatment is measured by improvement or a slowing of progression of weakness. In some embodiments, efficacy of treatment is measured using electromyogram, nerve conduction tests, or patient-reported outcomes.

[0467] In some embodiments, efficacy of treatment is measured by improvement or slowing of progression of symptoms of congestive heart failure or CHF. In some embodiments, efficacy of treatment is measured by an decrease or a slowing of increase in shortness of breath, trouble breathing, fatigue, or swelling in the ankles, feet, legs, abdomen, or veins in the neck. In some embodiments, efficacy of treatment is measured by improvement or a slowing of progression of fluid buildup in the body, which may be assessed by measures such as weight gain, frequent urination, or nighttime cough. In some embodiments, efficacy of treatment is measured using cardiac biomarker tests (such as B-type natriuretic peptide [BNP] or N-terminal pro b-type natriuretic peptide [NT-proBNP]), lung function tests, chest x-rays, or electrocardiography.

[0468] A. Combination Therapy

[0469] In some embodiments, the invention comprises combination therapies comprising any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein) together with an additional therapy suitable for alleviating symptoms of ATTR.

[0470] In some embodiments, the additional therapy for ATTR is a treatment for sensorimotor or autonomic neuropathy. In some embodiments, the treatment for sensorimotor or autonomic neuropathy is a nonsteroidal anti-inflammatory drug, antidepressant, anticonvulsant medication, antiarrythmic medication, or narcotic agent. In some embodiments, the antidepressant is a tricylic agent or a serotonin-norepinephrine reuptake inhibitor. In some embodiments, the antidepressant is amitriptyline, duloxetine, or venlafaxine. In some embodiments, the anticonvulsant agent is gabapentin, pregabalin, topiramate, or carbamazepine. In some embodiments, the additional therapy for sensorimotor neuropathy is transcutaneous electrical nerve stimulation.

[0471] In some embodiments, the additional therapy for ATTR is a treatment for restrictive cardiomyopathy or congestive heart failure (CHF). In some embodiments, the treatment for CHF is a ACE inhibitor, aldosterone antagonist, angiotensin receptor blocker, beta blocker, digoxin, diuretic, or isosorbide dinitrate/hydralazine hydrochloride. In some embodiments, the ACE inhibitor is enalapril, captopril, ramipril, perindopril, imidapril, or quinapril. In some embodiments, the aldosterone antagonist is eplerenone or spironolactone. In some embodiments, the angiotensin receptor blocker is azilsartan, cadesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan. In some embodiments, the beta blocker is acebutolol, atenolol, bisoprolol, metoprolol, nadolol, nebivolol, or propranolol. In some embodiments, the diuretic is chlorothiazide, chlorthalidone, hydrochlorothiazide, indapamide, metolazone, bumetanide, furosemide, torsemide, amiloride, or triameterene.

[0472] In some embodiments, the combination therapy comprises any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein) together with a siRNA that targets TTR or mutant TTR. In some embodiments, the siRNA is any siRNA capable of further reducing or eliminating the expression of wild type or mutant TTR. In some embodiments, the siRNA is the drug Patisiran (ALN-TTR02) or ALN-TTRsc02. In some embodiments, the siRNA is administered after any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein). In some embodiments, the siRNA is administered on a regular basis following treatment with any of the gRNA compositions provided herein.

[0473] In some embodiments, the combination therapy comprises any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein) together with antisense nucleotide that targets TTR or mutant TTR. In some embodiments, the antisense nucleotide is any antisense nucleotide capable of further reducing or eliminating the expression of wild type or mutant TTR. In some embodiments, the antisense nucleotide is the drug Inotersen (IONS-TTRRX). In some embodiments, the antisense nucleotide is administered after any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein). In some embodiments, the antisense nucleotide is administered on a regular basis following treatment with any of the gRNA compositions provided herein.

[0474] In some embodiments, the combination therapy comprises any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein) together with a small molecule stabilizer that promotes kinetic stabilization of the correctly folded tetrameric form of TTR. In some embodiments, the small molecule stabilizer is the drug tafamidis (Vyndaqel.RTM.) or diflunisal. In some embodiments, the small molecule stabilizer is administered after any one of the gRNAs comprising any one or more of the guide sequences disclosed in Table 1 or any one or more of the sgRNAs in Table 2 (e.g., in a composition provided herein). In some embodiments, the small molecule stabilizer is administered on a regular basis following treatment with any of the gRNA compositions provided herein.

[0475] B. Delivery of gRNA Compositions

[0476] In some embodiments, the guide RNA compositions described herein, alone or encoded on one or more vectors, are formulated in or administered via a lipid nanoparticle; see e.g., PCT/US2017/024973, filed Mar. 30, 2017 entitled "LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS," the contents of which are hereby incorporated by reference in their entirety. Any lipid nanoparticle (LNP) known to those of skill in the art to be capable of delivering nucleotides to subjects may be utilized with the guide RNAs described herein, as well as either mRNA encoding an RNA-guided DNA nuclease such as Cas or Cas9, or an RNA-guided DNA nuclease such as Cas or Cas9 protein itself.

[0477] Disclosed herein are various embodiments of LNP formulations for RNAs, including CRISPR/Cas cargoes. Such LNP formulations may include (i) a CCD lipid, such as an amine lipid, (ii) a neutral lipid, (iii) a helper lipid, and (iv) a stealth lipid, such as a PEG lipid. Some embodiments of the LNP formulations include an "amine lipid", along with a helper lipid, a neutral lipid, and a stealth lipid such as a PEG lipid. By "lipid nanoparticle" is meant a particle that comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces.

[0478] CCD Lipids

[0479] Lipid compositions for delivery of CRISPR/Cas mRNA and guide RNA components to a liver cell comprise a CCD Lipid.

[0480] In some embodiments, the CCD lipid is Lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy- )carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl- )oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

##STR00008##

[0481] Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86).

[0482] In some embodiments, the CCD lipid is Lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bi- s(decanoate), also called ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) bis(decanoate). Lipid B can be depicted as:

##STR00009##

[0483] Lipid B may be synthesized according to WO2014/136086 (e.g., pp. 107-09).

[0484] In some embodiments, the CCD lipid is Lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1- ,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate). Lipid C can be depicted as:

##STR00010##

[0485] In some embodiments, the CCD lipid is Lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate.

[0486] Lipid D can be depicted as:

##STR00011##

[0487] Lipid C and Lipid D may be synthesized according to WO2015/095340.

[0488] The CCD lipid can also be an equivalent to Lipid A, Lipid B, Lipid C, or Lipid D. In certain embodiments, the CCD lipid is an equivalent to Lipid A, an equivalent to Lipid B, an equivalent to Lipid C, or an equivalent to Lipid D.

[0489] Amine Lipids

[0490] In some embodiments, the LNP compositions for the delivery of biologically active agents comprise an "amine lipid", which is defined as Lipid A, Lipid B, Lipid C, Lipid D or equivalents of Lipid A (including acetal analogs of Lipid A), equivalents of Lipid B, equivalents of Lipid C, and equivalents of Lipid D.

[0491] In some embodiments, the amine lipid is Lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy- )carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl- )oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

##STR00012##

[0492] Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86). In certain embodiments, the amine lipid is an equivalent to Lipid A.

[0493] In certain embodiments, an amine lipid is an analog of Lipid A. In certain embodiments, a Lipid A analog is an acetal analog of Lipid A. In particular LNP compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11, and C12 acetal analog.

[0494] Amine lipids suitable for use in the LNPs described herein are biodegradable in vivo. The amine lipids have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg/kg). In certain embodiments, LNPs comprising an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipid include those where at least 50% of the mRNA or gRNA is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipid include those where at least 50% of the LNP is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example by measuring a lipid (e.g. an amine lipid), RNA (e.g. mRNA), or other component. In certain embodiments, lipid-encapsulated versus free lipid, RNA, or nucleic acid component of the LNP is measured.

[0495] Lipid clearance may be measured as described in literature. See Maier, M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 ("Maier"). For example, in Maier, LNP-siRNA systems containing luciferases-targeting siRNA were administered to six- to eight-week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolus injection via the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-dose. Mice were perfused with saline before tissue collection and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Further, Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, a luciferase-targeting siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours, about 1 mL of blood was obtained from the jugular vein of conscious animals and the serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy. Assessment of clinical signs, body weight, serum chemistry, organ weights and histopathology was performed. Although Maier describes methods for assessing siRNA-LNP formulations, these methods may be applied to assess clearance, pharmacokinetics, and toxicity of administration of LNP compositions of the present disclosure.

[0496] The amine lipids lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example the rate at which an amine lipid is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example the rate at which an mRNA or a gRNA is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high rate of clearance rate leads to a safety profile with no substantial adverse effects. The amine lipids reduce LNP accumulation in circulation and in tissues. In some embodiments, a reduction in LNP accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects.

[0497] The amine lipids of the present disclosure may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the amine lipids may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the amine lipids may not be protonated and thus bear no charge. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 10.

[0498] The ability of an amine lipid to bear a charge is related to its intrinsic pKa. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.2. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5. This may be advantageous as it has been found that cationic lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g. to the liver. Further, it has been found that cationic lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g. to tumors. See, e.g., WO2014/136086.

[0499] Additional Lipids

[0500] "Neutral lipids" suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine di stearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0501] "Helper lipids" include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.

[0502] "Stealth lipids" are lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, e.g., in WO 2006/007712.

[0503] In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG. Stealth lipids may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

[0504] In one embodiment, a stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly[N-(2-hydroxypropyl)methacrylamide].

[0505] In one embodiment, the PEG lipid comprises a polymer moiety based on PEG (sometimes referred to as poly(ethylene oxide)).

[0506] The PEG lipid further comprises a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some embodiments, the alkyl chail length comprises about C10 to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or assymetric.

[0507] Unless otherwise indicated, the term "PEG" as used herein means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the term includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment, about 150 to about 30,000, in a sub-embodiment, about 150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in a sub-embodiment, about 150 to about 10,000, in a sub-embodiment, about 150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in a sub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150 to about 3,000, in a sub-embodiment, about 300 to about 3,000, in a sub-embodiment, about 1,000 to about 3,000, and in a sub-embodiment, about 1,500 to about 2,500.

[0508] In certain embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a "PEG-2K," also termed "PEG 2000," which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art may be used, including, e.g., those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[0509] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-di stearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-di stearoylglycamide, PEG-cholesterol (1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- -[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Ala., USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Ala., USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound 5027, disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

[0510] LNP Formulations

[0511] The LNP may contain (i) an amine lipid for encapsulation and for endosomal escape, (ii) a neutral lipid for stabilization, (iii) a helper lipid, also for stabilization, and (iv) a stealth lipid, such as a PEG lipid.

[0512] In some embodiments, an LNP composition may comprise an RNA component that includes one or more of an RNA-guided DNA-binding agent, a Cas nuclease mRNA, a Class 2 Cas nuclease mRNA, a Cas9 mRNA, and a gRNA. In some embodiments, an LNP composition may include a Class 2 Cas nuclease and a gRNA as the RNA component. In certain embodiments, an LNP composition may comprise the RNA component, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the helper lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In additional embodiments, the stealth lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the LNP composition comprises Lipid A or an equivalent of Lipid A; a helper lipid; a neutral lipid; a stealth lipid; and a guide RNA. In certain compositions, the amine lipid is Lipid A. In certain compositions, the amine lipid is Lipid A or an acetal analog thereof; the helper lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG.

[0513] In certain embodiments, lipid compositions are described according to the respective molar ratios of the component lipids in the formulation. Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mol-% of the amine lipid may be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 40 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 45 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 55 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 55 mol-%. In one embodiment, the mol-% of the amine lipid may be about 50 mol-%. In one embodiment, the mol-% of the amine lipid may be about 55 mol-%. In some embodiments, the amine lipid mol-% of the LNP batch will be .+-.30%, .+-.25%, .+-.20%, .+-.15%, .+-.10%, .+-.5%, or .+-.2.5% of the target mol-%. In some embodiments, the amine lipid mol-% of the LNP batch will be .+-.4 mol-%, .+-.3 mol-%, .+-.2 mol-%, .+-.1.5 mol-%, .+-.1 mol-%, .+-.0.5 mol-%, or .+-.0.25 mol-% of the target mol-%. All mol-% numbers are given as a fraction of the lipid component of the LNP compositions. In certain embodiments, LNP inter-lot variability of the amine lipid mol-% will be less than 15%, less than 10% or less than 5%.

[0514] In one embodiment, the mol-% of the neutral lipid may be from about 5 mol-% to about 15 mol-%. In one embodiment, the mol-% of the neutral lipid may be from about 7 mol-% to about 12 mol-%. In one embodiment, the mol-% of the neutral lipid may be about 9 mol-%. In some embodiments, the neutral lipid mol-% of the LNP batch will be .+-.30%, .+-.25%, .+-.20%, .+-.15%, .+-.10%, .+-.5%, or .+-.2.5% of the target neutral lipid mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[0515] In one embodiment, the mol-% of the helper lipid may be from about 20 mol-% to about 60 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 55 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 30 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 30 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid, neutral lipid, and PEG lipid concentrations to bring the lipid component to 100 mol-%. In some embodiments, the helper mol-% of the LNP batch will be .+-.30%, .+-.25%, .+-.20%, .+-.15%, .+-.10%, .+-.5%, or .+-.2.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[0516] In one embodiment, the mol-% of the PEG lipid may be from about 1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 8 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2.5 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be about 3 mol-%. In one embodiment, the mol-% of the PEG lipid may be about 2.5 mol-%. In some embodiments, the PEG lipid mol-% of the LNP batch will be .+-.30%, .+-.25%, .+-.20%, .+-.15%, .+-.10%, .+-.5%, or .+-.2.5% of the target PEG lipid mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[0517] In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA-binding agent (e.g. a Cas nuclease, a Class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA. In one embodiment, an LNP composition may comprise a Lipid A or its equivalents. In some aspects, the amine lipid is Lipid A. In some aspects, the amine lipid is a Lipid A equivalent, e.g. an analog of Lipid A. In certain aspects, the amine lipid is an acetal analog of Lipid A. In various embodiments, an LNP composition comprises an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition may comprise a Lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, an LNP composition comprises an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from Lipid A, and an equivalent of Lipid A, including an acetal analog of Lipid A. In additional embodiments, an LNP composition comprises Lipid A, cholesterol, DSPC, and PEG2k-DMG.

[0518] Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N/P. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N/P ratio is about 3 to 10. In one embodiment, the N/P ratio may about 5-7. In one embodiment, the N/P ratio may about 4.5-8. In one embodiment, the N/P ratio may about 6. In one embodiment, the N/P ratio may be 6.+-.1. In one embodiment, the N/P ratio may about 6.+-.0.5. In some embodiments, the N/P ratio will be .+-.30%, .+-.25%, .+-.20%, .+-.15%, .+-.10%, .+-.5%, or .+-.2.5% of the target N/P ratio. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[0519] In some embodiments, the RNA component may comprise an mRNA, such as an mRNA disclosed herein, e.g., encoding a Cas nuclease. In one embodiment, RNA component may comprise a Cas9 mRNA. In some compositions comprising an mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises a Class 2 Cas nuclease mRNA and a gRNA.

[0520] In certain embodiments, an LNP composition may comprise an mRNA disclosed herein, e.g., encoding a Cas nuclease, such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain LNP compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In specific compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A.

[0521] In some embodiments, an LNP composition may comprise a gRNA. In certain embodiments, an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain LNP compositions comprising a gRNA, the helper lipid is cholesterol. In some compositions comprising a gRNA, the neutral lipid is DSPC. In additional embodiments comprising a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A.

[0522] In one embodiment, an LNP composition may comprise an sgRNA. In one embodiment, an LNP composition may comprise a Cas9 sgRNA. In one embodiment, an LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC. In additional embodiments comprising an sgRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A.

[0523] In certain embodiments, an LNP composition comprises an mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA. In one embodiment, an LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the helper lipid is cholesterol. In some compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease and a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A.

[0524] In certain embodiments, the LNP compositions include a Cas nuclease mRNA, such as a Class 2 Cas mRNA and at least one gRNA. In certain embodiments, the LNP composition includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 25:1 to about 1:25. In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 10:1 to about 1:10. In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 8:1 to about 1:8. As measured herein, the ratios are by weight. In some embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas mRNA from about 5:1 to about 1:5. In some embodiments, ratio range is about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the gRNA to mRNA ratio is about 3:1 or about 2:1 In some embodiments the ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease is about 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:25.

[0525] The LNP compositions disclosed herein may include a template nucleic acid. The template nucleic acid may be co-formulated with an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid may be co-formulated with a guide RNA. In some embodiments, the template nucleic acid may be co-formulated with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid may be formulated separately from an mRNA encoding a Cas nuclease or a guide RNA. The template nucleic acid may be delivered with, or separately from the LNP compositions. In some embodiments, the template nucleic acid may be single- or double-stranded, depending on the desired repair mechanism. The template may have regions of homology to the target DNA, or to sequences adjacent to the target DNA.

[0526] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of LNPs, may be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.7. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer comprises NaCl. In certain embodiments, NaCl is omitted. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary embodiments, compositions contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall formulation is maintained. For example, the final osmolality may be maintained at less than 450 mOsm/L. In further embodiments, the osmolality is between 350 and 250 mOsm/L. Certain embodiments have a final osmolality of 300+/-20 mOsm/L.

[0527] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, tangential flow filtration, or chromatography. The LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, an LNP composition is stored at 2-8.degree. C., in certain aspects, the LNP compositions are stored at room temperature. In additional embodiments, an LNP composition is stored frozen, for example at -20.degree. C. or -80.degree. C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0.degree. C. to about -80.degree. C. Frozen LNP compositions may be thawed before use, for example on ice, at 4.degree. C., at room temperature, or at 25.degree. C. Frozen LNP compositions may be maintained at various temperatures, for example on ice, at 4.degree. C., at room temperature, at 25.degree. C., or at 37.degree. C.

[0528] In some embodiments, an LNP composition has greater than about 80% encapsulation. In some embodiments, an LNP composition has a particle size less than about 120 nm. In some embodiments, an LNP composition has a pdi less than about 0.2. In some embodiments, at least two of these features are present. In some embodiments, each of these three features is present. Analytical methods for determining these parameters are discussed below in the general reagents and methods section.

[0529] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis or chromatography. The LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, the LNP compositions are stored at 2-8.degree. C., in certain aspects, the LNP compositions are stored at room temperature. In additional embodiments, the LNP composition is stored frozen, for example at -20.degree. C. or -80.degree. C. In other embodiments, the LNP composition is stored at a temperature ranging from 0.degree. C. to -80.degree. C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25.degree. C.

[0530] Dynamic Light Scattering ("DLS") can be used to characterize the polydispersity index ("pdi") and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero. In some embodiments, the pdi may range from 0.005 to 0.75. In some embodiments, the pdi may range from 0.01 to 0.5. In some embodiments, the pdi may range from 0.02 to 0.4. In some embodiments, the pdi may range from 0.03 to 0.35. In some embodiments, the pdi may range from 0.1 to 0.35.

[0531] In some embodiments, LNPs disclosed herein have a size of 1 to 250 nm. In some embodiments, the LNPs have a size of 10 to 200 nm. In further embodiments, the LNPs have a size of 20 to 150 nm. In some embodiments, the LNPs have a size of 50 to 150 nm. In some embodiments, the LNPs have a size of 50 to 100 nm. In some embodiments, the LNPs have a size of 50 to 120 nm. In some embodiments, the LNPs have a size of 75 to 150 nm. In some embodiments, the LNPs have a size of 30 to 200 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcts. The data is presented as a weighted-average of the intensity measure. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 50% to 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 50% to 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 70% to 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 90% to 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from 75% to 95%.

[0532] In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating ATTR. In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for reducing or preventing accumulation and aggregation of TTR in amyloids or amyloid fibrils in subjects having ATTR. In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for reducing serum TTR concentration. In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in treating ATTR in a subject, such as a mammal, e.g., a primate such as a human. In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in reducing or preventing accumulation and aggregation of TTR in amyloids or amyloid fibrils in subjects having ATTR, such as a mammal, e.g., a primate such as a human. In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in reducing serum TTR concentration in a subject, such as a mammal, e.g., a primate such as a human.

[0533] Electroporation is also a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and an RNA-guided DNA nuclease such as Cas9 or an mRNA encoding an RNA-guided DNA nuclease such as Cas9.

[0534] In some embodiments, the invention comprises a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is associated with an LNP or not associated with an LNP. In some embodiments, the gRNA/LNP or gRNA is also associated with an RNA-guided DNA nuclease such as Cas9 or an mRNA encoding an RNA-guided DNA nuclease such as Cas9.

[0535] In certain embodiments, the invention comprises DNA or RNA vectors encoding any of the guide RNAs comprising any one or more of the guide sequences described herein. In some embodiments, in addition to guide RNA sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9 or Cpf1. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9. In one embodiment, the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.

[0536] In some embodiments, the crRNA and the trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, the crRNA and the trRNA may be encoded by a contiguous nucleic acid. In some embodiments, the crRNA and the trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the trRNA are encoded by the same strand of a single nucleic acid.

[0537] In some embodiments, the vector may be circular. In other embodiments, the vector may be linear. In some embodiments, the vector may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0538] In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be genetically modified from its wild type counterpart. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some embodiments, the viral vector may have an enhanced transduction efficiency. In some embodiments, the immune response induced by the virus in a host may be reduced. In some embodiments, viral genes (such as, e.g., integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some embodiments, the viral vector may be replication defective. In some embodiments, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some embodiments, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as, e.g., viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell along with the vector system described herein. In other embodiments, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without any helper virus. In some embodiments, the vector system described herein may also encode the viral components required for virus amplification and packaging.

[0539] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In some embodiments, the viral vector is AAV2, AAV3, AAV3B, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAVrh10, or AAVLK03. In other embodiments, the viral vector may a lentivirus vector.

[0540] In some embodiments, the lentivirus may be non-integrating. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high-cloning capacity or "gutless" adenovirus, where all coding viral regions apart from the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal (`I`) are deleted from the virus to increase its packaging capacity. In yet other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper dependent, and in other embodiments it is helper independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30 kb-deleted HSV-1 vector that removes non-essential viral functions does not require helper virus. In additional embodiments, the viral vector may be bacteriophage T4. In some embodiments, the bacteriophage T4 may be able to package any linear or circular DNA or RNA molecules when the head of the virus is emptied. In further embodiments, the viral vector may be a baculovirus vector. In yet further embodiments, the viral vector may be a retrovirus vector. In embodiments using AAV or lentiviral vectors, which have smaller cloning capacity, it may be necessary to use more than one vector to deliver all the components of a vector system as disclosed herein. For example, one AAV vector may contain sequences encoding an RNA-guided DNA nuclease such as a Cas nuclease, while a second AAV vector may contain one or more guide sequences.

[0541] In some embodiments, the vector may be capable of driving expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.

[0542] In some embodiments, the vector may comprise a nucleotide sequence encoding an RNA-guided DNA nuclease such as a nuclease described herein. In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may comprise one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may comprise more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter.

[0543] In some embodiments, the promoter may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On.RTM. promoter (Clontech).

[0544] In some embodiments, the promoter may be a tissue-specific promoter, e.g., a promoter specific for expression in the liver.

[0545] The vector may further comprise a nucleotide sequence encoding the guide RNA described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be non-identical such that they target different target sequences, or may be identical in that they target the same target sequence. In some embodiments where the vectors comprise more than one guide RNA, each guide RNA may have other different properties, such as activity or stability within a complex with an RNA-guided DNA nuclease, such as a Cas RNP complex. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, a 3' UTR, or a 5' UTR. In one embodiment, the promoter may be a tRNA promoter, e.g., tRNA.sup.Lys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human H1 promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the trRNA may be driven by the same promoter. In some embodiments, the crRNA and trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA may be processed from the single transcript to form a double-molecule guide RNA. Alternatively, the crRNA and trRNA may be transcribed into a single-molecule guide RNA (sgRNA). In other embodiments, the crRNA and the trRNA may be driven by their corresponding promoters on the same vector. In yet other embodiments, the crRNA and the trRNA may be encoded by different vectors.

[0546] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding an RNA-guided DNA nuclease such as a Cas nuclease. In some embodiments, expression of the guide RNA and of the RNA-guided DNA nuclease such as a Cas protein may be driven by their own corresponding promoters. In some embodiments, expression of the guide RNA may be driven by the same promoter that drives expression of the RNA-guided DNA nuclease such as a Cas protein. In some embodiments, the guide RNA and the RNA-guided DNA nuclease such as a Cas protein transcript may be contained within a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the RNA-guided DNA nuclease such as a Cas protein transcript. In some embodiments, the guide RNA may be within the 5' UTR of the transcript. In other embodiments, the guide RNA may be within the 3' UTR of the transcript. In some embodiments, the intracellular half-life of the transcript may be reduced by containing the guide RNA within its 3' UTR and thereby shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be within an intron of the transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the RNA-guided DNA nuclease such as a Cas protein and the guide RNA from the same vector in close temporal proximity may facilitate more efficient formation of the CRISPR RNP complex.

[0547] In some embodiments, the compositions comprise a vector system. In some embodiments, the vector system may comprise one single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of the guide RNA are used, the vector system may comprise more than three vectors.

[0548] In some embodiments, the vector system may comprise inducible promoters to start expression only after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On.RTM. promoter (Clontech).

[0549] In additional embodiments, the vector system may comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.

[0550] The vector may be delivered by liposome, a nanoparticle, an exosome, or a microvesicle. The vector may also be delivered by a lipid nanoparticle (LNP); see e.g., U.S. Ser. No. 62/433,228, filed Dec. 12, 2016 and entitled "LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS," the contents of which are hereby incorporated by reference in their entirety. Any of the LNPs and LNP formulations described herein are suitable for delivery of the guides alone or together a cas nuclease or an mRNA encoding a cas nuclease. In some embodiments, an LNP composition is encompassed comprising: an RNA component and a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid; and wherein the N/P ratio is about 1-10.

[0551] In some instances, the lipid component comprises Lipid A or its acetal analog, cholesterol, DSPC, and PEG-DMG; and wherein the N/P ratio is about 1-10. In some embodiments, the lipid component comprises: about 40-60 mol-% amine lipid; about 5-15 mol-% neutral lipid; and about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In some embodiments, the lipid component comprises about 50-60 mol-% amine lipid; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-8. In some instances, the lipid component comprises: about 50-60 mol-% amine lipid; about 5-15 mol-% DSPC; and about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the LNP composition is about 3-8. In some instances, the lipid component comprises: 48-53 mol-% Lipid A; about 8-10 mol-% DSPC; and 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the LNP composition is 3-8.+-.0.2.

[0552] In some embodiments, the vector may be delivered systemically. In some embodiments, the vector may be delivered into the hepatic circulation.

[0553] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about," to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0554] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

Examples

[0555] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

Example 1. Materials and Methods

[0556] In Vitro Transcription ("IVT") of Nuclease mRNA

[0557] Capped and polyadenylated Streptococcus pyogenes ("Spy") Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter, a sequence for transcription according to SEQ ID NO: 1 or 2, and a 100 nt poly (A/T) region was linearized by incubating at 37.degree. C. for 2 hours with XbaI with the following conditions: 200 ng/.mu.L plasmid, 2 U/.mu.L XbaI (NEB), and 1.times. reaction buffer. The XbaI was inactivated by heating the reaction at 65.degree. C. for 20 min. The linearized plasmid was purified from enzyme and buffer salts using a silica maxi spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization. The IVT reaction to generate Cas9 modified mRNA was incubated at 37.degree. C. for 4 hours in the following conditions: 50 ng/.mu.L linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/.mu.L T7 RNA polymerase (NEB); 1 U/.mu.L Murine RNase inhibitor (NEB); 0.004 U/.mu.L Inorganic E. coli pyrophosphatase (NEB); and 1.times. reaction buffer. After the 4-hour incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U/.mu.L, and the reaction was incubated for an additional 30 minutes to remove the DNA template. The Cas9 mRNA was purified from enzyme and nucleotides using a MegaClear Transcription Clean-up kit per the manufacturer's protocol (ThermoFisher). Alternatively, the mRNA was purified through a precipitation protocol, which in some cases was followed by HPLC-based purification. Briefly, after the DNase digestion, the mRNA was precipitated by adding 0.21.times. vol of a 7.5 M LiCl solution and mixing, and the precipitated mRNA was pelleted by centrifugation. Once the supernatant was removed, the mRNA was reconstituted in water. The mRNA was precipitated again using ammonium acetate and ethanol. 5M Ammonium acetate was added to the mRNA solution for a final concentration of 2M along with 2.times. volume of 100% EtOH. The solution was mixed and incubated at -20.degree. C. for 15 min. The precipitated mRNA was again pelleted by centrifugation, the supernatant was removed, and the mRNA was reconstituted in water. As a final step, the mRNA was precipitated using sodium acetate and ethanol. 1/10 volume of 3 M sodium acetate (pH 5.5) was added to the solution along with 2.times. volume of 100% EtOH. The solution was mixed and incubated at -20.degree. C. for 15 min. The precipitated mRNA was again pelleted by centrifugation, the supernatant was removed, the pellet was washed with 70% cold ethanol and allowed to air dry. The mRNA was reconstituted in water. For HPLC purified mRNA, after the LiCl precipitation and reconstitution, the mRNA was purified by RP-IP HPLC (see, e.g., Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). The fractions chosen for pooling were combined and desalted by sodium acetate/ethanol precipitation as described above. The transcript concentration was determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript was analyzed by capillary electrophoresis by Bioanlayzer (Agilent).

[0558] When SEQ ID NOs: 1 and 2 are referred to below with respect to RNAs, it is understood that Ts should be replaced with Us (which were N1-methyl pseudouridines as described above). Cas9 mRNAs used in the Examples include a 5' cap and a 3' poly-A tail, e.g., up to 100 nts, and are identified by SEQ ID NO.

TABLE-US-00006 SEQ ID NO: 1: Cas9 sequence 1 for transcription. GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGA CTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATA CAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACA GCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACA GCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAG AAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAA AGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAA GAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGA AGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGC TGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACAT ACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCA AAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCT GATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGA TCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTG GCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCT GGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGG CAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTC AACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATA CGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGC AGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGA TACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTT CATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCA AGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGA AGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAG ACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAA AGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGA AACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACC GTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCA TCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTG CCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGAC AAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCG GAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAG GTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTT CGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGG GAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACT GTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACC TGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGA TGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAG CGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAA ACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATC CAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGC AAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCA AGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAAC ATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGG GAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAAC GAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCG TCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACA AGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGT CGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGA TCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTG AGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAG ACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAA AGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTG AAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGT CAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAG TCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTC GTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAG CGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACA TCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGA AAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGA CAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGG TCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAA AGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGA CTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAG AGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGA AAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGA AGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAC GGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGA ACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACT ACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTC GTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGA ATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGA GCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAAC ATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAA GTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAG TCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACA AGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAA GAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACC ATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTT TCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTC TTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAA AGAACCTCGAG SEQ ID NO: 2: Cas9 sequence 2 for transcription. GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT TGCAGGCCTTATTCGGATCCATGGATAAGAAGTACTCAATCGGGCTGGAT ATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGT GCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCA AGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAA GCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAA TCGCATCTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCG ACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGAC AAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGC GTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTG ACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCAT ATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGA TAACTCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACC AACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAGGCC ATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGC ACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTC TCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAG GACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAA TTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTA AGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACC GAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGA GCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGC CTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTACGCA GGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAA GCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGA ACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATT CCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGA GGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTC TGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCG CGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAA TTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAAC GAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAG CACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGT GAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAAC AGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACC

GTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTC AGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCT ATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAG GAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGA GGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCG ACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGC CGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAA AACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCA TGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAA GCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCT GGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGG TCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTG ATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTC CCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCC AGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAG CTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGA GCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCAC AGTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGC GACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAA GAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCC AGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAG CTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGAT TACCAAGCACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACG ACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGC AAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGA AATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCG GTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTAC GGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACA GGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGA ACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGG CCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGG CAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATA TTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATC CTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGA CCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCC TCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTC AAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAA CCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATC TGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGG AAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGC CTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAA AACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAG CAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTC AAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCT ACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATC CACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTT CGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGG ACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATC GATCTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAGAAGAG AAAGGTGTAATGAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCAT GAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTC TTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTT TAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAG AACCTCGAG

Human TTR Guide Design and Human TTR with Cynomolgus Monkey Homology Guide Design

[0559] Initial guide selection was performed in silico using a human reference genome (e.g., hg38) and user defined genomic regions of interest (e.g., TTR protein coding exons), for identifying PAMs in the regions of interest. For each identified PAM, analyses were performed and statistics reported. gRNA molecules were further selected and rank ordered based on a number of criteria (e.g., GC content, predicted on-target activity, and potential off-target activity).

A total of 68 guide RNAs were designed toward TTR (ENSG00000118271) targeting the protein coding regions within Exon 1, 2, 3 and 4. Of the total 68 guides, 33 were 100% homologous in cynomolgus monkey ("cyno"). In addition, for 10 of the human TTR guides which were not perfectly homologous in cyno, "surrogate" guides were designed and made in parallel to perfectly match the corresponding cyno target sequence. These "surrogate" or "tool" guides may be screened in cyno, e.g., to approximate the activity and function of the homologous human guide sequence. Guide sequences and corresponding genomic coordinates are provided (Table 1). All of the guide RNAs were made as dual guide RNAs, and a subset of the guide sequences were made as modified single guide RNA (Table 2). Guide ID alignment across dual guide RNA (dgRNA) IDs, modified single guide RNA (sgRNA) IDs, the number of mismatches to the cyno genome as well as the cyno exact matched IDs are provided (Table 3). Where dgRNAs are used in the experiments detailed throughout the Examples, SEQ ID NO: 270 was used. Cas9 mRNA and Guide RNA Delivery In Vitro

[0560] HEK293_Cas9 Cell Line.

[0561] The human embryonic kidney adenocarcinoma cell line HEK293 constitutively expressing Spy Cas9 ("HEK293_Cas9") was cultured in DMEM media supplemented with 10% fetal bovine serum and 500 .mu.g/ml G418. Cells were plated at a density of 10,000 cells/well in a 96-well plate 24 hours prior to transfection. Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) per the manufacturer's protocol. Cells were transfected with a lipoplex containing individual crRNA (25 nM), trRNA (25 nM), Lipofectamine RNAiMAX (0.3 .mu.L/well) and OptiMem.

[0562] HUH7 Cell Line.

[0563] The human hepatocellular carcinoma cell line HUH7 (Japanese Collection of Research Bioresources Cell Bank, Cat. JCRB0403) was cultured in DMEM media supplemented with 10% fetal bovine serum. Cells were plated on at a density of 15,000 cells/well in a 96-well plate 20 hours prior to transfection. Cells were transfected with Lipofectamine MessengerMAX (ThermoFisher, Cat. LMRNA003) per the manufacturer's protocol. Cells were sequentially transfected with a lipoplex containing Spy Cas9 mRNA (100 ng), MessengerMAX (0.3 .mu.L/well) and OptiMem followed by a separate lipoplex containing individual crRNA (25 nM), tracer RNA (25 nM), MessengerMAX (0.3 .mu.L/well) and OptiMem.

[0564] HepG2 Cell Line.

[0565] The human hepatocellular carcinoma cell line HepG2 (American Type Culture Collection, Cat. HB-8065) was cultured in DMEM media supplemented with 10% fetal bovine serum. Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 10,000 cells/well in a 96-well plate 24 hours prior to transfection. Cells were transfected with Lipofectamine 2000 (ThermoFisher, Cat. 11668019) per the manufacturer's protocol. Cells were sequentially transfected with lipoplex containing Spy Cas9 mRNA (100 ng), Lipofectamine 2000 (0.2 .mu.L/well) and OptiMem followed by a separate lipoplex containing individual crRNA (25 nM), tracer RNA (25 nM), Lipofectamine 2000 (0.2 .mu.L/well) and OptiMem.

[0566] Primary Liver Hepatocytes.

[0567] Primary human liver hepatocytes (PHH) and primary cynomolgus liver hepatocytes (PCH) (Gibco) were cultured per the manufacturer's protocol (Invitrogen, protocol 11.28.2012). In brief, the cells were thawed and resuspended in hepatocyte thawing medium with supplements (Gibco, Cat. CM7000) followed by centrifugation at 100 g for 10 minutes for human and 80 g for 4 minutes for cyno. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 33,000 cells/well for human or 60,000 cells/well for cyno (or 65,000 cells/well when assaying effects on TTR protein, described further below). Plated cells were allowed to settle and adhere for 6 or 24 hours in a tissue culture incubator at 37.degree. C. and 5% CO.sub.2 atmosphere. After incubation cells were checked for monolayer formation and media was replaced with hepatocyte culture medium with serum-free supplement pack (Invitrogen, Cat. A1217601 and CM4000).

[0568] Lipofectamine RNAiMax (ThermoFisher, Cat. 13778150) based transfections were conducted as per the manufacturer's protocol. Cells were sequentially transfected with a lipoplex containing Spy Cas9 mRNA (100 ng), Lipofectamine RNAiMax (0.4 .mu.L/well) and OptiMem followed by a separate lipoplex containing crRNA (25 nM) and tracer RNA (25 nM) or sgRNA (25 nM), Lipofectamine RNAiMax (0.4 .mu.L/well) and OptiMem.

[0569] Ribonucleotide formation was performed prior to electroporation or transfection of Spy Cas9 protein loaded with guide RNAs (RNPs) onto cells. For dual guide (dgRNAs), individual crRNA and trRNA was pre-annealed by mixing equivalent amounts of reagent and incubating at 95.degree. C. for 2 min and cooling to room temperature. Single guide (sgRNAs) were boiled at 95.degree. C. for 2 min and cooling to room temperature. The boiled dgRNA or sgRNA was incubated with Spy Cas9 protein in Optimem for 10 minutes at room temperature to form a ribonucleoprotein (RNP) complex.

[0570] For RNP electroporation into primary human and cyno hepatocytes, the cells are thawed and resuspended in Lonza electroporation Primary Cell P3 buffer at a concentration of 2500 cells per .mu.L for human hepatocytes and 3500 cells per .mu.L for cyno hepatocytes. A volume of 20 .mu.L of resuspended cells and 5 .mu.L of RNP are mixed together per guide. 20.mu.L of the mixture is placed into a Lonza electroporation plate. The cells were electroporated using the Lonza nucleofector with the preset protocol EX-147. Post electroporation, the cells are transferred into a Biocoat plate containing pre-warmed maintenance media and placed in a tissue culture incubator at 37.degree. C. and 5% CO.sub.2.

[0571] For RNP lipoplex transfections, cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) per the manufacturer's protocol. Cells were transfected with an RNP containing Spy Cas9 (10 nM), individual guide (10 nM), tracer RNA (10 nM), Lipofectamine RNAiMAX (1.0 .mu.L/well) and OptiMem. RNP formation was performed as described above.

[0572] LNPs were formed either by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr.TM. Benchtop Instrument, per the manufacturer's protocol, or cross-flow mixing.

[0573] LNP Formulation--NanoAssemblr

[0574] In general, the lipid nanoparticle components were dissolved in 100% ethanol with the lipid component of various molar ratios. The RNA cargos were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, with the ratio of mRNA to gRNA at 1:1 by weight.

[0575] The LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr.TM. Benchtop Instrument, according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were collected, diluted in water (approximately 1:1 v/v), held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v) before final buffer exchange. The final buffer exchange into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) was completed with PD-10 desalting columns (GE). If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 .mu.m sterile filter. The final LNP was stored at -80.degree. C. until further use.

LNP Formulation--Cross-Flow

[0576] For LNPs prepared using the cross-flow technique, the LNPs were formed by impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol is mixed through a mixing cross with the two volumes of RNA solution. A fourth stream of water is mixed with the outlet stream of the cross through an inline tee. (See WO2016010840 FIG. 2.) The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v). Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchanged by diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS). Alternatively, the final buffer exchange into TSS was completed with PD-10 desalting columns (GE). If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 .mu.m sterile filter. The final LNP was stored at 4.degree. C. or -80.degree. C. until further use.

Formulation Analytics

[0577] Dynamic Light Scattering ("DLS") is used to characterize the polydispersity index ("pdi") and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted 30.times. in PBS prior to being measured by DLS. Z-average diameter which is an intensity based measurement of average particle size was reported along with number average diameter and pdi. A Malvern Zetasizer instrument is also used to measure the zeta potential of the LNP. Samples are diluted 1:17 (50 uL into 800 uL) in 0.1.times.PBS, pH 7.4 prior to measurement.

[0578] A fluorescence-based assay (Ribogreen.RTM., ThermoFisher Scientific) is used to determine total RNA concentration and free RNA. Encapsulation efficiency is calculated as (Total RNA-Free RNA)/Total RNA. LNP samples are diluted appropriately with 1.times.TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1.times.TE buffer to determine free RNA. Standard curves are prepared by utilizing the starting RNA solution used to make the formulations and diluted in 1.times.TE buffer+/-0.2% Triton-X 100. Diluted RiboGreen.RTM. dye (according to the manufacturer's instructions) is then added to each of the standards and samples and allowed to incubate for approximately 10 minutes at room temperature, in the absence of light. A SpectraMax M5 Microplate Reader (Molecular Devices) is used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. Total RNA and free RNA are determined from the appropriate standard curves.

[0579] Encapsulation efficiency is calculated as (Total RNA-Free RNA)/Total RNA. The same procedure may be used for determining the encapsulation efficiency of a DNA-based cargo component. For single-strand DNA Oligreen Dye may be used, and for double-strand DNA, Picogreen Dye.

[0580] Typically, when preparing LNPs, encapsulation was >80%, particle size was <120 nm, and pdi was <0.2.

LNP Delivery In Vivo

[0581] Unless otherwise noted, CD-1 female mice, ranging from 6-10 weeks of age were used in each study. Animals were weighed and grouped according to body weight for preparing dosing solutions based on group average weight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram body weight). The animals were observed at approximately 6 hours post dose for adverse effects. Body weight was measured at twenty-four hours post-administration, and animals were euthanized at various time points by exsanguination via cardiac puncture under isoflourane anesthesia. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the median lobe or from three independent lobes (e.g., the right median, left median, and left lateral lobes) from each animal for DNA extraction and analysis.

Transthyretin (TTR) ELISA Analysis Used in Animal Studies

[0582] Blood was collected and the serum was isolated as indicated. The total mouse TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111); rat TTR serum levels were measured using a rat specific ELISA kit (Aviva Systems Biology catalog number OKIA00159); human TTR serum levels were measured using a human specific ELISA kit (Aviva Systems Biology catalog number OKIA00081); each according to manufacture's protocol. Briefly, sera were serial diluted with kit sample diluent to a final dilution of 10,000-fold, or 5,000-fold when measuring human TTR in mouse sera. 100 ul of the prepared standard curve or diluted serum samples were added to the ELISA plate, incubated for 30 minutes at room temperature then washed 3 times with provided wash buffer. 100 uL of detection antibody was then added to each well and incubated for 20 minutes at room temperature followed by 3 washes. 100 uL of substrate is added then incubated for 10 minutes at room temperature before the addition of 100 uL stop solution. The absorbance of the contents was measured on the Spectramax M5 plate reader with analysis using SoftmaxPro version 7.0 software. Serum TTR levels were quantitated off the standard curve using 4 parameter logistic fit and expressed as ug/mL of serum or percent knockdown relative control (vehicle treated) animals.

Genomic DNA Isolation

[0583] Transfected cells were harvested post-transfection at 24, 48, or 72 hours. The genomic DNA was extracted from each well of a 96-well plate using 50 .mu.L/well BuccalAmp DNA Extraction solution (Epicentre, Cat. QE09050) per manufacturer's protocol. All DNA samples were subjected to PCR and subsequent NGS analyses, as described herein.

Next-Generation Sequencing ("NGS") Analysis

[0584] To quantitatively determine the efficiency of editing at the target location in the genome, sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing.

[0585] Primers were designed around the target site within the gene of interest (e.g. TTR), and the genomic area of interest was amplified.

[0586] Additional PCR was performed per the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to a reference genome (e.g., the human reference genome (hg38), the cynomologus reference genome (mf5), the rat reference genome (rn6), or the mouse reference genome (mm10)) after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion, substitution, or deletion was calculated.

[0587] The editing percentage (e.g., the "editing efficiency" or "percent editing" or "indel frequency") is defined as the total number of sequence reads with insertions/deletions ("indels") or substitutions over the total number of sequence reads, including wild type.

Analysis of Secreted Transthyretin ("TTR") Protein by Western Blot

[0588] Secreted levels of TTR protein in media were determined using western blotting methods. HepG2 cells were transfected as previously described with select guides from Table 1. Media changes were performed every 3 days post transfection. Six days post-transfection, the media was removed, and cells were washed once with media that did not contain fetal bovine serum (FBS). Media without serum was added to the cells and incubated at 37.degree. C. After 4 hrs the media was removed and centrifuged to pellet any debris; cell number for each well was estimated based on the values obtained from a CTG assay on remaining cells and comparison to the plate average. After centrifugation, the media was transferred to a new plate and stored at -20.degree. C. An acetone precipitation of the media was performed to precipitate any protein that had been secreted into the media. Four volumes of ice cold acetone were added to one volume of media. The solution was mixed well and kept at -20.degree. C. for 90 min. The acetone:media mixture was centrifuged at 15,000.times.g and 4.degree. C. for 15 min. The supernatant was discarded and the retained pellet was air dried to eliminate any residual acetone. The pellet was resuspended in 154, RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, Cat. 11697498001) and 1 mM DTT. Lysates were mixed with Laemmli buffer and denatured at 95.degree. C. for 10 minutes. Western blots were run using the NuPage system on 12% Bis-Tris gels (ThermoFisher) per the manufacturer's protocol followed by wet transfer onto 0.45 .mu.m nitrocellulose membrane (Bio-Rad, Cat. 1620115). Blots were blocked using 5% Dry Milk in TBS for 30 minutes on a lab rocker at room temperature. Blots were rinsed with TBST and probed with rabbit .alpha.-TTR monoclonal antibody (Abcam, Cat. Ab75815) at 1:1000 in TBST. Alpha-1 antitrypsin was used as a loading control (Sigma, Cat. HPA001292) at 1:1000 in TBST and incubated simultaneously with the TTR primary antibody. Blots were sealed in a bag and kept overnight at 4.degree. C. on a lab rocker. After incubation, blots were rinsed 3 times for 5 min each in TBST and probed with secondary antibodies to Rabbit (ThermoFisher, Cat. PISA535571) at 1:25,000 in TBST for 30 min at room temperature. After incubation, blots were rinsed 3 times for 5 min each in TBST and 2 times with PBS. Blots were visualized and analyzed using a Licor Odyssey system.

Analysis of Intracellular TTR by Western Blot

[0589] The hepatocellular carcinoma cell line, HUH7, was transfected as previously described with select guides from Table 1. Six-days post-transfection, the media was removed and the cells were lysed with 50 .mu.L/well RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, Cat. 11697498001), 1 mM DTT, and 250 U/ml Benzonase (EMD Millipore, Cat. 71206-3). Cells were kept on ice for 30 minutes at which time NaCl (1 M final concentration) was added. Cell lysates were thoroughly mixed and retained on ice for 30 minutes. The whole cell extracts ("WCE") were transferred to a PCR plate and centrifuged to pellet debris. A Bradford assay (Bio-Rad, Cat. 500-0001) was used to assess protein content of the lysates. The Bradford assay procedure was completed per the manufacturer's protocol. Extracts were stored at minus 20.degree. C. prior to use. Western blots were performed to assess intracellular TTR protein levels. Lysates were mixed with Laemmli buffer and denatured at 95.degree. C. for 10 min. Western blots were run using the NuPage system on 12% Bis-Tris gels (ThermoFisher) per the manufacturer's protocol followed by wet transfer onto 0.45 .mu.m nitrocellulose membrane (Bio-Rad, Cat. 1620115). After transfer membranes were rinsed thoroughly with water and stained with Ponceau S solution (Boston Bio Products, Cat. ST-180) to confirm complete and even transfer. Blots were blocked using 5% Dry Milk in TBS for 30 minutes on a lab rocker at room temperature. Blots were rinsed with TBST and probed with rabbit .alpha.-TTR monoclonal antibody (Abcam, Cat. Ab75815) at 1:1000 in TBST. (3-actin was used as a loading control (ThermoFisher, Cat. AM4302) at 1:2500 in TBST and incubated simultaneously with the TTR primary antibody. Blots were sealed in a bag and kept overnight at 4.degree. C. on a lab rocker. After incubation, blots were rinsed 3 times for 5 minutes each in TBST and probed with secondary antibodies to Mouse and Rabbit (ThermoFisher, Cat. PI35518 and PISA535571) at 1:25,000 each in TBST for 30 min at room temperature. After incubation, blots were rinsed 3 times for 5 min each in TBST and 2 times with PBS. Blots were visualized and analyzed using a Licor Odyssey system.

Example 2. Screening of dgRNA Sequences

[0590] Cross Screening of TTR dgRNAs in Multiple Cell Types

[0591] Guides in dgRNA format targeting human TTR and the cynomologus matched sequences were delivered to HEK293_Cas9, HUH7 and HepG2 cell lines, as well as primary human hepatocytes and primary cynomolgus monkey hepatocytes as described in Example 1. Percent editing was determined for crRNAs comprising each guide sequence across each cell type and the guide sequences were then rank ordered based on highest % edit. The screening data for the guide sequences in Table 1 in all five cell lines are listed below (Table 4 through 11).

[0592] Table 4 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the TTR crRNAs in the human kidney adenocarcinoma cell line, HEK293_Cas9, which constitutively over expresses Spy Cas9 protein.

TABLE-US-00007 TABLE 4 TTR editing data in Hek_Cas9 cells transfected with dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR003335 26.59 4.73 4.73 0.65 21.87 4.09 CR003336 29.09 4.57 3.31 0.24 25.78 4.32 CR003337 42.72 1.72 5.24 1.62 37.48 0.70 CR003338 52.42 3.28 4.76 0.03 47.66 3.30 CR003339 56.37 4.13 49.39 3.23 6.98 0.91 CR003340 42.38 8.43 27.88 4.31 14.50 4.13 CR003341 20.04 5.26 6.73 1.86 13.31 3.41 CR003342 36.57 5.80 1.19 0.22 35.38 5.59 CR003343 24.36 1.51 4.82 0.43 19.53 1.39 CR003344 33.87 2.93 4.32 0.58 29.54 2.37 CR003345 35.02 7.05 19.00 3.58 16.01 3.48 CR003346 48.33 5.81 33.03 3.12 15.30 2.72 CR003347 21.45 5.57 0.95 0.33 20.50 5.26 CR003348 35.53 5.81 22.32 3.79 13.21 2.03 CR003349 13.19 4.46 8.03 2.81 5.16 1.66 CR003350 22.31 4.25 5.54 0.74 16.77 3.51 CR003351 49.67 3.77 28.42 1.69 21.24 2.22 CR003352 27.90 7.55 4.91 1.35 22.99 6.26 CR003353 25.03 5.16 3.71 0.75 21.32 4.42 CR003354 18.46 2.02 2.56 0.21 15.90 1.89 CR003355 30.60 2.53 6.99 0.80 23.61 1.75 CR003356 32.21 4.71 10.03 1.39 22.19 3.36 CR003357 43.23 6.71 5.38 0.87 37.85 5.88 CR003358 5.44 0.86 1.29 0.16 4.14 0.84 CR003359 37.75 7.50 18.35 3.73 19.40 3.78 CR003360 22.68 3.16 2.70 0.56 19.98 2.60 CR003361 34.45 8.97 8.66 1.66 25.78 7.32 CR003362 9.90 2.66 1.48 0.33 8.41 2.33 CR003363 31.03 10.74 14.77 4.21 16.26 6.54 CR003364 35.65 7.90 19.17 4.24 16.48 3.76 CR003365 36.43 6.20 11.83 1.88 24.61 4.45 CR003366 47.36 6.59 10.10 1.28 37.26 5.32 CR003367 47.11 15.43 28.44 9.11 18.67 6.33 CR003368 40.35 10.13 3.73 0.96 36.61 9.17 CR003369 33.10 7.26 9.06 1.12 24.04 6.16 CR003370 34.22 5.69 4.49 0.67 29.73 5.06 CR003371 25.60 8.33 3.84 1.41 21.76 6.92 CR003372 15.24 7.92 3.25 1.61 11.99 6.31 CR003373 13.55 2.40 1.31 0.21 12.25 2.19 CR003374 10.91 0.88 0.81 0.10 10.10 0.81 CR003375 11.63 3.18 0.78 0.17 10.85 3.05 CR003376 28.16 4.49 1.35 0.18 26.81 4.52 CR003377 24.70 4.44 2.71 0.54 21.99 3.91 CR003378 20.97 2.67 4.49 0.49 16.48 2.18 CR003379 26.32 2.91 5.34 0.61 20.98 2.30 CR003380 47.64 5.74 3.64 0.24 44.00 5.52 CR003381 22.04 5.74 3.82 1.26 18.23 4.64 CR003382 29.95 3.13 4.46 0.45 25.49 2.73 CR003383 40.47 0.64 25.12 0.45 15.35 0.66 CR003384 17.45 1.32 1.45 0.23 16.00 1.42 CR003385 26.19 5.62 7.36 1.57 18.82 4.06 CR003386 33.12 10.65 2.94 0.63 30.18 10.03 CR003387 24.68 5.93 7.75 1.99 16.92 3.94 CR003388 19.23 4.41 1.41 0.39 17.82 4.07 CR003389 34.18 5.09 10.30 2.12 23.87 3.02 CR003390 28.02 3.77 4.31 0.25 23.71 3.61 CR003391 44.81 4.67 0.61 0.07 44.19 4.63 CR003392 21.67 7.52 0.85 0.26 20.82 7.27

[0593] Table 5 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR crRNAs co-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in the human hepatocellular carcinoma cell line, HUH7.

TABLE-US-00008 TABLE 5 TTR editing data in HUH7 cells transfected with Spy Cas9 mRNA and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR003335 31.95 4.50 4.62 0.83 27.57 4.08 CR003336 30.05 4.25 4.14 1.07 26.56 3.55 CR003337 55.72 3.12 8.34 0.93 48.95 2.24 CR003338 75.64 2.03 10.22 1.42 67.06 2.79 CR003339 79.97 4.73 60.55 3.94 20.13 1.02 CR003340 46.93 7.12 33.33 6.01 14.23 1.65 CR003341 20.58 5.98 7.78 1.64 13.20 4.44 CR003342 45.14 7.16 1.23 0.91 44.66 7.68 CR003343 76.13 7.04 9.58 3.49 66.97 6.10 CR003344 64.02 3.33 10.76 1.35 54.40 2.71 CR003345 72.43 2.17 41.33 0.96 32.18 1.37 CR003346 18.07 1.02 13.17 1.39 6.97 3.06 CR003347 32.16 5.50 1.64 0.42 30.79 5.11 CR003348 57.14 10.98 36.08 6.97 22.71 4.42 CR003349 14.14 4.99 9.73 3.26 4.82 1.91 CR003350 52.91 7.61 13.43 2.00 41.64 6.03 CR003351 63.51 4.61 36.87 2.49 27.49 2.14 CR003352 39.68 9.53 7.62 7.42 32.79 7.37 CR003353 69.18 4.59 7.73 2.46 62.87 3.13 CR003354 12.27 3.38 1.25 0.40 11.46 3.23 CR003355 38.83 5.31 9.40 1.81 30.31 3.56 CR003356 49.63 5.55 18.98 2.67 31.31 3.04 CR003357 36.31 5.72 6.37 1.17 30.82 4.68 CR003358 36.50 6.17 10.53 1.56 26.60 4.49 CR003359 66.75 5.84 21.73 2.30 45.97 3.93 CR003360 58.62 8.73 5.01 0.60 55.13 8.19 CR003361 28.68 6.52 6.84 1.26 22.44 5.31 CR003362 26.43 0.83 3.43 0.32 23.76 0.85 CR003363 41.01 7.16 17.83 3.32 23.78 3.97 CR003364 47.13 10.61 24.68 5.15 23.03 5.74 CR003365 60.68 5.25 17.77 1.57 43.82 3.73 CR003366 69.98 8.84 20.77 3.10 50.32 5.69 CR003367 66.29 4.48 33.62 4.14 33.48 0.51 CR003368 31.57 11.73 3.08 0.92 29.69 11.32 CR003369 24.19 6.89 7.12 2.27 17.38 4.76 CR003370 39.16 11.59 4.83 1.79 35.55 10.35 CR003371 40.47 7.68 6.07 0.89 35.65 7.01 CR003372 21.52 6.02 4.89 1.66 17.25 4.58 CR003373 27.29 4.45 3.31 0.66 25.12 4.12 CR003374 3.10 0.68 0.45 0.24 2.87 0.54 CR003375 2.38 0.22 0.26 0.14 2.25 0.12 CR003376 19.42 5.60 1.37 0.45 18.55 5.28 CR003377 34.93 5.47 5.59 0.88 29.89 4.71 CR003378 40.73 4.63 9.73 1.85 32.27 2.91 CR003379 19.18 5.17 3.38 0.77 16.48 4.32 CR003380 31.76 5.81 3.29 0.57 29.29 5.42 CR003381 99.70 0.17 1.92 0.20 99.70 0.17 CR003382 34.47 5.71 0.14 0.16 34.47 5.71 CR003383 42.89 10.14 2.14 0.56 41.19 9.67 CR003384 17.03 1.95 0.84 0.30 16.29 1.84 CR003386 69.40 19.41 0.53 0.23 69.34 19.32 CR003387 25.64 3.69 0.23 0.07 25.55 3.62 CR003388 59.48 4.29 3.88 0.68 56.45 4.45 CR003389 62.32 1.97 13.19 1.18 50.90 1.02 CR003390 18.97 4.82 3.31 0.91 16.49 3.98 CR003391 61.31 13.21 2.10 0.51 59.70 12.76 CR003392 28.37 8.58 1.93 0.73 26.98 7.94

[0594] Table 6 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR and control crRNAs co-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in the human hepatocellular carcinoma cell line, HepG2.

TABLE-US-00009 TABLE 6 TTR editing data in HepG2 cells transfected with Spy Cas9 mRNA and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR001261 49.16 7.45 16.46 3.46 32.71 4.06 (control) CR001262 63.33 5.66 59.88 4.92 3.45 0.86 (control) CR001263 39.19 6.98 37.59 8.01 1.60 1.92 (control) CR001264 57.09 12.14 47.47 9.25 9.61 2.89 (control) CR003335 37.19 2.12 32.96 1.67 4.23 0.59 CR003336 31.31 5.47 30.48 5.10 0.83 0.75 CR003337 61.93 2.68 59.28 2.11 2.65 1.39 CR003338 68.00 6.09 65.40 6.78 2.60 1.17 CR003339 68.21 7.67 12.37 1.47 55.84 6.31 CR003340 37.76 6.01 6.12 1.95 31.65 4.07 CR003341 15.60 5.49 9.94 3.38 5.66 2.13 CR003342 11.06 6.71 10.78 6.69 0.28 0.03 CR003343 45.41 15.20 40.05 10.79 5.36 5.20 CR003344 33.43 6.11 29.81 5.09 3.62 1.13 CR003345 10.58 9.25 6.12 5.38 4.45 3.87 CR003346 0.13 0.05 0.07 0.02 0.05 0.03 CR003347 22.57 10.94 21.08 11.19 1.49 0.90 CR003348 38.44 10.45 17.04 5.04 21.40 5.89 CR003349 8.36 2.19 4.46 1.75 3.91 0.76 CR003350 29.60 5.17 25.16 4.56 4.44 0.67 CR003351 57.54 5.67 31.98 2.63 25.57 3.08 CR003352 44.28 8.71 39.51 7.10 4.77 1.79 CR003353 60.40 11.37 56.71 9.95 3.68 1.45 CR003354 5.36 3.94 4.84 3.41 0.53 0.71 CR003355 15.80 5.38 12.36 4.23 3.44 1.16 CR003356 9.39 1.82 5.67 1.03 3.72 0.92 CR003357 45.83 10.66 42.37 8.47 3.46 2.28 CR003358 35.93 7.34 28.66 7.76 7.27 1.77 CR003359 64.44 14.90 48.79 14.32 15.65 1.94 CR003360 41.31 12.23 38.94 10.60 2.38 1.78 CR003361 14.05 4.79 11.47 4.35 2.58 0.43 CR003362 17.44 4.34 16.50 4.86 0.94 0.52 CR003363 42.65 9.90 28.58 6.95 14.07 3.01 CR003364 51.88 7.67 31.03 2.67 20.85 5.03 CR003365 46.88 15.78 35.77 13.49 11.11 2.30 CR003366 54.69 9.10 46.20 8.98 8.49 1.11 CR003367 45.55 8.19 24.28 6.57 21.27 1.62 CR003368 51.55 8.60 48.34 9.87 3.22 1.36 CR003369 22.62 4.01 17.11 4.47 5.51 2.52 CR003370 28.51 6.94 24.88 6.17 3.62 1.45 CR003371 15.91 4.17 14.07 4.02 1.84 0.22 CR003372 14.57 2.47 12.14 2.08 2.42 0.40 CR003373 17.69 8.41 15.92 6.44 1.77 1.97 CR003374 5.43 0.53 5.12 0.62 0.31 0.36 CR003375 2.06 0.04 1.96 0.06 0.10 0.03 CR003376 14.41 3.01 14.16 2.93 0.24 0.10 CR003377 16.30 2.85 15.29 2.59 1.02 0.59 CR003378 8.16 3.83 6.82 3.43 1.34 0.61 CR003379 19.74 4.24 17.70 4.30 2.04 0.33 CR003380 17.08 2.48 14.78 1.18 2.30 1.36 CR003381 6.81 3.48 6.18 3.82 0.63 0.44 CR003382 1.73 0.14 1.58 0.12 0.15 0.03 CR003383 6.35 1.67 6.19 1.68 0.16 0.04 CR003384 3.37 0.88 3.12 0.94 0.25 0.09 CR003385 53.94 9.41 46.32 10.66 7.62 1.29 CR003386 2.71 0.76 2.15 0.77 0.56 0.53 CR003387 1.39 0.15 1.27 0.17 0.12 0.02 CR003388 9.33 4.47 7.76 4.56 1.56 0.10 CR003389 31.84 6.09 27.27 5.96 4.57 1.21 CR003390 24.88 4.96 22.44 3.41 2.44 2.25 CR003391 48.78 14.41 48.28 14.44 0.50 0.52 CR003392 14.64 5.25 14.32 4.95 0.33 0.36 CR005298 42.65 10.94 21.29 8.16 21.36 2.87 CR005299 38.61 5.57 36.32 3.99 2.30 2.11 CR005300 64.34 9.55 53.20 6.59 11.15 3.33 CR005301 37.04 5.32 33.39 3.85 3.65 1.89 CR005302 33.21 2.19 30.93 2.43 2.29 0.24 CR005303 21.63 6.05 20.55 5.80 1.08 0.25 CR005304 62.82 3.28 8.07 1.22 54.75 4.27 CR005305 13.51 3.58 12.30 3.49 1.21 0.84 CR005306 24.07 5.24 21.20 5.03 2.87 1.10 CR005307 22.03 3.86 7.70 1.35 14.33 4.15

[0595] Table 7 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR dgRNAs electroporated with Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE-US-00010 TABLE 7 TTR editing data in primary human hepatocytes electroporated with Spy Cas9 protein loaded with dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR003335 72.20 4.53 69.70 4.36 2.50 0.30 CR003336 39.17 3.04 38.43 3.20 0.70 0.17 CR003337 54.27 2.70 53.23 3.05 1.30 0.26 CR003338 83.03 4.84 80.87 4.63 2.13 0.25 CR003339 43.00 2.66 8.93 1.86 34.07 1.72 CR003340 12.03 1.55 5.60 1.32 6.50 0.53 CR003341 11.43 0.71 7.03 0.50 4.40 1.21 CR003342 32.77 3.63 31.87 3.28 0.90 0.35 CR003343 77.10 2.21 75.63 2.01 1.50 0.36 CR003344 39.40 3.86 33.30 2.52 6.10 1.31 CR003345 48.07 6.24 34.53 2.95 13.57 3.74 CR003346 35.67 1.80 20.83 1.65 14.83 1.66 CR003347 82.30 5.93 81.97 5.98 0.43 0.15 CR003348 28.53 1.79 11.30 2.46 17.27 0.86 CR003349 4.10 0.17 2.33 0.46 1.87 0.25 CR003350 28.13 3.50 22.40 2.41 5.73 1.22 CR003351 51.77 5.11 30.83 3.32 20.97 2.43 CR003352 29.83 4.18 25.63 3.67 4.30 0.56 CR003353 84.83 4.68 82.23 4.05 2.63 0.74 CR003354 2.50 0.36 2.43 0.32 0.03 0.06 CR003355 12.53 1.54 10.60 2.36 1.97 1.17 CR003356 9.97 2.68 7.80 2.01 2.23 0.85 CR003357 36.23 4.02 35.47 4.11 0.77 0.61 CR003358 5.70 1.42 4.93 1.36 0.80 0.26 CR003359 63.77 7.07 56.33 5.81 7.50 1.35 CR003360 32.23 3.09 31.67 2.97 0.63 0.31 CR003361 4.10 0.36 3.73 0.42 0.37 0.06 CR003362 7.03 1.30 6.87 1.20 0.20 0.20 CR003363 9.43 8.22 7.80 6.86 1.63 1.44 CR003364 23.30 5.20 16.93 4.96 6.53 0.55 CR003365 42.37 3.88 35.57 1.88 6.83 2.00 CR003366 34.70 3.26 31.63 2.98 3.10 1.15 CR003367 39.20 5.31 22.93 4.14 16.37 1.46 CR003368 28.47 3.29 27.63 2.90 0.80 0.66 CR003369 3.67 1.16 3.30 1.06 0.40 0.20 CR003370 15.27 1.75 14.43 1.72 0.90 0.20 CR003371 16.20 2.13 14.47 2.37 1.87 0.81 CR003372 12.17 2.69 10.47 2.63 1.77 0.12 CR003373 0.87 0.21 0.83 0.25 0.07 0.12 CR003374 0.80 0.17 0.70 0.26 0.10 0.10 CR003375 1.33 1.10 1.27 1.08 0.07 0.06 CR003376 1.90 1.06 1.87 1.00 0.03 0.06 CR003377 10.23 1.53 10.13 1.51 0.10 0.10 CR003378 4.60 1.92 3.87 1.19 0.73 0.67 CR003379 6.57 1.00 6.30 0.70 0.27 0.31 CR003380 5.37 2.57 5.27 2.54 0.10 0.10 CR003381 6.20 2.74 5.83 2.61 0.50 0.10 CR003382 8.40 2.07 8.10 1.87 0.43 0.21 CR003383 8.57 0.75 3.37 0.67 5.27 0.46 CR003384 1.87 0.67 1.73 0.57 0.23 0.12 CR003385 40.87 6.86 38.43 6.41 2.53 0.45 CR003386 4.90 1.20 4.47 1.14 0.47 0.25 CR003387 1.87 0.25 1.70 0.26 0.20 0.10 CR003388 5.70 0.40 5.47 0.40 0.27 0.12 CR003389 27.67 2.76 27.20 2.88 0.50 0.36 CR003390 15.97 3.86 15.80 3.99 0.23 0.15 CR003391 29.77 3.85 29.57 3.85 0.27 0.06 CR003392 4.13 1.21 4.00 1.15 0.17 0.06 CR005298 39.90 2.92 22.37 3.04 17.57 0.42 CR005299 8.65 0.78 8.30 0.99 0.35 0.21 CR005300 57.47 1.69 53.47 1.86 4.10 0.92 CR005301 25.37 1.65 24.00 2.26 1.60 0.82 CR005302 61.10 5.20 60.10 4.77 1.00 0.46 CR005303 53.57 8.52 53.07 8.36 0.53 0.47 CR005304 67.00 5.80 5.53 1.37 61.63 6.98 CR005305 3.83 0.78 3.53 0.61 0.40 0.17 CR005306 9.43 1.63 8.07 2.17 1.37 0.72 CR005307 8.17 1.20 5.20 0.87 3.00 0.82

[0596] Table 8 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR and control dgRNAs transfected with Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE-US-00011 TABLE 8 TTR editing data in primary human hepatocytes transfected with Spy Cas9 loaded with dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR001261 32.51 1.00 12.50 0.47 20.01 0.59 CR001262 50.09 1.48 45.25 1.69 4.83 0.31 CR001263 15.25 2.41 14.83 2.37 0.42 0.10 CR001264 45.30 3.48 23.87 2.09 21.43 1.68 CR003335 51.14 4.27 49.51 4.04 1.63 0.25 CR003336 30.70 2.41 30.11 2.48 0.58 0.11 CR003337 49.43 4.75 47.54 4.49 1.88 0.47 CR003338 61.34 3.55 59.13 3.44 2.22 0.11 CR003339 45.06 9.83 8.85 1.65 36.21 8.34 CR003340 10.44 2.44 5.94 1.34 4.50 1.16 CR003341 19.66 3.67 14.64 3.31 5.02 0.37 CR003342 20.66 2.55 19.85 2.54 0.81 0.15 CR003343 43.25 4.47 41.61 4.26 1.63 0.33 CR003344 35.45 13.12 30.97 11.72 4.48 1.51 CR003345 28.90 6.33 21.00 5.23 7.91 1.81 CR003346 4.11 1.36 2.27 0.53 1.84 0.85 CR003347 66.35 4.48 66.11 4.51 0.24 0.08 CR003348 23.18 2.16 13.74 1.17 9.44 0.99 CR003349 10.83 1.57 9.00 1.41 1.83 0.32 CR003350 24.84 2.74 19.77 1.91 5.07 0.89 CR003351 40.28 1.31 23.92 0.70 16.36 0.78 CR003352 30.48 1.93 27.27 2.31 3.21 0.38 CR003353 61.54 4.13 59.38 4.04 2.16 0.11 CR003354 10.31 1.47 10.07 1.50 0.23 0.11 CR003355 19.11 0.92 17.69 0.79 1.42 0.44 CR003356 7.53 1.78 6.24 1.51 1.29 0.32 CR003357 49.35 2.53 48.45 2.54 0.90 0.13 CR003358 31.62 5.97 25.95 5.03 5.67 1.04 CR003359 59.47 6.05 50.96 5.69 8.51 0.54 CR003360 31.47 4.12 30.27 4.21 1.19 0.22 CR003361 13.08 1.48 12.52 1.45 0.56 0.18 CR003362 11.65 1.24 11.10 1.06 0.56 0.36 CR003363 27.65 2.84 21.47 2.39 6.18 0.61 CR003364 35.29 3.50 23.93 2.63 11.36 1.16 CR003365 47.78 3.67 40.24 3.12 7.54 0.72 CR003366 42.74 3.41 37.95 2.88 4.79 0.60 CR003367 31.19 4.60 16.06 2.66 15.13 1.94 CR003368 34.83 5.05 33.83 5.09 1.00 0.10 CR003369 12.98 0.26 11.67 0.21 1.31 0.11 CR003370 20.06 1.79 18.80 1.65 1.26 0.28 CR003371 18.80 2.73 17.23 2.34 1.57 0.43 CR003372 17.56 2.26 15.74 2.16 1.81 0.10 CR003373 3.64 0.29 3.44 0.30 0.19 0.07 CR003374 2.65 0.33 2.52 0.33 0.14 0.02 CR003375 5.04 0.66 4.93 0.66 0.11 0.01 CR003376 5.00 1.10 4.86 1.10 0.14 0.03 CR003377 12.77 2.00 12.45 1.84 0.31 0.18 CR003378 8.66 1.90 8.24 1.74 0.42 0.19 CR003379 16.86 2.62 16.51 2.62 0.34 0.08 CR003380 8.17 1.42 7.71 1.47 0.46 0.10 CR003381 7.15 0.73 6.88 0.67 0.27 0.07 CR003382 2.44 0.06 2.28 0.05 0.15 0.03 CR003383 4.76 0.40 4.52 0.42 0.24 0.09 CR003384 3.56 0.26 3.39 0.26 0.17 0.01 CR003385 41.15 6.06 38.15 5.59 3.00 0.48 CR003386 3.22 0.25 2.97 0.27 0.25 0.02 CR003387 1.79 0.11 1.68 0.09 0.11 0.04 CR003388 5.43 1.03 4.38 1.00 1.05 0.25 CR003389 19.87 4.39 19.19 4.52 0.68 0.24 CR003390 16.09 2.84 15.85 2.91 0.24 0.09 CR003391 34.72 8.29 34.46 8.35 0.26 0.06 CR003392 10.07 1.06 9.93 1.02 0.14 0.04 CR005298 32.07 1.02 21.12 1.02 10.95 0.15 CR005299 19.37 0.61 18.79 0.51 0.58 0.13 CR005300 57.23 6.24 53.62 5.44 3.61 0.87 CR005301 31.37 3.02 29.53 2.88 1.84 0.15 CR005302 48.29 5.22 47.32 5.32 0.97 0.14 CR005303 36.45 4.83 36.06 4.72 0.39 0.12 CR005304 49.45 6.85 4.32 0.31 45.13 6.74 CR005305 7.07 1.43 6.73 1.30 0.34 0.17 CR005306 18.81 1.82 16.24 1.57 2.57 0.35 CR005307 18.73 1.68 10.18 0.92 8.55 0.88

[0597] Table 9 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR and control dgRNAs co-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in primary human hepatocytes.

TABLE-US-00012 TABLE 9 TTR editing data in primary human hepatocytes transfected with Spy Cas9 mRNA and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR001261 32.33 4.95 5.83 1.63 26.47 3.30 CR001262 41.50 4.71 34.43 3.31 7.13 1.42 CR001263 10.23 3.61 9.40 3.20 0.90 0.44 CR001264 42.80 0.50 11.90 1.32 30.90 1.80 CR003335 36.43 2.98 33.03 2.31 3.40 0.70 CR003336 16.93 3.78 16.20 3.41 0.80 0.44 CR003337 19.30 1.57 18.10 1.44 1.23 0.15 CR003338 36.30 9.55 33.73 9.27 2.73 0.49 CR003339 36.43 1.21 2.27 0.15 34.23 1.31 CR003340 24.97 2.78 1.83 0.23 23.17 2.66 CR003341 15.83 1.38 6.80 0.53 9.07 0.81 CR003342 22.10 1.27 20.60 0.57 1.50 0.71 CR003343 55.03 0.38 52.40 0.53 2.60 0.44 CR003344 31.50 1.30 22.40 1.31 9.20 0.10 CR003345 50.65 2.90 32.30 1.56 18.45 1.20 CR003346 19.97 1.94 5.63 0.55 14.33 1.72 CR003347 41.47 3.59 41.33 3.63 0.17 0.06 CR003348 18.00 0.87 2.30 0.66 15.80 0.61 CR003349 2.57 0.81 0.90 0.35 1.70 0.46 CR003350 26.63 4.25 16.33 2.45 10.33 1.75 CR003351 26.50 1.61 10.20 0.92 16.37 0.97 CR003352 16.80 5.03 11.73 3.86 5.07 1.14 CR003353 53.73 6.01 49.50 5.82 4.43 0.75 CR003354 2.97 0.95 2.87 0.85 0.13 0.12 CR003355 12.07 2.61 10.47 2.08 1.63 0.59 CR003356 7.27 0.72 4.70 0.53 2.67 0.21 CR003357 25.93 4.55 25.30 4.22 0.63 0.35 CR003358 3.90 0.79 2.73 0.45 1.17 0.51 CR003359 32.93 4.34 25.67 3.25 7.33 1.24 CR003360 14.90 4.85 14.13 4.66 0.90 0.52 CR003361 3.53 0.60 2.73 0.55 0.87 0.15 CR003362 6.60 1.47 6.17 1.45 0.47 0.21 CR003363 16.70 1.08 11.80 0.79 4.93 0.60 CR003364 15.63 2.45 6.73 0.81 8.93 1.70 CR003365 26.90 3.05 20.23 2.02 6.67 1.16 CR003366 24.53 1.26 20.47 1.45 4.07 0.23 CR003367 37.33 1.40 14.03 0.40 23.37 1.25 CR003368 11.10 1.91 10.53 1.90 0.60 0.10 CR003369 1.60 0.46 0.90 0.20 0.70 0.36 CR003370 2.83 0.57 2.33 0.40 0.50 0.17 CR003371 3.40 0.80 2.67 0.75 0.73 0.15 CR003372 1.77 0.75 1.13 0.57 0.63 0.23 CR003373 1.40 0.36 1.00 0.35 0.37 0.12 CR003374 0.27 0.21 0.27 0.21 0.03 0.06 CR003375 1.27 0.64 1.23 0.58 0.03 0.06 CR003376 2.83 0.81 2.73 0.81 0.13 0.06 CR003377 17.53 6.35 16.97 6.11 0.57 0.25 CR003378 9.80 1.37 8.50 1.21 1.37 0.15 CR003379 13.20 1.18 12.00 1.05 1.27 0.15 CR003380 2.93 0.58 2.47 0.57 0.47 0.15 CR003381 4.07 1.21 3.33 0.96 0.73 0.25 CR003382 0.97 0.25 0.97 0.25 0.00 0.00 CR003383 15.70 3.22 2.07 0.35 13.70 2.82 CR003384 1.70 0.62 1.50 0.56 0.20 0.10 CR003385 36.77 0.70 33.23 0.74 3.60 0.26 CR003386 8.27 1.63 8.20 1.57 0.13 0.06 CR003387 7.87 1.58 7.80 1.64 0.03 0.06 CR003388 12.97 1.30 11.87 1.21 1.17 0.25 CR003389 44.27 1.72 41.47 1.59 2.83 0.15 CR003390 20.23 2.08 18.73 1.92 1.60 0.17 CR003391 15.47 5.87 15.20 5.72 0.30 0.10 CR003392 2.43 0.55 2.37 0.59 0.07 0.06 CR005298 15.70 2.79 4.13 0.87 11.60 2.00 CR005299 9.43 0.68 8.93 0.68 0.60 0.00 CR005300 31.53 3.44 27.60 2.77 3.97 0.76 CR005301 6.77 1.44 5.47 0.96 1.40 0.61 CR005302 34.80 7.17 33.67 7.01 1.13 0.21 CR005303 35.50 5.90 35.00 5.81 0.50 0.10 CR005304 45.27 4.71 0.83 0.15 44.47 4.57 CR005305 7.53 1.06 5.93 1.10 1.60 0.10 CR005306 9.97 0.38 7.13 0.23 2.87 0.12 CR005307 12.90 2.43 3.67 0.61 9.30 1.80

[0598] Table 10 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR dgRNAs electroporated with Spy Cas9 protein (RNP) in primary cyno hepatocytes.

TABLE-US-00013 TABLE 10 TTR editing data in primary cyno hepatocytes electroporated with Spy Cas9 protein and dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR003336 8.18 1.93 8.10 1.94 0.07 0.01 CR003337 24.94 5.80 24.10 4.71 0.84 1.10 CR003338 44.94 9.99 44.89 9.97 0.05 0.01 CR003339 8.95 0.89 4.93 0.64 4.02 0.25 CR003340 12.53 2.22 7.72 0.13 4.80 2.09 CR003341 8.43 10.53 7.66 9.91 0.77 0.63 CR003344 35.72 4.67 33.81 5.29 1.91 0.61 CR003345 52.92 3.26 30.74 0.78 22.19 2.48 CR003346 1.91 0.86 1.82 0.82 0.09 0.04 CR003347 72.41 0.38 72.15 0.73 0.25 0.34 CR003352 1.25 0.20 1.16 0.21 0.09 0.01 CR003353 4.75 0.43 4.67 0.47 0.08 0.04 CR003358 20.47 0.30 19.01 0.51 1.46 0.21 CR003359 46.17 1.14 40.66 2.00 5.51 0.86 CR003360 29.47 0.63 29.05 1.00 0.42 0.37 CR003361 4.53 0.14 4.46 0.18 0.08 0.04 CR003362 4.59 0.80 4.36 0.77 0.22 0.03 CR003363 15.64 1.92 13.24 2.65 2.39 0.73 CR003364 19.62 2.54 14.27 2.72 5.35 0.17 CR003365 10.31 1.81 9.33 1.80 0.97 0.01 CR003366 18.52 0.71 17.62 0.33 0.90 0.39 CR003368 18.56 3.89 18.30 3.77 0.26 0.11 CR003369 1.53 0.25 1.28 0.40 0.25 0.15 CR003370 2.52 0.64 2.40 0.63 0.12 0.01 CR003371 1.83 0.38 1.69 0.41 0.14 0.03 CR003372 2.15 0.30 1.83 0.33 0.32 0.04 CR003382 10.86 2.04 8.54 1.93 2.33 0.11 CR003383 8.86 2.30 4.31 0.69 4.55 1.61 CR003384 3.75 0.35 2.50 0.37 1.25 0.02 CR003385 30.96 1.61 26.84 2.20 4.12 0.59 CR003386 5.54 1.42 3.51 1.26 2.03 0.15 CR003387 4.72 0.03 4.55 0.08 0.17 0.11 CR003388 6.81 0.17 6.59 0.28 0.22 0.11 CR003389 18.83 4.99 18.05 4.92 0.78 0.07 CR003390 16.87 3.88 16.49 3.48 0.39 0.39 CR003391 36.44 1.09 35.73 1.37 0.71 0.28 CR003392 7.02 0.97 6.63 0.59 0.38 0.37 CR005299 13.48 2.96 13.23 2.74 0.26 0.22 CR005301 46.76 1.75 46.34 2.19 0.42 0.44 CR005302 1.34 0.19 1.26 0.19 0.08 0.00 CR005303 59.28 1.05 58.72 1.06 0.56 0.00 CR005305 11.28 0.39 11.13 0.39 0.15 0.00 CR005307 4.56 0.71 2.01 0.49 2.55 0.21

[0599] Table 11 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested cyno specific TTR dgRNAs electroporated with Spy Cas9 protein (RNP) on primary cyno hepatocytes.

TABLE-US-00014 TABLE 11 TTR editing data in primary cyno hepatocytes electroporated with Spy Cas9 protein and cyno specific dgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion CR000689 24.41 1.67 18.11 2.41 6.30 0.93 CR005364 27.70 0.74 0.58 0.29 27.11 0.60 CR005365 64.94 2.03 0.10 0.04 64.85 2.05 CR005366 77.00 1.17 0.33 0.27 76.67 0.99 CR005367 50.79 0.53 0.53 0.25 50.26 0.36 CR005368 27.60 2.07 0.33 0.45 27.27 2.32 CR005369 42.01 0.33 8.09 0.55 33.92 0.31 CR005370 63.52 3.21 0.59 0.33 62.93 2.88 CR005371 8.42 0.69 0.31 0.12 8.10 0.57 CR005372 17.98 1.39 0.83 0.77 17.16 0.71

Example 3. Screening of sgRNA Sequences

[0600] Cross Screening of TTR sgRNAs in Multiple Cell Types

[0601] Guides in modified sgRNA format targeting human and/or cyno TTR were delivered to primary human hepatocytes and primary cyno hepatocytes as described in Example 1. Percent editing was determined for crRNAs comprising each guide sequence across each cell type and the guide sequences were then rank ordered based on highest % edit. The screening data for the guide sequences in Table 2 in both cell lines are listed below (Table 12 through 15).

[0602] Table 12 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAs transfected with Spy Cas9 protein (RNP) in primary human hepatocytes.

TABLE-US-00015 TABLE 12 TTR editing data in primary human hepatocytes transfected with Spy Cas9 protein and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 81.80 1.98 77.15 2.19 4.70 0.28 G000481 46.90 1.71 27.77 3.88 19.43 4.76 G000482 66.67 2.35 56.57 4.14 10.10 1.85 G000483 47.90 6.56 19.57 3.37 28.50 3.25 G000484 62.97 0.90 29.23 0.21 33.83 0.95 G000485 56.07 3.37 53.07 2.84 3.13 0.60 G000486 69.73 6.86 9.83 1.93 59.93 5.63 G000487 67.30 2.75 65.27 3.41 2.07 1.06 G000488 61.27 1.95 26.30 1.55 35.00 1.30 G000489 60.17 2.75 51.07 3.18 9.43 0.45 G000490 55.90 7.88 46.13 7.55 9.80 0.69 G000491 74.30 1.55 70.27 2.37 4.33 0.72 G000492 60.97 5.81 57.90 4.64 3.13 1.35 G000493 41.40 3.08 38.90 3.29 2.67 0.35 G000494 62.23 3.30 61.47 3.25 0.77 0.31 G000495 50.80 1.85 45.80 1.25 5.37 0.64 G000496 72.33 1.63 44.73 2.14 27.67 1.46 G000497 59.67 1.40 51.10 1.14 8.73 0.71 G000498 72.80 3.75 60.17 3.12 12.70 0.72 G000499 66.40 3.55 65.23 3.72 1.17 0.38 G000500 65.53 1.21 62.00 1.11 3.83 0.40 G000501 60.93 1.91 55.13 1.43 6.00 0.56

[0603] Table 13 shows the average and standard deviation at 12.5 nM for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAs co-transfected with Spy Cas9 mRNA (SEQ ID NO:2) in primary human hepatocytes.

TABLE-US-00016 TABLE 13 TTR editing data in primary human hepatocytes transfected with Spy Cas9 mRNA and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 73.28 0.61 59.85 0.13 13.47 0.51 G000481 34.30 5.26 14.62 2.59 19.77 2.72 G000482 40.93 3.95 27.70 2.92 13.25 0.97 G000483 27.82 2.93 4.05 0.51 23.85 2.43 G000484 43.37 6.79 13.98 2.61 29.48 4.15 G000485 30.82 5.76 28.87 5.50 1.97 0.28 G000486 59.13 5.62 2.82 0.86 56.37 4.92 G000487 49.57 0.99 47.38 0.89 2.27 0.24 G000488 49.40 5.05 11.98 1.40 37.48 3.68 G000489 24.25 2.82 14.17 2.01 10.28 1.38 G000490 24.72 2.35 19.38 2.04 5.38 0.41 G000491 45.93 1.22 42.42 1.06 3.60 0.33 G000492 34.65 2.21 32.45 2.01 2.22 0.25 G000493 11.55 1.35 10.65 1.58 0.97 0.30 G000494 26.22 4.03 25.17 3.89 1.07 0.15 G000495 47.77 1.88 43.40 1.91 4.45 0.17 G000496 63.30 2.60 11.08 2.10 52.25 0.67 G000497 40.33 3.32 34.48 2.71 5.85 0.61 G000498 60.02 5.42 45.20 4.34 14.90 1.08 G000499 39.30 6.04 38.58 5.86 0.77 0.12 G000500 35.50 0.61 32.47 0.49 3.10 0.18 G000501 40.32 1.50 33.82 2.04 6.62 0.55 G000567 27.28 7.59 17.35 4.72 10.02 2.94 G000568 43.75 5.83 43.00 5.81 0.80 0.18 G000570 68.42 3.64 68.08 3.61 0.35 0.00 G000571 20.47 3.41 14.47 2.72 6.13 0.78 G000572 55.42 8.13 41.62 6.48 13.85 1.60

[0604] Table 14 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAs electroporated with Spy Cas9 protein (RNP) on primary cyno hepatocytes. Note that guides G000480 and G000488 have one mismatch to cyno, which may compromise their editing efficiency in cyno cells.

TABLE-US-00017 TABLE 14 TTR editing data in primary cyno hepatocytes electroporated with Spy Cas9 protein and sgRNAs Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000480 10.20 0.56 9.83 0.81 0.37 0.25 G000481 69.13 8.62 33.73 2.67 35.50 11.23 G000482 75.17 2.34 55.23 2.00 20.03 0.85 G000485 22.93 0.95 22.00 0.82 1.07 0.21 G000486 79.90 0.79 11.90 0.85 68.07 0.35 G000488 9.63 0.50 5.37 0.38 4.27 0.35 G000489 67.53 1.15 53.53 1.56 14.17 0.64 G000490 61.67 0.72 54.47 1.10 7.27 1.23 G000491 66.20 1.11 64.37 0.47 1.90 0.70 G000493 50.13 0.74 48.07 1.69 2.10 0.98 G000494 81.53 0.71 79.57 0.49 2.07 0.67 G000498 91.37 1.48 68.50 1.64 22.87 1.50 G000499 83.40 0.36 82.00 0.20 1.43 0.55 G000500 45.20 3.66 42.60 3.80 2.63 0.25

[0605] Table 15 shows the average and standard deviation for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested cyno specific TTR sgRNAs electroporated with Spy Cas9 protein (RNP) on primary cyno hepatocytes.

TABLE-US-00018 TABLE 15 TTR editing data in primary cyno hepatocytes electroporated with Spy Cas9 protein and cyno specific sgRNAs (e.g., those having an analogous human gRNA, See Table 3) Std Std Std Avg Dev Avg Dev Avg Dev % % % % % % GUIDE ID Edit Edit Insert Insert Deletion Deletion G000502 95.10 0.96 13.97 1.69 81.27 2.60 G000503 58.53 2.40 52.07 1.68 6.50 2.46 G000504 77.17 0.96 69.73 1.29 7.53 0.57 G000505 95.53 1.06 95.50 1.01 0.10 0.10 G000506 89.43 1.36 86.90 1.64 3.07 0.42 G000507 71.17 3.22 67.03 2.39 4.60 1.65 G000508 45.63 3.01 41.57 2.95 4.17 0.91 G000509 93.03 0.81 43.60 1.30 49.73 1.76 G000510 90.80 0.53 89.13 0.40 1.77 0.12 G000511 62.77 1.63 60.87 1.55 2.00 0.35

Example 4. Screening of Lipid Nanoparticle (LNP) Formulations Containing Spy Ca9 mRNA and sgRNA

[0606] Cross screening of LNP formulated TTR sgRNAs with Spy Cas9 mRNA in primary human hepatocytes and primary cyno hepatocytes.

[0607] Lipid nanoparticle formulations of modified sgRNAs targeting human TTR and the cyno matched sgRNA sequences were tested on primary human hepatocytes and primary cyno hepatocytes in a dose response curve. Primary human and cyno hepatocytes were plated as described in Example 1. Both cell lines were incubated at 37.degree. C., 5% CO.sub.2 for 24 hours prior to treatment with LNPs. The LNPs used in the experiments detailed in Tables 16-19 were prepared using the Nanoassemblr.TM. procedure, each containing the specified sgRNA and Cas9 mRNA (SEQ ID NO:2), each having Lipid. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2 molar ratio, respectively, and had a N:P ratio of 4.5. LNPs were incubated in hepatocyte maintenance media containing 6% cyno serum at 37.degree. C. for 5 minutes. Post incubation the LNPs were added onto the primary human or cyno hepatocytes in an 8 point 2-fold dose response curve starting at 100 ng mRNA. The cells were lysed 72 hours post treatment for NGS analysis as described in Example 1. Percent editing was determined for crRNAs comprising each guide sequence across each cell type and the guide sequences were then rank ordered based on highest % editing at 12.5 ng mRNA input and 3.9 nM guide concentration. The dose response curve data for the guide sequences in both cell lines is shown in FIGS. 4 through 7. The % editing at 12.5 ng mRNA input and 3.9 nM guide concentration are listed below (Table 16 through 18).

[0608] Table 16 shows the average and standard deviation at 12.5 ng of cas9 mRNA for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAs formulated in lipid nanoparticles with Spy Cas9 mRNA on primary human hepatocytes as dose response curves. G000570 exhibited an uncharacteristic dose response curve compared to the other sgRNAs which may be an artifact of the experiment. The data are shown graphically in FIG. 4.

TABLE-US-00019 TABLE 16 TTR editing data in primary human hepatocytes treated with LNP formulated Spy Cas9 mRNA (SEQ ID NO: 2) and sgRNAs 12.5 ng mRNA, 3.9 nM sgRNA GUIDE ID Avg % Edit Std Dev % Edit G000480 59.33 0.73 G000481 24.37 0.37 G000482 19.10 2.64 G000483 7.37 0.67 G000484 16.67 1.23 G000485 14.23 2.36 G000486 61.33 2.59 G000487 17.37 0.95 G000488 44.80 3.00 G000489 16.85 0.06 G000490 10.53 1.90 G000491 31.60 2.33 G000492 15.87 0.44 G000493 7.33 0.73 G000494 6.37 1.07 G000495 23.97 1.66 G000496 30.73 3.76 G000497 15.10 3.30 G000498 24.43 1.30 G000499 16.07 1.67 G000500 23.57 2.44 G000501 32.30 2.49 G000567 48.95 1.06 G000568 54.60 3.68 G000570 88.30 1.84 G000572 55.45 1.20

[0609] Table 17 shows the average and standard deviation at 12.5 ng of mNRA and 3.9 nM guide concentration for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested TTR sgRNAs formulated in lipid nanoparticles with Spy Cas9 mRNA on primary cyno hepatocytes as dose response curves. The data are shown graphically in FIG. 5.

TABLE-US-00020 TABLE 17 TTR editing data in primary cyno hepatocytes treated with LNP formulated Spy Cas9 mRNA (SEQ ID NO: 2) and sgRNAs 12.5 ng mRNA, 3.9 nM sgRNA, GUIDE ID Avg % Edit Std Dev % Edit G000480 0.73 0.15 G000481 49.20 1.39 G000482 26.13 5.33 G000483 0.73 0.60 G000484 0.10 0.00 G000485 1.43 1.02 G000489 31.87 2.40 G000490 15.23 1.08 G000491 6.37 0.38 G000492 0.70 0.28 G000493 7.63 1.14 G000494 14.30 1.06 G000495 0.73 0.06 G000497 0.23 0.06 G000498 37.90 1.42 G000499 14.63 0.70 G000500 10.47 0.32 G000501 1.37 0.31 G000567 0.10 0.00 G000568 9.25 0.21 G000570 17.30 0.85 G000571 20.20 2.26 G000572 30.60 0.42

[0610] Table 18 shows the average and standard deviation at 12.5 ng of mRNA and 3.9 nM guide concentration for % Edit, % Insertion (Ins), and % Deletion (Del) for the tested cyno specific TTR sgRNAs formulated in lipid nanoparticles with Spy Cas9 mRNA on primary cyno hepatocytes as dose response curves. The data are shown graphically in FIG. 6.

TABLE-US-00021 TABLE 18 TTR editing data in primary cyno hepatocytes treated with LNP formulated Spy Cas9 mRNA (SEQ ID NO: 2) and cyno matched sgRNAs 12.5 ng mRNA, 3.9 nM sgRNA GUIDE ID % Edit Std Dev % Edit G000502 80.70 0.14 G000506 60.13 0.70 G000509 74.47 7.28 G000510 61.87 2.54

Cross Screening of LNP Formulated TTR sgRNAs with Spy Cas9 mRNA in Primary Human Hepatocytes and Primary Cyno Hepatocytes

[0611] Lipid nanoparticle formulations of modified sgRNAs targeting human TTR and the cyno matched sgRNA sequences were tested on primary human hepatocytes and primary cyno hepatocytes in a dose response curve. Primary human and cyno hepatocytes were plated as described in Example 1. Both cell lines were incubated at 37.degree. C., 5% CO.sub.2 for 24 hours prior to treatment with LNPs. The LNPs used in the experiments detailed in Tables 20-22 were prepared using the cross-flow procedure described above but purified using PD-10 columns (GE Healthcare Life Sciences) and concentrated using Amicon centrifugal filter units (Millipore Sigma), each containing the specified sgRNA and Cas9 mRNA (SEQ ID NO:1). The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively, and had a N:P ratio of 6.0. LNPs were incubated in hepatocyte maintenance media containing 6% cyno serum at 37.degree. C., 5% CO.sub.2 for 5 minutes. Post incubation the LNPs were added onto the primary human or cyno hepatocytes in an 8 point 3-fold dose response curve starting at 300 ng mRNA. The cells were lysed 72 hours post treatment for NGS analysis as described in Example 1. Percent editing was determined for crRNAs comprising each guide sequence across each cell type and the guide sequences were then rank ordered based on EC50 values and maximum editing percent. The dose response curve data for the guide sequences in both cell lines is shown in FIGS. 4 through 7. The EC 50 values and maximum editing percent are listed below (Table 19 through 22).

[0612] Table 19 shows the EC50 and maximum editing the tested human specific TTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNA on primary human hepatocytes as dose response curves. The data are shown graphically in FIG. 4.

TABLE-US-00022 TABLE 19 TTR editing data in primary human hepatocytes treated with LNP formulated Spy Cas9 mRNA and human specific sgRNAs GUIDE ID EC50 Max Editing G000480 0.10 98.69 G000481 1.43 87.05 G000482 0.65 97.02 G000483 1.88 77.39 G000484 0.95 94.14 G000488 0.72 95.83 G000489 1.38 86.33 G000490 1.52 94.16 G000493 2.42 63.95 G000494 1.28 75.70 G000499 0.63 96.31 G000500 0.39 88.70 G000568 0.78 95.72 G000570 0.23 98.22 G000571 2.21 71.28 G000572 0.42 97.94

[0613] Table 20 shows the EC50 and maximum editing the tested human specific TTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNA on primary cyno hepatocytes as dose response curves. The data are shown graphically in FIG. 16.

TABLE-US-00023 TABLE 20 TTR editing data in primary cyno hepatocytes treated with LNP formulated Spy Cas9 mRNA and human specific sgRNAs GUIDE ID EC50 Max Editing G000480 5.28 20.32 G000481 0.93 95.07 G000482 0.89 97.47 G000483 4.40 56.52 G000484 3.47 0.22 G000488 11.56 21.63 G000489 1.79 89.21 G000490 3.09 90.76 G000493 4.97 61.15 G000494 2.77 60.84 G000499 2.00 74.94 G000500 4.42 58.04 G000567 1.76 97.06 G000568 1.87 87.93 G000570 2.00 96.73 G000571 1.55 97.03 G000572 0.79 100.31

[0614] Table 21 shows the EC50 and maximum editing the tested cyno matched TTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNA on primary human hepatocytes as dose response curves. The data are shown graphically in FIG. 17.

TABLE-US-00024 TABLE 21 TTR editing data in primary human hepatocytes treated with LNP formulated Spy Cas9 mRNA and cyno specific sgRNAs GUIDE ID EC50 Max Editing G000502 0.70 91.50 G000504 5.16 7.16 G000505 3.57 13.48 G000506 1.26 89.49

[0615] Table 22 shows the EC50 and maximum editing the tested cyno matched TTR sgRNAs formulated in lipid nanoparticles with U-depleted Spy Cas9 mRNA on primary cyno hepatocytes as dose response curves. The data are shown graphically in FIG. 18.

TABLE-US-00025 TABLE 22 TTR editing data in primary cyno hepatocytes treated with LNP formulated Spy Cas9 mRNA and cyno specific sgRNAs GUIDE ID EC50 Max Editing G000502 0.26 100.05 G000503 2.26 83.41 G000504 1.42 98.04 G000505 1.10 99.97 G000506 0.66 99.18

Example 5. Off-Target Analysis of TTR dgRNAs and sgRNAs Off-Target Analysis of TTR Guides

[0616] An oligo insertion based assay (See, e.g., Tsai et al., Nature Biotechnology 33, 187-197; 2015) was used to determine potential off-target genomic sites cleaved by Cas9 targeting TTR. Forty-five dgRNAs from Table 1 (and two control guides with known off-target profiles) were screened in the HEK293_Cas9 cells. The human embryonic kidney adenocarcinoma cell line HEK293 constitutively expressing Spy Cas9 ("HEK293_Cas9") was cultured in DMEM media supplemented with 10% fetal bovine serum and 500 .mu.g/ml G418. Cells were plated at a density of 30,000 cells/well in a 96-well plate 24 hours prior to transfection. Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) per the manufacturer's protocol. Cells were transfected with a lipoplex containing individual crRNA (15 nM), trRNA (15 nM), and donor oligo with (10 nM) Lipofectamine RNAiMAX (0.3 .mu.L/well) and OptiMem. Cells were lysed 24 hours post transfection and genomic DNA was extracting using Zymo's Quick gDNA 96 Extraction kit (catalog # D3012) following the manufacturer's recommended protocol. The gDNA was quantified using the Qubit High Sensitivity dsDNA kit (Life Technologies). Libraries were prepared per the previously described method in Tsai et al, 2015 with minor modifications. Sequencing was performed on Illumina's MiSeq and HiSeq 2500. The assay identified potential off-target sites for some of the crRNAs which are plotted in FIG. 2.

[0617] Table 23 shows the number of off-target integration sites detected in HekCas9 cells transfected with TTR dgRNAs along with a double stranded DNA oligo donor sequence.

TABLE-US-00026 TABLE 23 Number of off-target integration sites detected for TTR dgRNAs via an oligo insertion based assay GUIDE ID # Sites CR003335 0 CR003336 2 CR003337 10 CR003338 2 CR003339 3 CR003340 0 CR003342 0 CR003343 2 CR003344 0 CR003345 0 CR003346 0 CR003347 1 CR003348 3 CR003351 1 CR003352 2 CR003353 2 CR003355 1 CR003356 4 CR003357 3 CR003359 6 CR003360 0 CR003363 4 CR003365 3 CR003366 1 CR003367 1 CR003368 2 CR003369 2 CR003377 0 CR003380 0 CR003382 34 CR003383 1 CR003385 3 CR003386 1 CR003387 6 CR003388 2 CR003389 2 CR003390 1 CR003391 0 CR003392 0 CR005298 0 CR005300 0 CR005301 0 CR005302 1 CR005303 1 CR005304 0

[0618] Additionally, a subset of the guides was assessed for off-target potential as modified sgRNAs in the Hek_Cas9 cells via the oligo based insertion method described above. The off-target results were plotted in FIG. 4.

[0619] Table 24 shows the number of off-target integration sites detected in HekCas9 cells transfected with TTR sgRNAs along with a double stranded DNA oligo donor sequence.

TABLE-US-00027 TABLE 24 Number of off-target integration sites detected for TTR sgRNAs via an insertion detection method GUIDE ID # Sites G000480 11 G000481 3 G000482 13 G000483 5 G000484 7 G000485 22 G000486 12 G000487 14 G000488 0 G000489 19 G000490 12 G000491 28 G000492 97 G000493 7 G000494 4 G000495 13 G000496 1 G000497 26 G000498 82 G000499 4 G000500 46 G000501 4 G000567 9 G000568 937 G000570 19 G000571 16 G000572 15

Example 6. Targeted Sequencing for Validating Potential Off-Target Sites

[0620] The HEK293_Cas9 cells used in Example 5 for detecting potential off-targets constitutively overexpress Cas9, leading to a higher number of potential off-target "hits" as compared to a transient delivery paradigm in various cell types. Further, when delivering sgRNAs (as opposed to dgRNAs), the number of potential off-target hits may be further inflated as sgRNA molecules are more stable than dgRNAs (especially when chemically modified). Accordingly, potential off-target sites identified by an oligo insertion method as used in Example 5 may be validated using targeted sequencing of the identified potential off-target sites.

[0621] In one approach, primary hepatocytes are treated with LNPs comprising Cas9 mRNA and a sgRNA of interest (e.g., a sgRNA having potential off-target sites for evaluation). The primary hepatocytes are then lysed and primers flanking the potential off-target site(s) are used to generate an amplicon for NGS analysis. Identification of indels at a certain level may validate potential off-target site, whereas the lack of indels found at the potential off-target site may indicate a false positive in the HEK293_Cas9 cell assay.

Example 7. Phenotypic Analysis

Western Blot Analysis of Secreted TTR

[0622] The hepatocellular carcinoma cell line, HepG2, was transfected as described in Example 1 with select guides from Table 1 in triplicate. Two days post-transfection, one replicate was harvested for genomic DNA and analysis by NGS sequencing for editing efficiency. Five days post-transfection, media without serum was replaced on one replicate. After 4 hrs the media was harvested for analysis of secreted TTR by WB as previously described. The data for % edit for each guide and reduction of extracellular TTR is provided in FIG. 7.

Western Blot Analysis of Intracellular TTR

[0623] The hepatocellular carcinoma cell line, HUH7, was transfected as described in Example 1 with crRNA comprising the guides from Table 1. The transfected pools of cells were retained in tissue culture and passaged for further analysis. At seven days post-transfection, cells were harvested and whole cell extracts (WCEs) were prepared and subjected to analysis by Western Blot as previously described.

[0624] WCEs were analyzed by Western Blot for reduction of TTR protein. Full length TTR protein has a predicted molecular weight of -16 kD. A band at this molecular weight was observed in the control lanes in the Western Blot.

[0625] Percent reduction of TTR protein was calculated using the Licor Odyssey Image Studio Ver 5.2 software. GAPDH was used as a loading control and probed simultaneously with TTR. A ratio was calculated for the densitometry values for GAPDH within each sample compared to the total region encompassing the TTR band. Percent reduction of TTR protein was determined after the ratios were normalized to control lanes. Results are shown in FIG. 8.

Example 8. LNP Delivery to Humanized TTR Mice and Mice Having Wt (Murine) TTR

[0626] Mice humanized with respect to the TTR gene were dosed with LNP formulations 701-704 containing the guides indicated in Table 25 (5 mice per formulation). These humanized TTR mice were engineered such that a region of the endogenous murine TTR locus was deleted and replaced with an orthologous human TTR sequence so that the locus encodes a human TTR protein. For comparison, 6 mice with murine TTR were dosed with LNP700, containing a guide (G000282) targeting murine TTR. LNPs with Formulation Numbers 1-5 in Table 25 were prepared using the Nanoassemblr.TM. procedure as described above while LNPs with Formulation Numbers 6-16 were prepared using the cross-flow procedure described above but purified using PD-10 columns (GE Healthcare Life Sciences) and concentrated using Amicon centrifugal filter units (Millipore Sigma). As negative controls, mice of the corresponding genotype were dosed with vehicle alone (Tris-saline-sucrose buffer (TSS)). The background of the humanized TTR mice administered LNPs with Formulation Numbers 2-5 in Table 25 was 50% 12956/SvEvTac 50% C57BL/6NTac; the background of the humanized TTR mice administered LNPs having Formulation Numbers 6-16 in Table 25 as well as the mice with murine TTR (administered LNP700, Formulation Number 1) was 75% C57BL/6NTac 25% 12956/SvEvTac.

TABLE-US-00028 TABLE 25 LNP formulations for dosing humanized TTR mice. Molar Ratios (Lipid A, RNA Cholesterol, concen- DSPC, and Formulation tration N:P PEG2k-DMG, Number LNP Guide (mg/ml) Ratio respectively) 1 LNP700 G000282 0.53 4.5 45:44:9:2 2 LNP701 G000481 0.46 4.5 45:44:9:2 3 LNP702 G000489 0.61 4.5 45:44:9:2 4 LNP703 G000494 0.57 4.5 45:44:9:2 5 LNP704 G000499 0.59 4.5 45:44:9:2 6 LNP1148 G000481 0.73 4.5 45:44:9:2 7 LNP1152 G000499 0.45 6.0 50:38:9:3 8 LNP1153 G000482 0.53 6.0 50:38:9:3 9 LNP1155 G000571 0.70 6.0 50:38:9:3 10 LNP1156 G000572 0.58 6.0 50:38:9:3 11 LNP1157 G000480 0.84 6.0 50:38:9:3 12 LNP1159 G000488 0.79 6.0 50:38:9:3 13 LNP1160 G000493 0.71 6.0 50:38:9:3 14 LNP1161 G000500 0.66 6.0 50:38:9:3 15 LNP1162 G000567 0.69 6.0 50:38:9:3 16 LNP1163 G000570 0.66 6.0 50:38:9:3

[0627] LNPs having Formulation numbers 1-5 contained Cas9 mRNA of SEQ ID NO:2 and LNPs having Formulation Numbers 6-16 contained Cas9 mRNA of SEQ ID NO: 1, all in a 1:1 ratio by weight to the guide. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in the molar ratios recited in Table 25, respectively. Dosing with LNPs having Formulation Numbers 1-5 was at 2 mg/kg (total RNA content) and dosing with LNPs having Formulation Numbers 6-16 was at 1 mg/kg (total RNA content). Liver editing results were determined using primers designed to amplify the region of interest for NGS analysis. Liver editing results for Formulation Numbers 1-5 are shown in FIG. 9 and indicate editing of the human TTR sequence with each of the four guides tested at a level >35% editing (mean values) with G000494 and G000499 providing values near 60%. Liver editing results for formulation numbers 6-8, 10-13, and 15-16 are shown in FIG. 13 and Table 26, which show efficient editing of the human TTR sequence with each of the formulations tested. Greater than 38% editing was seen for all formulations, with several formulations providing editing values greater than 60%. Formulations 9 and 14 are not shown due to the design of the PCR amplicon and a resulting low number of sequencing reads.

[0628] The level of human TTR in serum was measured in the mice provided formulation numbers 6-8, 10-13, and 15-16. See FIG. 14B. FIG. 14A is a repeat of FIG. 13 provided for comparison purposes. Knockdown of serum human TTR was detected for each formulation tested, which correlated with the amount of editing detected in liver (See FIG. 14A vs 14B, Table 26).

TABLE-US-00029 TABLE 26 GUIDE ID % Editing Serum TTR(% TSS) TSS (vehicle) 0.06 100 G481 61.28 10.52 G499 65.66 8.39 G482 70.86 4.65 G572 73.52 2.11 G480 77.34 3.48 G488 59.125 27.78 G493 38.55 49.73 G567 47.525 44.24 G570 45.5 41.73 G571 33.88 11.39 G500 44.44 34.28

[0629] In another set of experiments, humanized TTR mice were dosed with LNP formulations across a range of doses with guides G000480, G000488, G000489 and G000502. The formulations contained Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively, and having a N:P ratio of 6. Dosing was at 1, 0.3, 0.1, or 0.03 mg/kg (n=5/group). The LNPs were prepared using the cross-flow procedure described above and purified and concentrated using PD-10 columns and Amicon centrifugal filter units, respectively. Liver editing results were determined using primers designed to amplify the region of interest for NGS analysis and serum human TTR levels were measured as described above. Results for liver editing are shown in FIG. 26A and serum human TTR levels in FIG. 26B-C. A dose response for both editing and serum TTR levels was evident.

[0630] In another set of experiments, humanized TTR mice were dosed with LNP formulations across a range of doses with guides G000481, G000482, G000486 and G000499. The formulations contained Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively, and had an N:P ratio of 6. Dosing was at 1, 0.3, or 0.1 mg/kg (n=5/group). The LNPs were prepared using the cross-flow procedure described above and purified and concentrated using PD-10 columns and Amicon centrifugal filter units, respectively. Liver editing results were determined using primers designed to amplify the region of interest for NGS analysis and serum human TTR levels were measured as described above. Results for liver editing are shown in FIG. 27A and serum human TTR levels in FIG. 27B-C. A dose response for both editing and serum TTR levels was evident.

[0631] In another set of experiments, humanized TTR mice were dosed with LNP formulations across a range of doses with guides G000480, G000481, G000486, G000499 and G000502. The formulations contained Cas9 mRNA (SEQ ID NO: 1) in a 1:2 ratio by weight to the guide. The LNPs contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio, respectively, and had an N:P ratio of 6. Dosing was at 1, 0.3, or 0.1 mg/kg (n=5/group). The LNPs were prepared using the cross-flow procedure described above and purified and concentrated using PD-10 columns and Amicon centrifugal filter units, respectively. Liver editing results were determined using primers designed to amplify the region of interest for NGS analysis and serum human TTR levels were measured as described above. Results for liver editing are shown in FIG. 28A and serum human TTR levels in FIG. 28B-C. A dose response for both editing and serum TTR levels was evident.

[0632] In separate experiments using wild type CD-1 mice, an LNP formulation comprising guide G000502, which is cross homologous between mouse and cyno, was tested in a dose response study. The formulation contained Cas9 mRNA (SEQ ID NO: 1) in a 1:1 ratio by weight to the guide. The LNP contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2 molar ratio, respectively, and having a N:P ratio of 6. Dosing was at 1, 0.3, 0.1, 0.03, or 0.01 mg/kg (n=5/group). Liver editing results were determined using primers designed to amplify the region of interest for NGS analysis. Results for liver editing are shown in FIG. 15A and serum mouse TTR levels in FIG. 15B. A dose response for both editing and serum TTR levels was evident.

Example 9. LNP Delivery to Mice in Multiple Doses

[0633] Mice (females from Charles River Laboratory, aged approximately 6-7 weeks) were dosed with an LNP formulation LNP705, prepared using cross-flow and TFF procedures as described above containing G000282 ("G282") and Cas9 mRNA (SEQ ID NO: 2) in a 1:1 ratio by weight and a total RNA concentration of 0.5 mg/ml. The LNP had an N:P ratio of 4.5 and contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 45:44:9:2 molar ratio, respectively. Groups were dosed either once weekly up to one, two, three, or four weeks (QWx1-4) or once monthly up to two or three months (QMx2-3). Dosages were 0.5 mg/kg or 1 mg/kg (total RNA content). Control groups received a single dose on day 1 of 0.5, 1, or 2 mg/kg. Each group contained 5 mice. Serum TTR was analyzed by ELISA and at necropsy the liver, spleen and muscle were each collected for NGS editing analysis. Groups are shown in Table 27. X=sacrifice and necropsy. MPK=mg/kg.

TABLE-US-00030 TABLE 27 Study Groups Total Duration/ Dose Dose Dose Dose Dose NX Dose NX Dose Dose (MPK) Day Day Day Day Day Day Day Group Regimen (MPK) Given 1 8 15 22 28 43 49 1 4 Week 0 (TSS 0 X X X X X Multi control) Dose/ QW .times. 4 2 2 Month 1 3 X X X X 3 Multi 0.5 1.5 X X X X Dose/ QM .times. 3 4 1 Month 1 2 X X X 5 Multi 0.5 1 X X X Dose/ QM .times. 2 6 4 Week 1 4 X X X X X 7 Multi 0.5 2 X X X X X Dose/ QW .times. 4 8 3 Week 1 3 X X X X 9 Multi 0.5 1.5 X X X X Dose/ QW .times. 3 10 2 Week 1 2 X X X 11 Multi 0.5 1 X X X Dose/ QW .times. 2 12 Single 1 1 X X 13 Dose/ 0.5 0.5 X X 14 QW .times. 1 2 2 Day Day 26 32

[0634] Table 28 and FIGS. 10A-11B show serum TTR level results (% KD=% knockdown). Table 29 and FIGS. 12A-C show liver editing results.

TABLE-US-00031 TABLE 28 Serum TTR Results. Serum TTR Serum TTR Time Regimen Dose (.mu.g/mL) (% KD) QWx4 TSS 1190.7 -- QMx3 0.5 245.01 79.42 QMx2 0.5 776.73 34.77 QWx4 0.5 347.43 70.82 QWx3 0.5 405.70 65.93 QWx2 0.5 432.25 63.70 QWx1 0.5 804.06 32.47 QMx3 1 91.95 92.28 QMx2 1 176.81 85.15 QWx4 1 119.52 89.96 QWx3 1 167.15 85.96 QWx2 1 130.98 89.00 QWx1 1 573.02 51.88 QWx1 2 219.07 81.60

TABLE-US-00032 TABLE 29 Liver Editing Results. Time Regimen Dose Liver Editing (%) QWx4 TSS 0.38 QMx3 0.5 48.18 QMx2 0.5 36.66 QWx4 0.5 56.03 QWx3 0.5 51.35 QWx2 0.5 34.77 QWx1 0.5 24.16 QMx3 1 63.40 QMx2 1 57.37 QWx4 1 62.89 QWx3 1 59.22 QWx2 1 60.12 QWx1 1 35.16 QWx1 2 60.57

[0635] The results show that it is possible to build up a cumulative dose and effect with multiple administrations over time, including at weekly or monthly intervals, to achieve increasing editing levels and % KD of TTR.

Example 10. RNA Cargo: Varying mRNA and gRNA Ratios

[0636] This study evaluated in vivo efficacy in mice of different ratios of gRNA to mRNA. CleanCap.TM. capped Cas9 mRNAs with the ORF of SEQ ID NO: 4, HSD 5' UTR, human albumin 3' UTR, a Kozak sequence, and a poly-A tail were made by IVT synthesis as indicated in Example 1 with N1-methylpseudouridine triphosphate in place of uridine triphosphate.

[0637] LNP formulations prepared from the mRNA described and G282 (SEQ ID NO: 124) as described in Example 1 with Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio and with an N:P ratio of 6. The gRNA:Cas9 mRNA weight ratios of the formulations were as shown in FIGS. 19A and 19B.

[0638] For in vivo characterization, the LNPs were administered to mice at 0.1 mg total RNA (mg guide RNA+mg mRNA) per kg (n=5 per group). At 7-9 days post-dose, animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured as described in Example 1. Serum TTR and liver editing results are shown in FIGS. 19A and 19B. Negative control mice were dosed with TSS vehicle.

[0639] In addition, the above LNPs were administered to mice at a constant mRNA dose of 0.05 mg mRNA per kg (n=5 per group), while varying the gRNA dose from 0.06 mg per kg to 0.4 mg per kg. At 7-9 days post-dose, animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured. Serum TTR and liver editing results are shown in FIG. 19C and FIG. 19D. Negative control mice were dosed with TSS vehicle.

Example 11. Off-Target Analysis of TTR sgRNAs in Primary Human Hepatocyes

[0640] Off-target analysis of sgRNAs targeting TTR was performed in primary human hepatocytes (PHH) as described in Example 5, with the following modifications. PHH were plated at a density of 33,000 cells per well on collagen-coated 96-well plates as described in Example 1. Twenty-four hours post plating, cells were washed with media and transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) as described in Example 1. Cells were transfected with a lipoplex containing 100 ng Cas9 mRNA, immediately followed by the addition of another lipoplex containing 25 nM of the sgRNA and 12.5 nM of the donor oligo (0.3 .mu.L/well). Cells were lysed 48 hours post-transfection and gDNA was extracted and analyzed as further described in Example 5. The data is graphically represented in FIG. 20.

[0641] Table 30 shows the number of off-target integration sites detected in PHH, and compares to the number of sites that were detected in the HekCas9 cells used in Example 5. Fewer sites were detected in PHH for every guide tested as compared to the HekCas9 cell line, with no unique sites detected in PHH alone.

TABLE-US-00033 TABLE 30 Number of off-target integration sites detected for TTR sgRNAs in PHH via an oligo insertion based assay # Sites in # HekCas9 cells GUIDE ID Sites in PHH (Example 5) G000480 2 11 G000481 0 3 G000482 2 13 G000483 0 5 G000484 0 7 G000485 3 22 G000486 0 12 G000487 0 14 G000488 0 0 G000489 2 19 G000490 0 12 G000491 7 28 G000492 5 97 G000493 1 7 G000494 0 4 G000495 1 13 G000496 0 1 G000497 3 26 G000498 19 82 G000499 1 4 G000500 12 46 G000501 0 4 G000567 0 9 G000568 11 936 G000570 1 19 G000571 1 16 G000572 2 15

[0642] Following the identification of potential off-target sites in PHH via the oligo insertion assay, certain potential sites were further evaluated by targeted amplicon sequencing, e.g., as described in Example 6. In addition to the potential off-target sites identified by the oligo insertion strategy, additional potential off-target sites identified by in silico prediction were included in the analysis.

[0643] To this end, PHH were treated with LNPs comprising 100 ng of Cas9 mRNA (SEQ ID NO:1) and the gRNA of interest at 14.68 nM (in a 1:1 ratio by weight), as described in Example 4. The LNPs were prepared using the cross-flow procedure described above and purified and concentrated using PD-10 columns and Amicon centrifugal filter units, respectively. The LNPs were formulated with an N:P ratio of 6.0 and contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:2 molar ratio, respectively. Following LNP treatment, isolated genomic DNA was analyzed by NGS (e.g., as described in Examples 1 and 6) to determine whether indels could be detected at the potential off-target site, which would be indicative of a Cas9-mediated cleavage event. Tables 31 and 32 show the potential off-target sites that were evaluated for the gRNAs G000480 and G000486, respectively.

[0644] As shown in FIGS. 21A-B and 22A-B and Table 33 below, indels were detected at low levels for only two of the potential off-target sites identified by the oligo insertion assay for G000480, and only one for G000486. No indels were detected at any of the in silico predicted sites for either guide. Further, indels were only detected at these sites using a near-saturating dose of LNP, as the indel rates observed at the on-target sites for G000480 and G000486 were .about.97% and .about.91%, respectively (See Table 33). The genomic coordinates of these sites are also reported in Tables 31 and 32, and each correspond to sequences that do not code for any protein.

[0645] A dose response assay was then performed in order to determine the highest dose of LNP in which no off-targets were detected. PHH were treated with LNPs comprising either G000480 or G000486 as described in Example 4. The doses ranged across 11 points with respect to gRNA concentration (0.001 nM, 0.002 nM, 0.007 nM, 0.02 nM, 0.06 nM, 0.19 nM, 0.57 nM, 1.72 nM, 5.17 nM, 15.51 nM, and 46.55 nM). As represented by the dashed vertical line in FIGS. 21A-B and 22A-B, the highest concentrations (with respect to the concentration of gRNA) at which the potential off-target sites were no longer detected for G000480 and G000486 were 0.57 nM and 15.51 nM, respectively, which resulted in on-target indel rates of 84.60% and 89.50%, respectively.

TABLE-US-00034 TABLE 31 Identified potential off target sites via insertion detection and in silico prediction for G000480 evaluated via targeted amplicon sequencing Off-target Chromosomal Coordinates GUIDE ID (OT) Site ID Assay Used (hg38) Strand G000480 INS-OT.1 Insertion Detection chr7: 94767406-94767426 + G000480 INS-OT.2 Insertion Detection chr2: 192658562-192658582 + G000480 INS-OT.3 Insertion Detection chr7: 4834390-4834410 + G000480 INS-OT.4 Insertion Detection chr20: 9216118-9216138 - G000480 INS-OT.5 Insertion Detection chr10: 12547071-12547091 + G000480 INS-OT.6 Insertion Detection chr6: 168377978-168377998 - G000480 INS-OT.7 Insertion Detection chr12: 114144669-114144689 - G000480 INS-OT.8 Insertion Detection chr10: 7376755-7376775 + G000480 INS-OT.9 Insertion Detection chr2: 52950299-52950319 + G000480 INS-OT.10 Insertion Detection chr8: 56579165-56579185 - G000480 INS-OT.11 Insertion Detection chr1: 189992255-189992275 + G000480 PRED-OT.1 in silico prediction chr10:12547071-12547091 + G000480 PRE-DOT.2 in silico prediction chrX: 119702782-119702802 + G000480 PRED-OT.3 in silico prediction chr1: 116544586-116544606 + G000480 PRED-OT.4 in silico prediction chr6: 88282884-88282904 + G000480 PRED-OT.6 in silico prediction chr5: 121891868-121891888 + G000480 PRED-OT.7 in silico prediction chr3: 52544945-52544965 + G000480 PRED-OT.8 in silico prediction chr15: 36949639-36949659 + G000480 PRED-OT.9 in silico prediction chr5: 33866486-33866506 + G000480 PRED-OT.10 in silico prediction chr5: 159755754-159755774 + G000480 PRED-OT.11 in silico prediction chr5: 31349859-31349879 + G000480 PRED-OT.12 in silico prediction chr11: 79485652-79485672 + G000480 PRED-OT.13 in silico prediction chr15: 29448864-29448884 + G000480 PRED-OT.14 in silico prediction chr5: 171153565-171153585 + G000480 PRED-OT.15 in silico prediction chr9: 84855273-84855293 + G000480 PRED-OT.16 in silico prediction chr6: 159953060-159953080 + G000480 PRED-OT.17 in silico prediction chr16: 51849024-51849044 + G000480 PRED-OT.18 in silico prediction chr3: 24108809-24108829 + G000480 PRED-OT.19 in silico prediction chr18: 41118310-41118330 + G000480 PRED-OT.20 in silico prediction chr10: 108975241-108975261 + G000480 PREDO-T.21 in silico prediction chr1: 44683633-44683653 + G000480 PRED-OT.22 in silico prediction chr2: 196214849-196214869 + G000480 PRED-OT.23 in silico prediction chr9: 117353544-117353564 + G000480 PRED-OT.24 in silico prediction chr1: 55583322-55583342 + G000480 PRED-OT.25 in silico prediction chr12: 28246827-28246847 + G000480 PRED-OT.26 in silico prediction chr4: 54545361-54545381 + G000480 PRED-OT.27 in silico prediction chr13: 22364836-22364856 + G000480 PRED-OT.28 in silico prediction chr13: 80816049-80816069 + G000480 PRED-OT.29 in silico prediction chr7: 39078622-39078642 + G000480 PRED-OT.30 in silico prediction chr2: 59944386-59944406 + "INS-OT.N" refers to an off-target site ID detected by oligo insertion, where N is an integer specified above; "PRED-OT.N" refers to an off-target site ID predicted via in silico methods, where N is an integer specified above.

TABLE-US-00035 TABLE 32 Identified potential off target sites via insertion detection and in silico prediction for G000486 evaluated via targeted amplicon sequencing Off-target Chromosomal Coordinates GUIDE ID (OT) Site ID Assay Used (hg38) Strand G000486 INS-OT.1 Insertion Detection chr14: 77332157-77332177 + G000486 INS-OT.2 Insertion Detection chr14: 54672059-54672079 - G000486 INS-OT.3 Insertion Detection chr4: 108513169-108513189 - G000486 INS-OT.4 Insertion Detection chr5: 91397023-91397043 - G000486 INS-OT.5 Insertion Detection chr9:116626135-116626155 - G000486 INS-OT.6 Insertion Detection chr6: 73201226-73201246 + G000486 INS-OT.7 Insertion Detection chr16: 89368352-89368372 - G000486 INS-OT.8 Insertion Detection chr7: 56308371-56308391 - G000486 INS-OT.9 Insertion Detection chr21:43605667-43605687 + G000486 INS-OT.10 Insertion Detection chr5: 26758030-26758050 + G000486 INS-OT.11 Insertion Detection chr17: 30656428-30656448 + G000486 INS-OT.12 Insertion Detection chr8: 130486452-130486472 + G000486 PRED-OT.1 in silico prediction chr11: 44707064-44707084 + G000486 PRED-OT.2 in silico prediction chr5: 50775396-50775416 + G000486 PRED-OT.3 in silico prediction chr4: 141623949-141623969 + G000486 PRED-OT.4 in silico prediction chr1: 223481186-223481206 + G000486 PRED-OT.5 in silico prediction chr6: 39951487-39951507 + G000486 PRED-OT.6 in silico prediction chrY: 5456047-5456067 + G000486 PRED-OT.8 in silico prediction chr6: 129868719-129868739 + G000486 PRED-OT.9 in silico prediction chrX: 80450312-80450332 + G000486 PRED-OT.10 in silico prediction chr7: 27256771-27256791 + G000486 PRED-OT.11 in silico prediction chr3: 181416528-181416548 + G000486 PRED-OT12 in silico prediction chr7: 146425020-146425040 + G000486 PRED-OT.13 in silico prediction chr3: 16980977-16980997 + G000486 PRED-OT.14 in silico prediction chr7: 118161002-118161022 + G000486 PRED-OT.15 in silico prediction chr6: 102220539-102220559 + G000486 PRED-OT.16 in silico prediction chr12: 127278991-127279011 + G000486 PRED-OT.17 in silico prediction chr2: 67686631-67686651 + G000486 PRED-OT.18 in silico prediction chr1: 114467665-114467685 + G000486 PRED-OT.19 in silico prediction chr3: 194514436-194514456 + G000486 PRED-OT.20 in silico prediction chr14: 31767581-31767601 + G000486 PRED-OT.21 in silico prediction chr16: 28706209-28706229 + G000486 PRED-OT.22 in silico prediction chr8: 110526279-110526299 + G000486 PRED-OT.23 in silico prediction chr19: 2899814-2899834 + G000486 PRED-OT.25 in silico prediction chr3: 130760261-130760281 + G000486 PRED-OT.26 in silico prediction chr11: 2506046-2506066 + G000486 PRED-OT.27 in silico prediction chr2: 153918318-153918338 + G000486 PRED-OT.28 in silico prediction chr14: 40590226-40590246 + G000486 PRED-OT.29 in silico prediction chr18: 806650-806670 + G000486 PRED-OT.30 in silico prediction chr2: 117707480-117707500 + "INS-OT.N" refers to an off-target site ID detected by oligo insertion, where N is an integer specified above; "PRED-OT.N" refers to an off-target site ID predicted via in silico methods, where N is an integer specified.

TABLE-US-00036 TABLE 33 Detected Off Target sites in PHH treated with LNP containing 100 ng mRNA and 31.03 nM gRNA Indel Frequency (using LNP with 100 ng Cas9 Indel Off-target mRNA and 14.68 Frequency GUIDE ID (OT) Site ID Site Type nM gRNA) std. dev. G000480 n/a On-Target 97.33% 1.10% G000480 INS-OT.2 Off-Target 1.43% 0.40% G000480 INS-OT.4 Off-Target 0.97% 0.25% G000486 n/a On-Target 91.33% 1.97% G000486 INS-OT.4 Off-Target 0.47% 0.06%

Example 12. LNP Delivery to Humanized Mouse Model of ATTR

[0646] A well-established humanized transgenic mouse model of hereditary ATTR amyloidosis that expresses the V30M pathogenic mutant form of human TTR protein was used in this Example. This mouse model recapitulates the TTR deposition phenotype in tissues observed in ATTR patients, including within the peripheral nervous system and gastrointestinal (GI) tract (See Santos et al., Neurobiol Aging. 2010 February; 31(2):280-9).

[0647] Mice (aged approximately 4-5 months) were dosed with LNP formulations prepared using the cross-flow and TFF procedures as described in Example 1. The LNPs were formulated with an N:P ratio of 6.0 and contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:2 molar ratio, respectively. The LNPs contained Cas9 mRNA (SEQ ID NO: 1) and either G000481 ("G481") or a non-targeting control guide G000395 ("G395"; SEQ ID NO: 273), in a 1:1 ratio of gRNA:mRNA by weight.

[0648] Mice were injected via the lateral tail vein as described in Example 1 with a single 1 mg/kg (of total RNA content) dose of LNP with an n=10/group. At 8 weeks post treatment, the mice were euthanized for sample collection. Human TTR protein levels were measured in serum and cerebrospinal fluid (CSF) by ELISA as previously described by Butler et al., Amyloid. 2016 June; 23(2):109-18. Liver tissue was assayed for editing levels as described in Example 1. Other tissues (stomach, colon, sciatic nerve, dorsal root ganglion (DRG)) were collected and processed for semi-quantitative immunohistochemistry as previously described by Goncalves et al., Amyloid. 2014 September; 21(3): 175-184. Statistical analysis for the immunohistochemistry data was performed using Mann Whitney test with a p-value<0.0001.

[0649] As shown in FIG. 23A-B, robust editing (49.4%) of TTR was observed in livers of the humanized mice following the single dose of LNP comprising G481, with no editing detected in the control group. Analysis of the editing events demonstrated that 96.8% of the events were insertions, with the remainder deletions.

[0650] As shown in FIG. 24A-B, TTR protein levels were decreased in plasma but not in CSF from the treated mice, with greater than 99% knockdown of TTR plasma levels observed (p<0.001).

[0651] The near complete knockdown of TTR observed in the plasma of treated animals correlated with the clearance of TTR protein amyloid deposition in the assayed tissues. As shown in FIG. 25, control mice exhibited amyloid staining in tissues which resembles the pathophysiology observed in human subjects with ATTR. Decreasing circulating TTR by editing the HuTTR V30M locus resulted in a dramatic decrease of amyloid deposition in tissues. Approximately 85% or better reduction in TTR staining was observed across the treated tissues 8 weeks post-treatment (FIG. 25).

Example 13. TTR mRNA Knockdown in Primary Human Hepatocytes (PHH)

[0652] In one experiment, PHH were cultured and treated with LNPs comprising Cas9 mRNA (SEQ ID NO:1) and a gRNA of interest (See FIG. 29, Table 34), as described in Example 4. The LNPs were prepared using the cross-flow procedure described above and purified and concentrated using PD-10 columns and Amicon centrifugal filter units, respectively. The LNPs were formulated with an N:P ratio of 6.0 and contained Lipid A, Cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:2 molar ratio, respectively. The LNPs comprised a gRNA:mRNA ratio of 1:2, and the cells were treated at a dose of 300 ng (with respect to the amount of mRNA cargo delivered).

[0653] Ninety-six (96) hours following LNP treatment (with biological triplicates for each condition), mRNA was purified from PHH cells using the Dynabeads mRNA DIRECT Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Reverse Transcription (RT) was performed with Maxima reverse transcriptase (ThermoFisher Scientific) and a poly-dT primer. The resulting cDNA was purified with Ampure XP Beads (Agencourt). For Quantitative PCR, 2% of the purified cDNA was amplified with Taqman Fast Advanced Mastermix and 3 Taqman probe sets, TTR (Assay ID: Hs00174914_m1), GAPDH (Assay ID: Hs02786624_g1), and PPIB (Assay ID: Hs00168719_m1). The assays were run on the QuantStudio 7 Flex Real Time PCR System according to the manufacturer's instructions (Life Technologies). Relative expression of TTR mRNA was calculated by normalizing to the endogenous controls (GAPDH and PPIB) individually, and then averaged.

[0654] As shown in FIG. 29 and reproduced numerically in Table 34 below, each of the LNP formulations tested resulted in knockdown of TTR mRNA, as compared to the negative (untreated) control. The groups in FIG. 29 and Table 34 are identified by the gRNA ID used in each LNP preparation. Relative expression of TTR mRNA is plotted in FIG. 29, whereas the percent knockdown of TTR mRNA is provided in Table 34.

TABLE-US-00037 TABLE 34 GUIDE ID Avg % Knockdown Std Dev G000480 95.19 1.68 G000481 91.39 2.39 G000482 82.31 4.51 G000483 68.78 13.45 G000484 75.22 9.05 G000488 92.77 3.76 G000489 91.85 2.77 G000490 78.34 5.76 G000493 87.53 4.54 G000494 91.15 3.63 G000499 91.38 1.71 G000500 92.90 3.15 G000567 90.89 5.39 G000568 53.44 20.20 G000570 93.38 2.66 G000571 96.17 2.07 G000572 55.92 24.53

[0655] In a separate experiment, TTR mRNA knockdown was evaluated following treatment with LNPs comprising G000480, G000486, and G000502. The LNPs were formulated and PHH were cultured and treated with the LNPs, each as described in the experiment above in this Example with the exception that the cells were treated at a dose of 100 ng (with respect to the amount of mRNA cargo delivered).

[0656] Ninety-six (96) hours following LNP treatment (single treatment for each condition), mRNA was purified from PHH cells using the Dynabeads mRNA DIRECT Kit (ThermoFisher Scientific) according to the manufacturer's protocol. Reverse Transcription (RT) was performed with the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions. For Quantitative PCR, 2% of the cDNA was amplified with Taqman Fast Advanced Mastermix and 3 Taqman probe sets, TTR (Assay ID: Hs00174914_m1), GAPDH (Assay ID: Hs02786624_g1), and PPIB (Assay ID: Hs00168719_m1). The assays were run on the QuantStudio 7 Flex Real Time PCR System according to the manufacturer's instructions (Life Technologies). Relative expression of TTR mRNA was calculated by normalizing to the endogenous controls (GAPDH and PPIB) individually, and then averaged.

[0657] As shown in FIG. 30 and reproduced numerically in Table 35 below, each of the LNP formulations tested resulted in knockdown of TTR mRNA, as compared to the negative (untreated) control. The groups in FIG. 30 and Table 35 are identified by the gRNA ID used in each LNP preparation. Relative expression of TTR mRNA is plotted in FIG. 30, whereas the percent knockdown of TTR mRNA is provided in Table 35.

TABLE-US-00038 TABLE 35 GUIDE ID Avg % Knockdown Std Dev G000480 95.61 0.92 G000486 97.36 0.63 G000502 90.94 2.63

TABLE-US-00039 Sequence Table SEQ Description Sequence ID No. Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 1 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with 5' UTR GGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA of HSD, ORF GGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT corresponding CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA to SEQ ID AGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA NO: 204, CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA Kozak CGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA sequence, GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA and 3' UTR CCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG of ALB CACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG CGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG CGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC GGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGG ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCT GCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGC ACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCT GACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGA AATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGG AGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGAT GGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAG AAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGG AGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTA CGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAA GAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCT GCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCAC AGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAA GCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTT CAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAA GAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATT CAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCT GACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGAC ATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAG ATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGA CAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGA AGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACA CATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGAC AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGA AAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACA GAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAA CGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAG AAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCAC ACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGA ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAAC AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAAT CGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTT CGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAA GACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTA CGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAA GGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGG AATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCT GGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCC GAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGA AGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGA CGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCC GATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCT GGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAG ATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCAT CACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 2 transcript GCAGGCCTTATTCGGATCCATGCCTAAGAAAAAGCGGAAGGTCGACGGGGA comprising TAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGC Cas9 ORF AGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGG corresponding GAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTT to SEQ ID TGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCG NO: 205 ACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATCTTTTC using codons GAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAAGAATC with TTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGGAAA generally CATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCT high GCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTA expression CTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGAAGG in humans CGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACTGGT GCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGT CGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGA AAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAA CTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGA CCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGA CTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCT TGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGT GAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTA CGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCA ACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGGTA CGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCAT CAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCT GAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCAT TCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGA GGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCT GACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCG CTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTT CGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAAT GACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAG CCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATA CGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAA AGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCA GCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAAT CAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCT CCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGA CATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGAT GATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCAT GAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAA GCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTT CCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATCCACGA CGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACA GGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGAT TAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGT CATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAA CCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGAT CGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGT GGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAA TGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGA TTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGAT CGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAA TGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCT CCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGC CGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCA GCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTC CCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAA GGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCA GTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATA CCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGA ATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGAT AGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTA CTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGA AATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGT GTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCC GCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAA GGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAA AGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATA CTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAA ATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGA GAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAA GGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGG TCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCT GGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGA AAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGA GCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTC AAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTA CAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCA CTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGA TACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGC GACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCT GTCGCAGCTGGGTGGCGATTGATAGTCTAGCCATCACATTTAAAAGCATCT CAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCA TCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACAT AAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAA ATGGAAAGAACCTCGAG modified mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAm 3 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA sequence mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (''N'' may be any natural or non- natural nucleotide) 30/30/39 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAAAAAAAAAAAAA 3 poly-A AAAAAAAAAAAACCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA sequence AAA CR003335 CUGCUCCUCCUCUGCCUUGC 5 gRNA targeting Human TTR (Exon 1) CR003336 CCUCCUCUGCCUUGCUGGAC 6 gRNA targeting Human TTR (Exon 1) CR003337 CCAGUCCAGCAAGGCAGAGG 7 gRNA targeting Human TTR (Exon 1) CR003338 AUACCAGUCCAGCAAGGCAG 8 gRNA targeting Human TTR (Exon 1) CR003339 ACACAAAUACCAGUCCAGCA 9 gRNA targeting Human TTR (Exon 1) CR003340 UGGACUGGUAUUUGUGUCUG 10 gRNA targeting Human TTR (Exon 1) CR003341 CUGGUAUUUGUGUCUGAGGC 11 gRNA targeting Human TTR (Exon 1) CR003342 CUUCUCUACACCCAGGGCAC 12 gRNA targeting Human TTR (Exon 2) CR003343 CAGAGGACACUUGGAUUCAC 13 gRNA targeting Human TTR (Exon 2) CR003344 UUUGACCAUCAGAGGACACU 14 gRNA

targeting Human TTR (Exon 2) CR003345 UCUAGAACUUUGACCAUCAG 15 gRNA targeting Human TTR (Exon 2) CR003346 AAAGUUCUAGAUGCUGUCCG 16 gRNA targeting Human TTR (Exon 2) CR003347 CAUUGAUGGCAGGACUGCCU 17 gRNA targeting Human TTR (Exon 2) CR003348 AGGCAGUCCUGCCAUCAAUG 18 gRNA targeting Human TTR (Exon 2) CR003349 UGCACGGCCACAUUGAUGGC 19 gRNA targeting Human TTR (Exon 2) CR003350 CACAUGCACGGCCACAUUGA 20 gRNA targeting Human TTR (Exon 2) CR003351 AGCCUUUCUGAACACAUGCA 21 gRNA targeting Human TTR (Exon 2) CR003352 GAAAGGCUGCUGAUGACACC 22 gRNA targeting Human TTR (Exon 2) CR003353 AAAGGCUGCUGAUGACACCU 23 gRNA targeting Human TTR (Exon 2) CR003354 ACCUGGGAGCCAUUUGCCUC 24 gRNA targeting Human TTR (Exon 2) CR003355 CCCAGAGGCAAAUGGCUCCC 25 gRNA targeting Human TTR (Exon 2) CR003356 GCAACUUACCCAGAGGCAAA 26 gRNA targeting Human TTR (Exon 2) CR003357 UUCUUUGGCAACUUACCCAG 27 gRNA targeting Human TTR (Exon 2) CR003358 AUGCAGCUCUCCAGACUCAC 28 gRNA targeting Human TTR (Exon 3) CR003359 AGUGAGUCUGGAGAGCUGCA 29 gRNA targeting Human TTR (Exon 3) CR003360 GUGAGUCUGGAGAGCUGCAU 30 gRNA targeting Human TTR (Exon 3) CR003361 GCUGCAUGGGCUCACAACUG 31 gRNA targeting Human TTR (Exon 3) CR003362 GCAUGGGCUCACAACUGAGG 32 gRNA targeting Human TTR (Exon 3) CR003363 ACUGAGGAGGAAUUUGUAGA 33 gRNA targeting Human TTR (Exon 3) CR003364 CUGAGGAGGAAUUUGUAGAA 34 gRNA targeting Human TTR (Exon 3) CR003365 UGUAGAAGGGAUAUACAAAG 35 gRNA targeting Human TTR (Exon 3) CR003366 AAAUAGACACCAAAUCUUAC 36 gRNA targeting Human TTR (Exon 3) CR003367 AGACACCAAAUCUUACUGGA 37 gRNA targeting Human TTR (Exon 3) CR003368 AAGUGCCUUCCAGUAAGAUU 38 gRNA targeting Human TTR (Exon 3) CR003369 CUCUGCAUGCUCAUGGAAUG 39 gRNA targeting Human TTR (Exon 3) CR003370 CCUCUGCAUGCUCAUGGAAU 40 gRNA targeting Human TTR (Exon 3) CR003371 ACCUCUGCAUGCUCAUGGAA 41 gRNA targeting Human TTR (Exon 3) CR003372 UACUCACCUCUGCAUGCUCA 42 gRNA targeting Human TTR (Exon 3) CR003373 GUAUUCACAGCCAACGACUC 43 gRNA targeting Human TTR (Exon 4) CR003374 GCGGCGGGGGCCGGAGUCGU 44 gRNA targeting Human TTR (Exon 4) CR003375 AAUGGUGUAGCGGCGGGGGC 45 gRNA targeting Human TTR (Exon 4) CR003376 CGGCAAUGGUGUAGCGGCGG 46 gRNA targeting Human TTR (Exon 4) CR003377 GCGGCAAUGGUGUAGCGGCG 47 gRNA targeting Human TTR (Exon 4) CR003378 GGCGGCAAUGGUGUAGCGGC 48 gRNA targeting Human TTR (Exon 4) CR003379 GGGCGGCAAUGGUGUAGCGG 49 gRNA targeting Human TTR (Exon 4) CR003380 GCAGGGCGGCAAUGGUGUAG 50 gRNA targeting Human TTR (Exon 4) CR003381 GGGGCUCAGCAGGGCGGCAA 51 gRNA targeting Human TTR (Exon 4) CR003382 GGAGUAGGGGCUCAGCAGGG 52 gRNA targeting Human TTR (Exon 4) CR003383 AUAGGAGUAGGGGCUCAGCA 53 gRNA targeting Human TTR (Exon 4) CR003384 AAUAGGAGUAGGGGCUCAGC 54 gRNA targeting Human TTR (Exon 4) CR003385 CCCCUACUCCUAUUCCACCA 55 gRNA targeting Human TTR (Exon 4) CR003386 CCGUGGUGGAAUAGGAGUAG 56

gRNA targeting Human TTR (Exon 4) CR003387 GCCGUGGUGGAAUAGGAGUA 57 gRNA targeting Human TTR (Exon 4) CR003388 GACGACAGCCGUGGUGGAAU 58 gRNA targeting Human TTR (Exon 4) CR003389 AUUGGUGACGACAGCCGUGG 59 gRNA targeting Human TTR (Exon 4) CR003390 GGGAUUGGUGACGACAGCCG 60 gRNA targeting Human TTR (Exon 4) CR003391 GGCUGUCGUCACCAAUCCCA 61 gRNA targeting Human TTR (Exon 4) CR003392 AGUCCCUCAUUCCUUGGGAU 62 gRNA targeting Human TTR (Exon 4) CR005298 UCCACUCAUUCUUGGCAGGA 63 gRNA targeting Human TTR (Exon 1) CR005299 AGCCGUGGUGGAAUAGGAGU 64 gRNA targeting Human TTR (Exon 4) CR005300 UCACAGAAACACUCACCGUA 65 gRNA targeting Human TTR (Exon 1) CR005301 GUCACAGAAACACUCACCGU 66 gRNA targeting Human TTR (Exon 1) CR005302 ACGUGUCUUCUCUACACCCA 67 gRNA targeting Human TTR (Exon 2) CR005303 UGAAUCCAAGUGUCCUCUGA 68 gRNA targeting Human TTR (Exon 2) CR005304 GGCCGUGCAUGUGUUCAGAA 69 gRNA targeting Human TTR (Exon 2) CR005305 UAUAGGAAAACCAGUGAGUC 70 gRNA targeting Human TTR (Exon 3) CR005306 AAAUCUUACUGGAAGGCACU 71 gRNA targeting Human TTR (Exon 3) CR005307 UGUCUGUCUUCUCUCAUAGG 72 gRNA targeting Human TTR (Exon 4) CR000689 ACACAAAUACCAGUCCAGCG 73 gRNA targeting Cyno TTR CR005364 AAAGGCUGCUGAUGAGACCU 74 gRNA targeting Cyno TTR CR005365 CAUUGACAGCAGGACUGCCU 75 gRNA targeting Cyno TTR CR005366 AUACCAGUCCAGCGAGGCAG 76 gRNA targeting Cyno TTR CR005367 CCAGUCCAGCGAGGCAGAGG 77 gRNA targeting Cyno TTR CR005368 CCUCCUCUGCCUCGCUGGAC 78 gRNA targeting Cyno TTR CR005369 AAAGUUCUAGAUGCCGUCCG 79 gRNA targeting Cyno TTR CR005370 ACUUGUCUUCUCUAUACCCA 80 gRNA targeting Cyno TTR CR005371 AAGUGACUUCCAGUAAGAUU 81 gRNA targeting Cyno TTR CR005372 AAAAGGCUGCUGAUGAGACC 82 gRNA targeting Cyno TTR Not Used 83 Not Used 84 Not Used 85 Not Used 86 G000480 mA*mA*mA*GGCUGCUGAUGACACCUGUUUUAGAmGmCmUmAmGmAmAmAm 87 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000481 mU*mC*mU*AGAACUUUGACCAUCAGGUUUUAGAmGmCmUmAmGmAmAmAm 88 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000482 mU*mG*mU*AGAAGGGAUAUACAAAGGUUUUAGAmGmCmUmAmGmAmAmAm 89 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000483 mU*mC*mC*ACUCAUUCUUGGCAGGAGUUUUAGAmGmCmUmAmGmAmAmAm 90 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000484 mA*mG*mA*CACCAAAUCUUACUGGAGUUUUAGAmGmCmUmAmGmAmAmAm 91 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000485 mC*mC*mU*CCUCUGCCUUGCUGGACGUUUUAGAmGmCmUmAmGmAmAmAm 92 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000486 mA*mC*mA*CAAAUACCAGUCCAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 93 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000487 mU*mU*mC*UUUGGCAACUUACCCAGGUUUUAGAmGmCmUmAmGmAmAmAm 94 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000488 mA*mA*mA*GUUCUAGAUGCUGUCCGGUUUUAGAmGmCmUmAmGmAmAmAm 95 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000489 mU*mU*mU*GACCAUCAGAGGACACUGUUUUAGAmGmCmUmAmGmAmAmAm 96 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000490 mA*mA*mA*UAGACACCAAAUCUUACGUUUUAGAmGmCmUmAmGmAmAmAm 97 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000491 mA*mU*mA*CCAGUCCAGCAAGGCAGGUUUUAGAmGmCmUmAmGmAmAmAm 98 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000492 mC*mU*mU*CUCUACACCCAGGGCACGUUUUAGAmGmCmUmAmGmAmAmAm 99 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000493 mA*mA*mG*UGCCUUCCAGUAAGAUUGUUUUAGAmGmCmUmAmGmAmAmAm 100

sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000494 mG*mU*mG*AGUCUGGAGAGCUGCAUGUUUUAGAmGmCmUmAmGmAmAmAm 101 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000495 mC*mA*mG*AGGACACUUGGAUUCACGUUUUAGAmGmCmUmAmGmAmAmAm 102 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000496 mG*mG*mC*CGUGCAUGUGUUCAGAAGUUUUAGAmGmCmUmAmGmAmAmAm 103 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000497 mC*mU*mG*CUCCUCCUCUGCCUUGCGUUUUAGAmGmCmUmAmGmAmAmAm 104 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000498 mA*mG*mU*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmAm 105 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000499 mU*mG*mA*AUCCAAGUGUCCUCUGAGUUUUAGAmGmCmUmAmGmAmAmAm 106 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000500 mC*mC*mA*GUCCAGCAAGGCAGAGGGUUUUAGAmGmCmUmAmGmAmAmAm 107 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000501 mU*mC*mA*CAGAAACACUCACCGUAGUUUUAGAmGmCmUmAmGmAmAmAm 108 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000567 mG*mA*mA*AGGCUGCUGAUGACACCGUUUUAGAmGmCmUmAmGmAmAmAm 109 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000568 mG*mG*mC*UGUCGUCACCAAUCCCAGUUUUAGAmGmCmUmAmGmAmAmAm 110 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000570 mC*mA*mU*UGAUGGCAGGACUGCCUGUUUUAGAmGmCmUmAmGmAmAmAm 111 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000571 mG*mU*mC*ACAGAAACACUCACCGUGUUUUAGAmGmCmUmAmGmAmAmAm 112 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000572 mC*mC*mC*CUACUCCUAUUCCACCAGUUUUAGAmGmCmUmAmGmAmAmAm 113 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Human TTR G000502 mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAm 114 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000503 mA*mA*mA*AGGCUGCUGAUGAGACCGUUUUAGAmGmCmUmAmGmAmAmAm 115 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000504 mA*mA*mA*GGCUGCUGAUGAGACCUGUUUUAGAmGmCmUmAmGmAmAmAm 116 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000505 mC*mA*mU*UGACAGCAGGACUGCCUGUUUUAGAmGmCmUmAmGmAmAmAm 117 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000506 mA*mU*mA*CCAGUCCAGCGAGGCAGGUUUUAGAmGmCmUmAmGmAmAmAm 118 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000507 mC*mC*mA*GUCCAGCGAGGCAGAGGGUUUUAGAmGmCmUmAmGmAmAmAm 119 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000508 mC*mC*mU*CCUCUGCCUCGCUGGACGUUUUAGAmGmCmUmAmGmAmAmAm 120 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000509 mA*mA*mA*GUUCUAGAUGCCGUCCGGUUUUAGAmGmCmUmAmGmAmAmAm 121 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000510 mA*mC*mU*UGUCUUCUCUAUACCCAGUUUUAGAmGmCmUmAmGmAmAmAm 122 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000511 mA*mA*mG*UGACUUCCAGUAAGAUUGUUUUAGAmGmCmUmAmGmAmAmAm 123 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Cyno TTR G000282 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 124 sgRNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA modified mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sequence targeting Mouse TTR Not used 125 to 200 DNA coding ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGA 201 sequence of TGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTC Cas9 using CTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTG the CTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCA thymidine AGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATC analog of TTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA the minimal GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTC uridine GGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTAC codons CACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTG listed in ATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATC Table 3, GAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAG with start CTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC and stop GGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGA codons CTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTC GGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAAC TTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGAC GACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTG TTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTG AGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAG AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAAC GGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAG TTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTC AAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGA AGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGA CAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAG ATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAAC AGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGG AACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAA AGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAG CACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTC AAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAG AAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTC AAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTC GAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCAC GACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAAC GAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGA GAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAG GTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGC AGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTG GACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGC GGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCG GCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTC AAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTG CAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTG AGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGAC AGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGA CAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACA AAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAG AGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTG GACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAA GTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGAC GCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAG CTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAG ATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAAC GGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAA ATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGC ATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTC AGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGA AAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTC GCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAG CTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGC TTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTC AAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAA AACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAAC GAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCAC

TACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTC GTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGC GCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATC ATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTAC TTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTG GACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATC GACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGTCTAG DNA coding ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGT 202 sequence of TGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC Cas9 using CTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTG codons with CTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCG generally AGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATC high TTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAA expression GAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTT in humans GGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTAC CATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTG ATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATC GAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAA CTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGC GGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGC CTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTC GGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAAT TTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGAC GACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTG TTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTG CGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAG CGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGA CAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAAT GGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAG TTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTC AAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGA TCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGC CAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAA ATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAAT TCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGG AATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAA CGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAG CACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTG AAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAG AAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTC AAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTG GAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCAT GATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAAC GAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGC GAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAG GTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCC CGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTG GATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGATC CACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTCC GGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCG GCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTG AAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGA GAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAG CGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCAC CCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTG CAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTG TCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGAC TCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCA GATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGG CAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACT AAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAA CGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTG GACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAA GTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGAC TTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGAC GCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAA CTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAG ATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTC TTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAAT GGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAA ATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCT ATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTT TCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGC AAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTC GCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAG CTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCG TTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTG AAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAA AATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAAT GAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCAC TACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTC GTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAG TTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCG GCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATT ATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTAC TTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCTG GACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATC GATCTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAGAAGAGA AAGGTGTAATGA Amino acid MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 203 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 with ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL one nuclear IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS localization GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLTPNEKSN signal FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL (1xNLS) as RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN the C- GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminal 7 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acids SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAELSGEQKKAIVDLLEKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTEKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENITHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV Cas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 204 ORF using UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC minimal CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG uridine CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA codons, with AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC start and UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA stop codons GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAG Cas9 mRNA AUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAAUUCCGUGGGU 205 ORF using UGGGCAGUGAUCACGGAUGAAUACAAAGUGCCGUCCAAGAAGUUCAAGGUC codons with CUGGGGAACACCGAUAGACACAGCAUCAAGAAAAAUCUCAUCGGAGCCCUG generally CUGUUUGACUCCGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCG high AGGCGACGCUACACCCGGCGGAAGAAUCGCAUCUGCUAUCUGCAAGAGAUC expression UUUUCGAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACCGCCUGGAA in humans, GAAUCUUUCCUGGUGGAGGAGGACAAGAAGCAUGAACGGCAUCCUAUCUUU with start GGAAACAUCGUCGACGAAGUGGCGUACCACGAAAAGUACCCGACCAUCUAC and stop CAUCUGCGGAAGAAGUUGGUUGACUCAACUGACAAGGCCGACCUCAGAUUG codons AUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCCGCGGACACUUCCUGAUC GAAGGCGAUCUGAACCCUGAUAACUCCGACGUGGAUAAGCUUUUCAUUCAA CUGGUGCAGACCUACAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGC GGCGUCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGCGGCGC CUCGAAAACCUGAUCGCACAGCUGCCGGGAGAGAAAAAGAACGGACUUUUC GGCAACUUGAUCGCUCUCUCACUGGGACUCACUCCCAAUUUCAAGUCCAAU UUUGACCUGGCCGAGGACGCGAAGCUGCAACUCUCAAAGGACACCUACGAC GACGACUUGGACAAUUUGCUGGCACAAAUUGGCGAUCAGUACGCGGAUCUG UUCCUUGCCGCUAAGAACCUUUCGGACGCAAUCUUGCUGUCCGAUAUCCUG CGCGUGAACACCGAAAUAACCAAAGCGCCGCUUAGCGCCUCGAUGAUUAAG CGGUACGACGAGCAUCACCAGGAUCUCACGCUGCUCAAAGCGCUCGUGAGA CAGCAACUGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAAU GGGUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUAAG UUCAUCAAGCCAAUCCUGGAAAAGAUGGACGGAACCGAAGAACUGCUGGUC AAGCUGAACAGGGAGGAUCUGCUCCGGAAACAGAGAACCUUUGACAACGGA UCCAUUCCCCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCGC CAGGAGGACUUUUACCCAUUCCUCAAGGACAACCGGGAAAAGAUCGAGAAA AUUCUGACGUUCCGCAUCCCGUAUUACGUGGGCCCACUGGCGCGCGGCAAU UCGCGCUUCGCGUGGAUGACUAGAAAAUCAGAGGAAACCAUCACUCCUUGG AAUUUCGAGGAAGUUGUGGAUAAGGGAGCUUCGGCACAAAGCUUCAUCGAA CGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGUGCUUCCUAAG CACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAACUGACUAAAGUG AAAUACGUUACUGAAGGAAUGAGGAAGCCGGCCUUUCUGUCCGGAGAACAG AAGAAAGCAAUUGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUC AAGCAGCUUAAAGAGGACUACUUCAAGAAGAUCGAGUGUUUCGACUCAGUG GAAAUCAGCGGGGUGGAGGACAGAUUCAACGCUUCGCUGGGAACCUAUCAU GAUCUCCUGAAGAUCAUCAAGGACAAGGACUUCCUUGACAACGAGGAGAAC GAGGACAUCCUGGAAGAUAUCGUCCUGACCUUGACCCUUUUCGAGGAUCGC GAGAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCUCUUCGACGAUAAG GUCAUGAAACAACUCAAGCGCCGCCGGUACACUGGUUGGGGCCGCCUCUCC CGCAAGCUGAUCAACGGUAUUCGCGAUAAACAGAGCGGUAAAACUAUCCUG GAUUUCCUCAAAUCGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUC CACGACGACAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACAAGUGUCC GGACAGGGAGACUCACUCCAUGAACACAUCGCGAAUCUGGCCGGUUCGCCG GCGAUUAAGAAGGGAAUUCUGCAAACUGUGAAGGUGGUCGACGAGCUGGUG AAGGUCAUGGGACGGCACAAACCGGAGAAUAUCGUGAUUGAAAUGGCCCGA GAAAACCAGACUACCCAGAAGGGCCAGAAAAACUCCCGCGAAAGGAUGAAG

CGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCAGAUCCUGAAAGAGCAC CCGGUGGAAAACACGCAGCUGCAGAACGAGAAGCUCUACCUGUACUAUUUG CAAAAUGGACGGGACAUGUACGUGGACCAAGAGCUGGACAUCAAUCGGUUG UCUGAUUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGGAUGAC UCGAUCGAUAACAAGGUGUUGACUCGCAGCGACAAGAACAGAGGGAAGUCA GAUAAUGUGCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUACUGGCGG CAGCUCCUGAAUGCGAAGCUGAUUACCCAGAGAAAGUUUGACAAUCUCACU AAAGCCGAGCGCGGCGGACUCUCAGAGCUGGAUAAGGCUGGAUUCAUCAAA CGGCAGCUGGUCGAGACUCGGCAGAUUACCAAGCACGUGGCGCAGAUCUUG GACUCCCGCAUGAACACUAAAUACGACGAGAACGAUAAGCUCAUCCGGGAA GUGAAGGUGAUUACCCUGAAAAGCAAACUUGUGUCGGACUUUCGGAAGGAC UUUCAGUUUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGCGCAUGAC GCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAAAAAGUACCCUAAA CUUGAAUCGGAGUUUGUGUACGGAGACUACAAGGUCUACGACGUGAGGAAG AUGAUAGCCAAGUCCGAACAGGAAAUCGGGAAAGCAACUGCGAAAUACUUC UUUUACUCAAACAUCAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCAAU GGAGAAAUCAGGAAGAGGCCACUGAUCGAAACUAACGGAGAAACGGGCGAA AUCGUGUGGGACAAGGGCAGGGACUUCGCAACUGUUCGCAAAGUGCUCUCU AUGCCGCAAGUCAAUAUUGUGAAGAAAACCGAAGUGCAAACCGGCGGAUUU UCAAAGGAAUCGAUCCUCCCAAAGAGAAAUAGCGACAAGCUCAUUGCACGC AAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUUCGCCGACUGUC GCAUACUCCGUCCUCGUGGUGGCCAAGGUGGAGAAGGGAAAGAGCAAAAAG CUCAAAUCCGUCAAAGAGCUGCUGGGGAUUACCAUCAUGGAACGAUCCUCG UUCGAGAAGAACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUG AAGAAGGAUCUGAUCAUCAAACUCCCCAAGUACUCACUGUUCGAACUGGAA AAUGGUCGGAAGCGCAUGCUGGCUUCGGCCGGAGAACUCCAAAAAGGAAAU GAGCUGGCCUUGCCUAGCAAGUACGUCAACUUCCUCUAUCUUGCUUCGCAC UACGAAAAACUCAAAGGGUCACCGGAAGAUAACGAACAGAAGCAGCUUUUC GUGGAGCAGCACAAGCAUUAUCUGGAUGAAAUCAUCGAACAAAUCUCCGAG UUUUCAAAGCGCGUGAUCCUCGCCGACGCCAACCUCGACAAAGUCCUGUCG GCCUACAAUAAGCAUAGAGAUAAGCCGAUCAGAGAACAGGCCGAGAACAUU AUCCACUUGUUCACCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUAC UUCGAUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAAGUUCUG GACGCGACCCUGAUCCACCAAAGCAUCACUGGACUCUACGAAACUAGGAUC GAUCUGUCGCAGCUGGGUGGCGAUGGCGGUGGAUCUCCGAAAAAGAAGAGA AAGGUGUAAUGA Cas9 nickase MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 206 (D10A) amino LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE acid ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL sequence IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV Cas9 nickase AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 207 (D10A) mRNA UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC ORF CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAG dCas9 (D10A MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 208 H840A) amino LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE acid ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL sequence IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKY FDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGGGSPKKKR KV dCas9 (D10A AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 209 H840A) mRNA UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC ORF CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC

UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCUAG Cas9 mRNA GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 210 coding GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequence GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG using UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA minimal AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC uridine AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA codons (no AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA start or AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC stop codons; CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC suit able for UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA inclusion in GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG fusion GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA protein GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG coding GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA sequence) AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC Cas9 nickase GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 211 coding GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequence GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG using UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA minimal AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC uridine AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA codons (no AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA start or AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC stop codons; CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC suitable for UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA inclusion in GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG fusion GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA protein GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG coding GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA sequence) AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC dCas9 coding GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 212 sequence GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG using GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG minimal UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA uridine AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC codons (no AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA start or AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA stop codons; AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC suitable for CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC inclusion in UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA fusion GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG protein GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA coding GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG sequence) GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC

AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUC Amino acid MDKKYSIGLDIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 213 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (without IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS) GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 214 ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 213 using CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimal AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridine UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, with GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start and GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codons CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACUAG Cas9 coding GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 215 sequence GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQ GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 213 UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA using AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimal AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridine AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (no AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start or CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons; UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable for GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion in GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusion GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG protein GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA coding AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence) GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGAC Amino acid MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 216 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 nickase ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (without IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS) GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9 nickase AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 217 mRNA ORF UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC encoding SEQ CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG ID NO: 216 CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA using AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC minimal UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA uridine GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC codons as GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC listed in CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG Table 3, AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC with start GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG and stop CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC codons GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC

AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACUAG Cas9 nickase GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 218 coding GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequence GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG encoding SEQ UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA ID NO: 216 AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC using AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA minimal AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA uridine AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC codons as CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC listed in UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA Table 3 (no GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG start or GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA stop codons; GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG suitable for GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA inclusion in AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC fusion GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC protein GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC coding CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA sequence) GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGAC Amino acid MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 219 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE dCas9 ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL (without IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS NLS) GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD dCas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 220 ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 219 using CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimal AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridine UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons as GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC listed in GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC Table 3, CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG with start AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC and stop GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG codons CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACUAG

dCas9 coding GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 221 sequence GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQ GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 219 UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA using AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimal AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridine AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons as AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC listed in CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC Table 3 (no UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA start or GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG stop codons; GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA suitable for GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG inclusion in GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA fusion AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC protein GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC coding GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC sequence) CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGC Amino acid MDKKYSIGLDIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 222 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 with ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL two nuclear IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS localization GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLIPNFKSN signals FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL (2xNLS) as RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN the C- GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminal SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acids SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKINRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLIKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSG Cas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGA 223 ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 222 using CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimal AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridine UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, with GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start and GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codons CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGUCCCGAAGAAGAAGAGAAAGGUC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAG AAGAGAAAGGUCGACAGCGGAUAG Cas9 coding GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAUGG 224 sequence SEQ GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 222 UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA using AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimal AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridine AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (no AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start or CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons; UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable for GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion in GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusion GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG protein GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA coding AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence) GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC

AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAG GUCCCGAAGAAGAAGAGAAAGGUC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAG AAGAGAAAGGUCGACAGCGGA Amino acid MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 225 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE Cas9 nickase ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL with two IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS nuclear GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN localization FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL signals as RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN the C- GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG terminal SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN amino acids SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKY FDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSG Cas9 nickase AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 226 mRNA ORF UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC encoding SEQ CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG ID NO: 25 CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA using AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC minimal UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA uridine GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC codons as GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC listed in CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG Table 3, AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC with start GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG and stop CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC codons GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGA AAGGUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG Cas9 nickase GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 227 coding GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG sequence GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG encoding SEQ UUCGACAGGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA ID NO: 25 AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC using AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA minimal AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA uridine AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC codons (no CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC start or UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA stop codons; GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG suitable for GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA inclusion in GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG fusion GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA protein AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC coding GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC sequence) GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAG AAGAGAAAGGUCGACAGCGGA Amino acid MDKKYSIGLAIGINSVGWAVITDEYKVPSKKFKVLGNIDRHSIKKNLIGAL 228 sequence of LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE dCas9 with ESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL two nuclear IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS localization GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGNLIALSLGLIPNEKSN signals as FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL the C- RVNTEITKAPLSASMIKRYDEHHQDLILLKALVRQQLPEKYKEIFFDQSKN terminal GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG amino acids SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLEKTNRKVIV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLILTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLIFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLIRSDKNRGKS DNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLIKAERGGLSELDKAGFIK RQLVETRQIIKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGIALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIAR KKDWDPKKYGGFDSPIVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE

FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLINLGAPAAFKY FDTTIDRKRYISTKEVLDATLIHQSITGLYETRIDLSQLGGDGSGSPKKKR KVDGSPKKKRKVDSG dCas9 mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGA 229 ORF encoding UGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUC SEQ ID NO: CUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUG 228 using CUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCA minimal AGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUC uridine UUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA codons, with GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUC start and GGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUAC stop codons CACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUG AUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUC GAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAG CUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGC GGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGA CUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUC GGAAACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGAC GACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUG UUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUG AGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAG AGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGA CAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAAC GGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAG UUCAUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUC AAGCUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGA AGCAUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGA CAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAG AUCCUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAAC AGCAGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGG AACUUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAG CACAGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUC AAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUC AAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUC GAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCAC GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAAC GAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAG GUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGC AGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUG GACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUC CACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGC GGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCG GCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUC AAGGUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAG AGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACAC CCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUG CAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUG AGCGACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGAC AGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGA CAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACA AAGGCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAG AGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUG GACAGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAA GUCAAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGAC UUCCAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGAC GCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAG CUGGAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAG AUGAUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUC UUCUACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAAC GGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAA AUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGC AUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUC AGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUC GCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAG CUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAA AACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAAC GAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCAC UACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAA UUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGC GCAUACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUC AUCCACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUG GACGCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUC GACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGAAG AAGAGAAAGGUCGACAGCGGAUAG dCas9 coding GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAUGG 230 sequence GCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGUCCUG encoding SEQ GGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCACUGCUG ID NO: 228 UUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGA using AGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGAAAUCUUC minimal AGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAAGAA uridine AGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA codons (no AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC start or CUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUC stop codons; UACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAA suitable for GGAGACCUGAACCCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUG inclusion in GUCCAGACAUACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA fusion GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAGAAGACUG protein GAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGAAGAACGGACUGUUCGGA coding AACCUGAUCGCACUGAGCCUGGGACUGACACCGAACUUCAAGAGCAACUUC sequence) GACCUGGCAGAAGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGAC GACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGACCUGUUC CUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGCGACAUCCUGAGA GUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGA UACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAG CAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGA UACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUC AUCAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAG CUGAACAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGC AUCCCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAG GAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUC CUGACAUUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGC AGAUUCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAAC UUCGAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGA AUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAG UACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAG AAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCGAA AUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAUACCACGAC CUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAACGAA GACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGAA AUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUC AUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGA AAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGAC UUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGA CAGGGAGACAGCCUGCACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCA AUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUCGUCGACGAACUGGUCAAG GUCAUGGGAAGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAAUGAAGAGA AUCGAAGAAGGAAUCAAGGAACUGGGAAGCCAGAUCCUGAAGGAACACCCG GUCGAAAACACACAGCUGCAGAACGAAAAGCUGUACCUGUACUACCUGCAG AACGGAAGAGACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGGACGACAGC AUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAAGAGCGAC AACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAGACAG CUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGA CAGCUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGAC AGCAGAAUGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUC AAGGUCAUCACACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUC CAGUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCA UACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUG GAAAGCGAAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUG AUCGCAAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUC UACAGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGA GAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAACAGGAGAAAUC GUCUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGC AAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAAAG AAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGUCGCA UACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAGAAGCUG AAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUC GAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAAC GGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAA CUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUAC GAAAAGCUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUC GAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUC AGCAAGAGAGUCAUCCUGGCAGACGCAAACCUGGACAAGGUCCUGAGCGCA UACAACAAGCACAGAGACAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUC CACCUGUUCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGAAGUCCUGGAC GCAACACUGAUCCACCAGAGCAUCACAGGACUGUACGAAACAAGAAUCGAC CUGAGCCAGCUGGGAGGAGACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAG GUCGACGGAAGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGA T7 Promoter TAATACGACTCACTATA 231 Human beta- ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC 232 globin 5' UTR Human beta- GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAG 233 globin 3' TCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTC UTR TGCCTAATAAAAAACATTTATTTTCATTGC Human alpha- CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCCCACAGACT 234 globin 5' CAGAGAGAACCCACC UTR Human alpha- GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCC 235 globin 3' CTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAG UTR TGGGCGGC Xenopus AAGCTCAGAATAAACGCTCAACTTTGGCC 236 laevis beta- globin 5' UTR Xenopus ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACT 237 laevis beta- TACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCC globin 3' TAATAAAAAGAAAGTTTCTTCACATTCT UTR Bovine CAGGGTCCTGTGGACAGCTCACCAGCT 238 Growth Hormone 5' UTR Bovine TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA 239 Growth AGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA Hormone 3' UTR Mus musculus GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTG 240 hemoglobin CACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG alpha, adult chain 1 (Hba-a1), 3' UTR HSD17B4 5' TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCA 241 UTR GGCCTTATTC G282 single mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAm 242 guide RNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targeting mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the mouse TTR gene Not used 243 Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 244 transcript GCAGGCCTTATTCGGATCCATGGACAAGAAGTACAGCATCGGACTGGACAT with 5' UTR CGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCC of HSD, ORF GAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAA corresponding GAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAAC to SEQ ID AAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAAT NO: 204, and CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAG 3' UTR of CTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCA ALB CGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGA AAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGA CAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTT CAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGT CGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGA AAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAG ACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGA AAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGAC ACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCT GAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGG AGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAAT CCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCT GAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACT GCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTT CTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAG CCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGG

AACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCA GAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACT GCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAA CAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGA AGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAG CGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAA CGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTA CAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGC ATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGAC AAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGAT CGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGC AAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTT CCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACT GACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGC ACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACAC AGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAG AAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACAT CCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGC AAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAA GGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACAT CGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAA CAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAG CCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAA GCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGA ACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGA CAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAA GATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAG AAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGA CAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAA GCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAA CGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGT CAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAA CTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACT GATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAA GGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAA GGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGAC AGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAAC AAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAAC AGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGA AGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAG CGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGG ATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGA AAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGC AAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTA CAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTT CCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAA CGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAAT CATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAA CCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAG AGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGC ACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACAC AAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGG AAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCAT CTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATT CATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAA AAATGGAAAGAACCTCGAG Alternative ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTAATTCCGTGGGT 245 Cas9 ORF TGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC with 19.36% CTGGGGAACACCGATAGACACAGCATCAAGAAGAATCTCATCGGAGCCCTG U content CTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCG AGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAAATC TTTTCGAACGAAATGGCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAA GAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTT GGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTAC CATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTG ATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATC GAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAA CTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAGC GGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGC CTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAAGAACGGACTTTTC GGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAAT TTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGAC GACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTG TTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTG CGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAG CGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGA CAGCAACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAAT GGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAG TTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTC AAGCTGAACAGGGAGGATCTGCTCCGCAAACAGAGAACCTTTGACAACGGA AGCATTCCACACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGC CAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAA ATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAAT TCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGG AATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTTCATCGAA CGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAG CACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGTG AAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAG AAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTC AAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTG GAAATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCAT GATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAAC GAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGC GAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAG GTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCC CGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTG GATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATC CACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGC GGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCG GCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGTG AAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGA GAAAACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAG CGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCAC CCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTG CAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTG TCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGAC TCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCA GATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGG CAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACT AAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAA CGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTG GACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAA GTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGAC TTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGAC GCATACCTCAACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAA CTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAG ATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTC TTTTACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAAT GGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAA ATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCT ATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTT TCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGC AAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTC GCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAG CTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCG TTCGAGAAGAACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTG AAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAA AATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAAT GAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCAC TACGAGAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTC GTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAG TTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCG GCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATT ATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTAC TTCGATACTACTATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTG GACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATC GATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGAC GTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTG TGATAG Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 246 transcript GCAGGCCTTATTCGGATCTGCCACCATGGATAAGAAGTACTCGATCGGGCT with 5' UTR GGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAA of HSD, ORF AGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCAT corresponding CAAGAAGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGA to SEQ ID AGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAA NO: 245, TCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGA Kozak CGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAA sequence, GAAGCATGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTA and 3' UTR CCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTC of ALB AACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGAT CAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTC CGACGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTT CGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTC CGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCC GGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGG ACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCT GCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACA AATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGA CGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGC GCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCT CACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGA GATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGG CGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGAT GGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCG CAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGGG TGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAA GGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTA CGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAA ATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGG AGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCT CCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCAC TGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAA GCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTT CAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAA GAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATT CAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAA GGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCT GACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGAC CTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCG GTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGA TAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGC TAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTAAGGA GGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACA CATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAAC TGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGA GAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCA GAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCT GGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAA CGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGA CCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGT TCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCG CAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGT GAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTAC CCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGA GCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGAT TACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGA CGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAA ACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAAT CAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCAC CGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGA CTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAAT CGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTCTT CAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGAT CGAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTT CGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAA AACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAG AAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTA CGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAA GGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGG GATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCT GGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCC CAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTC GGCCGGAGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGT CAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGA AGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGA TGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGA CGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCC GATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCT GGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAG ATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCAT CACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGG TGGCGGTGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGG TGGCCCCAAGAAGAAACGGAAGGTGTGATAGCTAGCCATCACATTTAAAAG CATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTT ATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAA AACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAAT AAAAAATGGAAAGAACCTCGAG Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 247 transcript GCAGGCCTTATTCGGATCTATGGATAAGAAGTACTCGATCGGGCTGGATAT with 5' UTR CGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCC of HSD, ORF GTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGAA corresponding GAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGAC to SEQ ID CCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCAT NO: 245, and CTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGACGACAG 3' UTR of CTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCA ALB TGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCACGA AAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGA CAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATT CCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGT GGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGA AAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCG

GCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGA GAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCAC TCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACT CTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAAATTGG CGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAAT CTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCT TAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCT GCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATTTT CTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGGCGCCAG CCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGG AACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACA GAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGGGTGAGCT GCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAA CCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGG CCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGA GGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTC GGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAA CGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTA CAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGC CTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGAC CAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGAT CGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATTCAACGC TTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTT CCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTT GACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGC TCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACAC TGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACA GAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCG TAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACAT CCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATCGC GAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAA GGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATAT CGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAGAA CTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAG CCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAA GCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGA GCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACA GTCCTTTCTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCGCAGCGA CAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAA GATGAAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAG AAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGA TAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAA GCACGTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGACGAGAA CGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGT GTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAA CTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCCT GATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAA GGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAA AGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGAC TGAAATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAAC TAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAAC TGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGA AGTGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAG CGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGG ATTCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGA GAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTAC CATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGC GAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCCCAAGTA CTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGG AGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTT CCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGATAA CGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAAT CATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAA CCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAG AGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGC TCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACAC GTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGG ACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGTGGCGG TGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCC CAAGAAGAAACGGAAGGTGTGATAGCTAGCCATCACATTTAAAAGCATCTC AGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCAT CTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATA AATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAA TGGAAAGAACCTCGAG Not used 248 Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 249 transcript GCAGGCCTTATTCGGATCCGCCACCATGCCTAAGAAAAAGCGGAAGGTCGA comprising CGGGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGG Kozak TTGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGT sequence CCTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCT with Cas9 GCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGC ORF using GAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGAT codons with CTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGA generally AGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTT high TGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTA expression CCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATT in humans GATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGAT CGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCA ACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAG CGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCG CCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTT CGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAA TTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGA CGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCT GTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCT GCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAA GCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAG ACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAA TGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAA GTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGT CAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGG ATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCG CCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAA AATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAA TTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTG GAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGA ACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAA GCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAGT GAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACA GAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGT CAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGT GGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCA TGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAA CGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCG CGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAA GGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTC CCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCT GGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGAT CCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGTC CGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCC GGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGT GAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCG AGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAA GCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCA CCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTT GCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGTT GTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGA CTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTC AGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCG GCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCAC TAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAA ACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTT GGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGA AGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGA CTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGA CGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAA ACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAA GATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTT CTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACGCTGGCCAA TGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGA AATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTC TATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATT TTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACG CAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGT CGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAA GCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTC GTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGT GAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAACTGGA AAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAA TGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCA CTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTT CGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGA GTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTC GGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACAT TATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTA CTTCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAGTTCT GGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGAT CGATCTGTCGCAGCTGGGTGGCGATTGATAGTCTAGCCATCACATTTAAAA GCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCT TATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAA AAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAA TAAAAAATGGAAAGAACCTCGAG Cas9 ORF ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGA 250 with splice TGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTC junctions CTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTG removed; CTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCA 12.75% U AGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATC content TTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGGAA GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTC GGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTAC CACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTG ATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATC GAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAG CTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGC GGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGA CTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTC GGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAAC TTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGAC GACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTG TTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTG AGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAG AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAAC GGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAG TTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTC AAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGA AGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGA CAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAG ATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAAC AGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGG AACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAA AGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAG CACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTC AAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAG AAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTC AAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTC GAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCAC GACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAAC GAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGA GAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAG GTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGC AGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTG GACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGC GGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCG GCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTC AAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGA GAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTG CAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTG AGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGAC AGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGA CAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACA AAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAG AGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTG GACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAA GTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGAC GCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAG CTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAG ATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAAC GGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAA ATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGC ATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTC AGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGA AAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTC GCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAG CTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGC TTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTC AAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAA AACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAAC GAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCAC TACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTC GTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGC GCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATC ATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTAC TTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTG GACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATC GACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGA AAGGTCTAG Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 251 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with 5' UTR GGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA

of HSD, ORF GGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT corresponding CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA to SEQ ID AGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA NO: 250, CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA Kozak CGACAGCTTCTTCCACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA sequence, GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA and 3' UTR CCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG of ALB CACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG CGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG CGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC GGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGG ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCT GCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGC ACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCT GACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGA AATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGG AGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGAT GGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAG AAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGG AGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTA CGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAA GAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCT GCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCAC AGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAA GCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTT CAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAA GAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATT CAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCT GACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGAC ATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAG ATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGA CAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGA AGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACA CATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGAC AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGA AAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACA GAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAA CGAAAAGCTGTACCTGTACTACCTGCAaAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAG AAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCAC ACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGA ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAAC AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAAT CGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTT CGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAA GACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTA CGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAA GGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGG AATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCT GGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCC GAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGA AGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGA CGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCC GATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCT GGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAG ATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCAT CACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG Cas9 ORF ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGC 252 with minimal TGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTG uridine CTGGGCAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCCCTG codons CTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCC frequently AGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAGATC used in TTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAG humans in GAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACACCCCATCTTC general; GGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTAC 12.75% U CACCTGAGAAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTG content ATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCTGATC GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAG CTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGC GGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGA CTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTC GGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAAC TTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGAC GACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTG TTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTG AGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAG AGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGA CAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAAC GGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAG TTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTG AAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGC AGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGA CAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAG ATCCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAAC AGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGACCATCACCCCCTGG AACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAG AGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAG CACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTG AAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAG AAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTG AAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTG GAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTACCAC GACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAAC GAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACAGA GAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAG GTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGC AGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGACCATCCTG GACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGC GGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCC GCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTG AAGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGA GAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAG AGAATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCAC CCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTG CAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTG AGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGAC AGCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGC GACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGA CAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACC AAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAG AGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATCCTG GACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAGAGAG GTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGAC GCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAG CTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGAGAAAG ATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAAC GGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGCGAG ATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGC ATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTC AGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGA AAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTG GCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAG CTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGC TTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTG AAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAG AACGGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAAC GAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCAC TACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTC GTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAG TTCAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGC GCCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATC ATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTAC TTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCTG GACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAATC GACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGA AAGGTGTGA Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 253 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGCCT with 5' UTR GGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAA of HSD, ORF GGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGCAT corresponding CAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGA to SEQ ID GGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAGAAGAAAGAA NO: 252, CAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGA Kozak CGACAGCTTCTTCCACAGACTGGAGGAGAGCTTCCTGGTGGAGGAGGACAA sequence, GAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTA and 3' UTR CCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGACAG of ALB CACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGCCCACATGAT CAAGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAG CGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTT CGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAG CGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCC CGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGG CCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCT GCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCA GATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGA CGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGC CCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAGCACCACCAGGACCT GACCCTGCTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGA GATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGG CGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGAT GGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAG AAAGCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGG CGAGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAA GGACAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTA CGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAA GAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGG CGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAACCT GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCAC CGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAA GCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTT CAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAA GAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACAGATT CAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCT GACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACTGAAGAC CTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAG ATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGA CAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGC CAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGA GGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCA CATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGAC CGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGA GAACATCGTGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCCA GAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCT GGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAA CGAGAAGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGA CCAGGAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGT GCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAG AAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCAC CCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA GCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGAT CACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGA CGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAGCAA GCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGAGAT CAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCAC CGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGAGAT CGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACCGAGATCACCCTGGCCAACGGCGAGATCAGAAAGAGACCCCTGAT CGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTT CGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAA GACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAG AAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTA CGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAA GGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGG CATCACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCT GGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCC CAAGTACAGCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAG CGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGT GAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGA GGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGA CGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGA CGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCC CATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCT GGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAG ATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT CACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGACGG CGGCGGCAGCCCCAAGAAGAAGAGAAAGGTGTGACTAGCCATCACATTTAA

AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG Cas9 ORF ATGGACAAAAAATACAGCATAGGGCTAGACATAGGGACGAACAGCGTAGGG 254 with minimal TGGGCGGTAATAACGGACGAATACAAAGTACCGAGCAAAAAATTCAAAGTA uridine CTAGGGAACACGGACCGACACAGCATAAAAAAAAACCTAATAGGGGCGCTA codons CTATTCGACAGCGGGGAAACGGCGGAAGCGACGCGACTAAAACGAACGGCG infrequently CGACGACGATACACGCGACGAAAAAACCGAATATGCTACCTACAAGAAATA used in TTCAGCAACGAAATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAGAA humans in GAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGACACCCGATATTC general; GGGAACATAGTAGACGAAGTAGCGTACCACGAAAAATACCCGACGATATAC 12.75% U CACCTACGAAAAAAACTAGTAGACAGCACGGACAAAGCGGACCTACGACTA content ATATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTCCTAATA GAAGGGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCATACAA CTAGTACAAACGTACAACCAACTATTCGAAGAAAACCCGATAAACGCGAGC GGGGTAGACGCGAAAGCGATACTAAGCGCGCGACTAAGCAAAAGCCGACGA CTAGAAAACCTAATAGCGCAACTACCGGGGGAAAAAAAAAACGGGCTATTC GGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAAC TTCGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACACGTACGAC GACGACCTAGACAACCTACTAGCGCAAATAGGGGACCAATACGCGGACCTA TTCCTAGCGGCGAAAAACCTAAGCGACGCGATACTACTAAGCGACATACTA CGAGTAAACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATAAAA CGATACGACGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTACGA CAACAACTACCGGAAAAATACAAAGAAATATTCTTCGACCAAAGCAAAAAC GGGTACGCGGGGTACATAGACGGGGGGGCGAGCCAAGAAGAATTCTACAAA TTCATAAAACCGATACTAGAAAAAATGGACGGGACGGAAGAACTACTAGTA AAACTAAACCGAGAAGACCTACTACGAAAACAACGAACGTTCGACAACGGG AGCATACCGCACCAAATACACCTAGGGGAACTACACGCGATACTACGACGA CAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAGAAAAAATAGAAAAA ATACTAACGTTCCGAATACCGTACTACGTAGGGCCGCTAGCGCGAGGGAAC AGCCGATTCGCGTGGATGACGCGAAAAAGCGAAGAAACGATAACGCCGTGG AACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTCATAGAA CGAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAGTACTACCGAAA CACAGCCTACTATACGAATACTTCACGGTATACAACGAACTAACGAAAGTA AAATACGTAACGGAAGGGATGCGAAAACCGGCGTTCCTAAGCGGGGAACAA AAAAAAGCGATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAACGGTA AAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAGCGTA GAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGGGACGTACCAC GACCTACTAAAAATAATAAAAGACAAAGACTTCCTAGACAACGAAGAAAAC GAAGACATACTAGAAGACATAGTACTAACGCTAACGCTATTCGAAGACCGA GAAATGATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGACAAA GTAATGAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACTAAGC CGAAAACTAATAAACGGGATACGAGACAAACAAAGCGGGAAAACGATACTA GACTTCCTAAAAAGCGACGGGTTCGCGAACCGAAACTTCATGCAACTAATA CACGACGACAGCCTAACGTTCAAAGAAGACATACAAAAAGCGCAAGTAAGC GGGCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGGGAGCCCG GCGATAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACGAACTAGTA AAAGTAATGGGGCGACACAAACCGGAAAACATAGTAATAGAAATGGCGCGA GAAAACCAAACGACGCAAAAAGGGCAAAAAAACAGCCGAGAACGAATGAAA CGAATAGAAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAAGAACAC CCGGTAGAAAACACGCAACTACAAAACGAAAAACTATACCTATACTACCTA CAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGACATAAACCGACTA AGCGACTACGACGTAGACCACATAGTACCGCAAAGCTTCCTAAAAGACGAC AGCATAGACAACAAAGTACTAACGCGAAGCGACAAAAACCGAGGGAAAAGC GACAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTACTGGCGA CAACTACTAAACGCGAAACTAATAACGCAACGAAAATTCGACAACCTAACG AAAGCGGAACGAGGGGGGCTAAGCGAACTAGACAAAGCGGGGTTCATAAAA CGACAACTAGTAGAAACGCGACAAATAACGAAACACGTAGCGCAAATACTA GACAGCCGAATGAACACGAAATACGACGAAAACGACAAACTAATACGAGAA GTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCGAAAAGAC TTCCAATTCTACAAAGTACGAGAAATAAACAACTACCACCACGCGCACGAC GCGTACCTAAACGCGGTAGTAGGGACGGCGCTAATAAAAAAATACCCGAAA CTAGAAAGCGAATTCGTATACGGGGACTACAAAGTATACGACGTACGAAAA ATGATAGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAAATACTTC TTCTACAGCAACATAATGAACTTCTTCAAAACGGAAATAACGCTAGCGAAC GGGGAAATACGAAAACGACCGCTAATAGAAACGAACGGGGAAACGGGGGAA ATAGTATGGGACAAAGGGCGAGACTTCGCGACGGTACGAAAAGTACTAAGC ATGCCGCAAGTAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGGTTC AGCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAATAGCGCGA AAAAAAGACTGGGACCCGAAAAAATACGGGGGGTTCGACAGCCCGACGGTA GCGTACAGCGTACTAGTAGTAGCGAAAGTAGAAAAAGGGAAAAGCAAAAAA CTAAAAAGCGTAAAAGAACTACTAGGGATAACGATAATGGAACGAAGCAGC TTCGAAAAAAACCCGATAGACTTCCTAGAAGCGAAAGGGTACAAAGAAGTA AAAAAAGACCTAATAATAAAACTACCGAAATACAGCCTATTCGAACTAGAA AACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACTACAAAAAGGGAAC GAACTAGCGCTACCGAGCAAATACGTAAACTTCCTATACCTAGCGAGCCAC TACGAAAAACTAAAAGGGAGCCCGGAAGACAACGAACAAAAACAACTATTC GTAGAACAACACAAACACTACCTAGACGAAATAATAGAACAAATAAGCGAA TTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAAGTACTAAGC GCGTACAACAAACACCGAGACAAACCGATACGAGAACAAGCGGAAAACATA ATACACCTATTCACGCTAACGAACCTAGGGGCGCCGGCGGCGTTCAAATAC TTCGACACGACGATAGACCGAAAACGATACACGAGCACGAAAGAAGTACTA GACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGAAACGCGAATA GACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCGAAAAAAAAACGA AAAGTATGA Cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 255 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAAAAATACAGCATAGGGCT with 5' UTR AGACATAGGGACGAACAGCGTAGGGTGGGCGGTAATAACGGACGAATACAA of HSD, ORF AGTACCGAGCAAAAAATTCAAAGTACTAGGGAACACGGACCGACACAGCAT corresponding AAAAAAAAACCTAATAGGGGCGCTACTATTCGACAGCGGGGAAACGGCGGA to SEQ ID AGCGACGCGACTAAAACGAACGGCGCGACGACGATACACGCGACGAAAAAA NO: 254, CCGAATATGCTACCTACAAGAAATATTCAGCAACGAAATGGCGAAAGTAGA Kozak CGACAGCTTCTTCCACCGACTAGAAGAAAGCTTCCTAGTAGAAGAAGACAA sequence, AAAACACGAACGACACCCGATATTCGGGAACATAGTAGACGAAGTAGCGTA and 3' UTR CCACGAAAAATACCCGACGATATACCACCTACGAAAAAAACTAGTAGACAG of ALB CACGGACAAAGCGGACCTACGACTAATATACCTAGCGCTAGCGCACATGAT AAAATTCCGAGGGCACTTCCTAATAGAAGGGGACCTAAACCCGGACAACAG CGACGTAGACAAACTATTCATACAACTAGTACAAACGTACAACCAACTATT CGAAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAAG CGCGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGCAACTACC GGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAATAGCGCTAAGCCTAGG GCTAACGCCGAACTTCAAAAGCAACTTCGACCTAGCGGAAGACGCGAAACT ACAACTAAGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGCGCA AATAGGGGACCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAGCGA CGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAATAACGAAAGC GCCGCTAAGCGCGAGCATGATAAAACGATACGACGAACACCACCAAGACCT AACGCTACTAAAAGCGCTAGTACGACAACAACTACCGGAAAAATACAAAGA AATATTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGACGGGGG GGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCGATACTAGAAAAAAT GGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAGAAGACCTACTACG AAAACAACGAACGTTCGACAACGGGAGCATACCGCACCAAATACACCTAGG GGAACTACACGCGATACTACGACGACAAGAAGACTTCTACCCGTTCCTAAA AGACAACCGAGAAAAAATAGAAAAAATACTAACGTTCCGAATACCGTACTA CGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGACGCGAAA AAGCGAAGAAACGATAACGCCGTGGAACTTCGAAGAAGTAGTAGACAAAGG GGCGAGCGCGCAAAGCTTCATAGAACGAATGACGAACTTCGACAAAAACCT ACCGAACGAAAAAGTACTACCGAAACACAGCCTACTATACGAATACTTCAC GGTATACAACGAACTAACGAAAGTAAAATACGTAACGGAAGGGATGCGAAA ACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGACCTACTATT CAAAACGAACCGAAAAGTAACGGTAAAACAACTAAAAGAAGACTACTTCAA AAAAATAGAATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAAGACCGATT CAACGCGAGCCTAGGGACGTACCACGACCTACTAAAAATAATAAAAGACAA AGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAGTACT AACGCTAACGCTATTCGAAGACCGAGAAATGATAGAAGAACGACTAAAAAC GTACGCGCACCTATTCGACGACAAAGTAATGAAACAACTAAAACGACGACG ATACACGGGGTGGGGGCGACTAAGCCGAAAACTAATAAACGGGATACGAGA CAAACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCGACGGGTTCGC GAACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTTCAAAGA AGACATACAAAAAGCGCAAGTAAGCGGGCAAGGGGACAGCCTACACGAACA CATAGCGAACCTAGCGGGGAGCCCGGCGATAAAAAAAGGGATACTACAAAC GGTAAAAGTAGTAGACGAACTAGTAAAAGTAATGGGGCGACACAAACCGGA AAACATAGTAATAGAAATGGCGCGAGAAAACCAAACGACGCAAAAAGGGCA AAAAAACAGCCGAGAACGAATGAAACGAATAGAAGAAGGGATAAAAGAACT AGGGAGCCAAATACTAAAAGAACACCCGGTAGAAAACACGCAACTACAAAA CGAAAAACTATACCTATACTACCTACAAAACGGGCGAGACATGTACGTAGA CCAAGAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCACATAGT ACCGCAAAGCTTCCTAAAAGACGACAGCATAGACAACAAAGTACTAACGCG AAGCGACAAAAACCGAGGGAAAAGCGACAACGTACCGAGCGAAGAAGTAGT AAAAAAAATGAAAAACTACTGGCGACAACTACTAAACGCGAAACTAATAAC GCAACGAAAATTCGACAACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGA ACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAACGCGACAAAT AACGAAACACGTAGCGCAAATACTAGACAGCCGAATGAACACGAAATACGA CGAAAACGACAAACTAATACGAGAAGTAAAAGTAATAACGCTAAAAAGCAA ACTAGTAAGCGACTTCCGAAAAGACTTCCAATTCTACAAAGTACGAGAAAT AAACAACTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTAGGGAC GGCGCTAATAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATACGGGGA CTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAGCGAACAAGAAAT AGGGAAAGCGACGGCGAAATACTTCTTCTACAGCAACATAATGAACTTCTT CAAAACGGAAATAACGCTAGCGAACGGGGAAATACGAAAACGACCGCTAAT AGAAACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGACTT CGCGACGGTACGAAAAGTACTAAGCATGCCGCAAGTAAACATAGTAAAAAA AACGGAAGTACAAACGGGGGGGTTCAGCAAAGAAAGCATACTACCGAAACG AAACAGCGACAAACTAATAGCGCGAAAAAAAGACTGGGACCCGAAAAAATA CGGGGGGTTCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAA AGTAGAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAGAACTACTAGG GATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCCGATAGACTTCCT AGAAGCGAAAGGGTACAAAGAAGTAAAAAAAGACCTAATAATAAAACTACC GAAATACAGCCTATTCGAACTAGAAAACGGGCGAAAACGAATGCTAGCGAG CGCGGGGGAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCAAATACGT AAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAAGGGAGCCCGGA AGACAACGAACAAAAACAACTATTCGTAGAACAACACAAACACTACCTAGA CGAAATAATAGAACAAATAAGCGAATTCAGCAAACGAGTAATACTAGCGGA CGCGAACCTAGACAAAGTACTAAGCGCGTACAACAAACACCGAGACAAACC GATACGAGAACAAGCGGAAAACATAATACACCTATTCACGCTAACGAACCT AGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGACCGAAAACG ATACACGAGCACGAAAGAAGTACTAGACGCGACGCTAATACACCAAAGCAT AACGGGGCTATACGAAACGCGAATAGACCTAAGCCAACTAGGGGGGGACGG GGGGGGGAGCCCGAAAAAAAAACGAAAAGTATGACTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG Cas9 AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 256 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with AGG as GGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA first three GGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT nucleotides CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA for use with AGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA CleanCap .TM., CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA 5' UTR of CGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA HSD, ORF GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA corresponding CCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG to SEQ ID CACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT NO: 204, CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG Kozak CGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT sequence, CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG and 3' UTR CGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC of ALB GGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGG ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCT GCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGC ACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCT GACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGA AATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGG AGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGAT GGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAG AAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGG AGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTA CGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAA GAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCT GCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCAC AGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAA GCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTT CAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAA GAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATT CAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCT GACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGAC ATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAG ATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGA CAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGA AGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACA CATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGAC AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGA AAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACA GAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAA CGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAG AAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCAC ACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGA ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAAC AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAAT CGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTT CGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAA GACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTA CGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAA GGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGG AATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCT GGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCC GAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGA

AGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGA CGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCC GATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCT GGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAG ATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCAT CACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG Cas9 GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGA 257 transcript CACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGG with 5' UTR ATTCCCCGTGCCAAGAGTGACTCACCGTCCTTGACACGGCCACCATGGACA from CMV, AGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG ORF TCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA corresponding ACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG to SEQ ID ACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA NO: 204, GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA Kozak ACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT sequence, TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA and 3' UTR TCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA of ALB GAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACC TGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCC AGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAA ACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACC TGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACC TGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACC TGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGG CAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACG ACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGC TGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACG CAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCA AGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCC CGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAG ACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGA CATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGAT TCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCG AAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCC TGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACG TCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGC TGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACA TCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGA TCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGC TGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCC TGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACG ACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGG GAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCA AGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCA TGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACC AGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCG AAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCG AAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACG GAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCG ACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGC TGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAG AGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGC TGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGT TCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACC TGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAA GCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCG CAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAA TCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCT GGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGC AGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGG AAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGG ACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGA GCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAA AGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAA GAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGG CACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAAC AGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCA AGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACA ACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACC TGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACA CAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAA CACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGA GCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCT AGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAA AGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGT AAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTC TTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 GGGACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACA 258 transcript CCGGATCTGCCACCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAA with 5' UTR CAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCA from HBB, AGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC ORF TGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGAC corresponding TGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCT to SEQ ID ACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCT NO: 204, TCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAA Kozak GACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGT sequence, ACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGG and 3' UTR CAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAG of HBB GACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACA AGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACC CGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGA GCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGA AGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGA ACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCA AGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACC AGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGC TGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCG CAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGA AGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCG ACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGG AAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAG AAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAA CATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACG CAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAG AAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGC TGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAA CAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCAC AGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAA AGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACG AACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCC TGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACA GAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCC TGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGG ACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACAC TGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACC TGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGAT GGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCG GAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACT TCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGA AGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACC TGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCG TCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCA GAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGA TCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGT ACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGG ACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCT TCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGA AGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGT TCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACG TCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACA AGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCG ACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACC ACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCA AGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCT ACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAA CAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAA TCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACG GAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCA GAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCC AGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACA AGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCG ACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGG GAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCA TGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCC TGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAAC TGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGT ACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAAC AGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCG AACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGG ACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGG CAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCA CAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGT ACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCC CGAAGAAGAAGAGAAAGGTCTAGCTAGCGCTCGCTTTCTTGCTGTCCAATT TCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATA TTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTT TCATTGCCTCGAG Cas9 GGGAAGCTCAGAATAAACGCTCAACTTTGGCCGGATCTGCCACCATGGACA 259 transcript AGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG with 5' UTR TCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA from XBG, ACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG ORF ACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA corresponding GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA to SEQ ID ACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT NO: 204, TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA Kozak TCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA sequence, GAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACC and 3' UTR TGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG of XBG ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCC AGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAA ACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACC TGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACC TGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACC TGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGG CAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACG ACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGC TGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACG CAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCA AGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCC CGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAG ACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGA CATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGAT TCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCG AAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCC TGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACG TCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGC TGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACA TCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGA TCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGC TGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCC TGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACG ACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGG GAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCA AGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCA TGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACC AGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCG AAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCG AAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACG GAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCG ACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGC TGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAG AGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGC TGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGT TCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACC TGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAA GCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCG CAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA

GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAA TCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCT GGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGC AGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGG AAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGG ACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGA GCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAA AGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAA GAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGG CACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAAC AGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCA AGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACA ACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACC TGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACA CAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAA CACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGA GCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCT AGCTAGCACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAAT ACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTAT CTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTCTCGAG Cas9 AGGAAGCTCAGAATAAACGCTCAACTTTGGCCGGATCTGCCACCATGGACA 260 transcript AGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAG with AGG as TCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAA first three ACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCG nucleotides ACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA for use with GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCA CleanCap .TM., ACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT 5' UTR from TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA XBG, ORF TCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA corresponding GAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACC to SEQ ID TGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG NO: 204, ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCC Kozak AGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCG sequence, ACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAA and 3' UTR ACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACC of XBG TGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACC TGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACC TGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGG CAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACG ACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGC TGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACG CAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCA AGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCC CGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAG ACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGA CATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGAT TCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCG AAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCC TGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACG TCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGC TGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACA TCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGA TCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGA AGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGC TGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCC TGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACG ACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGG GAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCA AGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCA TGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACC AGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCG AAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCG AAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACG GAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCG ACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGC TGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAG AGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGC TGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGT TCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACC TGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAA GCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCG CAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAA TCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCT GGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGC AGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGG AAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGG ACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACA GCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGA GCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAA AGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAA GAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGG CACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAA AGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAAC AGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCA AGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACA ACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACC TGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACA CAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAA CACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGA GCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCT AGCTAGCACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAAT ACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTAT CTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTCTCGAG Cas9 AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTT 261 transcript GCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACT with AGG as GGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAA first three GGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT nucleotides CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGA for use with AGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAA CleanCap .TM., CAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGA 5' UTR from CGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAA HSD, ORF GAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATA corresponding CCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAG to SEQ ID CACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGAT NO: 204, CAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAG Kozak CGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTT sequence, CGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG and 3' UTR CGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCC of ALB GGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGG ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAGCT GCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGC ACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCT GACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGA AATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGG AGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGAT GGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAG AAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGG AGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTA CGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAA GAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCT GCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCAC AGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAA GCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTT CAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAA GAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATT CAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCT GACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGAC ATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAG ATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGA CAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGC AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGA AGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACA CATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGAC AGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGA AAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACA GAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAA CGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGA CCAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAG AAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGT CAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCAC ACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGA ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAAC AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAAT CGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTT CGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAA GACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTA CGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAA GGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGG AATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCT GGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCC GAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGA AGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGA CGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCC GATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCT GGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAG ATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCAT CACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAG CTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATT AATAAAAAATGGAAAGAACCTCGAG 30/30/39 Not used 262 poly-A sequence poly-A 100 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 263 sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA G209 single AAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 264 guide RNA targeting the mouse TTR gene ORF encoding ATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATC 265 Neisseria GGAATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAAC meningitidis CCGATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTC Cas9 using CCGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTC minimal AGAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTG uridine CTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTG codons, with ATCAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGAC start and AGAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAG stop codons CACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAG GAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAG ACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAG GAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCG AGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAA TTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTG CTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTG GGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATAC ACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTG GAACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATG GACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTG CTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGAC AACGCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCG AGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTG TCGCCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACA GACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTG GAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTG AAGGCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGAC GAAGCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAA GAAAAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTC GTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGA AGATACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGA AAGTCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGA AAGGACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTC GTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAG CAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTG AACGAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACA TGGGACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAG AACAAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCG AGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGA TCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAG GAAAGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTC

GTCGCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCA TCGAACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAG GTCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCA TGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAG GAAATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTC CTGCACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAA GTCATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAA GCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCG AGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCA CCGAACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCA AAGAGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTG AAGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTG TACGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAG GCATTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAG CAGGTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTC AGAAACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTC TTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTC GCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAA GACTGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCG AACGACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTC GCATCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTG GACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACA GCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGA CCGTGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCA GACGGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGA ORF encoding GCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGGA 266 Neisseria ATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAACCCG meningitidis ATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCCG Cas9 using AAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGA minimal AGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGCTG uridine AAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTGATC codons (no AAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGACAGA start or AAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCAC stop codons; AGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAA suitable for CTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAGACA inclusion in GGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGAA fusion TCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCGAGA protein AAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTC coding GGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTG sequence) ATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTGGGA CACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATACACA GCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAA CAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATGGAC GAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGCTG GGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGACAAC GCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCGAGA GCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCG CCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGAC GAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTGGAA GCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTGAAG GCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGACGAA GCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGAA AAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTC CTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGAAGA TACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGAAAG TCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAG GACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTC GGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCAG CACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTGAAC GAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACATGG GACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAAC AAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGA GAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGATCG AAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAGGAA AGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTCGTC GCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCG AACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTC AGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCATGC TCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAGGAA ATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTCCTG CACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTC ATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCA GACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCGAGA CCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCACCG AACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCAAAG AGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAG CTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTAC GAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGCA TTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAGCAG GTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGA AACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTC GAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCA AAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGAC TGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAAC GACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCA TCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGAC CACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCA CTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCG TGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGAC GGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAA Transcript GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGATCCGCCACCAT 267 comprising GGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGG SEQ ID NO: AATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAACCC 265 GATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCC (encoding GAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAG Neisseria AAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGCT meningitidis GAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGACTGAT Cas9) CAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTGGACAG AAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCA CAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGA ACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGCAGAC AGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTTCGAAAAGGA ATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACACATTCTCGAG AAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATT CGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCT GATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGATGCTGGG ACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAAAGAACACATACAC AGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACCTGAGAATCCTGGA ACAGGGATCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATGGA CGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGCT GGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACGGAAAGGACAA CGCAGAAGCATCGACACTGATGGAAATGAAGGCATACCACGCAATCTCGAG AGCACTGGAAAAGGAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTC GCCGGAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGA CGAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCCTGGA AGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCAGATCTCGCTGAA GGCACTGAGAAGAATCGTCCCGCTGATGGAACAGGGAAAGAGATACGACGA AGCATGCGCAGAAATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGA AAAGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGT CCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGTCAGAAG ATACGGATCGCCGGCAAGAATCCACATCGAAACAGCAAGAGAAGTCGGAAA GTCGTTCAAGGACAGAAAGGAAATCGAAAAGAGACAGGAAGAAAACAGAAA GGACAGAGAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGT CGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCA GCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGGAAGACTGAA CGAAAAGGGATACGTCGAAATCGACCACGCACTGCCGTTCTCGAGAACATG GGACGACTCGTTCAACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAA CAAGGGAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAG AGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGATC GAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGACGGATTCAAGGA AAGAAACCTGAACGACACAAGATACGTCAACAGATTCCTGTGCCAGTTCGT CGCAGACAGAATGAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATC GAACGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGT CAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCGCATG CTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCGTCAGATACAAGGA AATGAACGCATTCGACGGAAAGACAATCGACAAGGAAACAGGAGAAGTCCT GCACCAGAAGACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGT CATGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGC AGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTCGTCGAG ACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTCGTCTCGAGAGCACC GAACAGAAAGATGTCGGGACAGGGACACATGGAAACAGTCAAGTCGGCAAA GAGACTGGACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAA GCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTA CGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCGGCAAAGGC ATTCGCAGAACCGTTCTACAAGTACGACAAGGCAGGAAACAGAACACAGCA GGTCAAGGCAGTCAGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAG AAACCACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTT CGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGC AAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGA CTGGCAGCTGATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAA CGACCTGGTCGAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGC ATCGTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGA CCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGC ACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACC GTGCAGACTGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGA CGGATCGGAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGATAGCT AGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTG CATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGG Amino acid MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEV 268 sequence of PKTGDSLAMARRLARSVRRLIRRRAHRLLRIRRLLKREGVLQAANFDENGL Neisseria IKSLPNTPWQLRAAALDRKLIPLEWSAVLLHLIKHRGYLSQRKNEGETADK meningitidis ELGALLKGVAGNAHALQTGDFRIPAELALNKFEKESGHIRNQRSDYSHIFS Cas9 RKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKML GHCIFEPAEPKAAKNTYTAERFIWLIKLNNLRILEQGSERPLIDTERAILM DEPYRKSKLIYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAIS RALEKEGLKDKKSPLNLSPELQDEIGTAFSLEKTDEDITGRLKDRIQPEIL EALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVG KSFKDRKEIEKRQEENRKDREKAAAKFREYFPNEVGEPKSKDILKLRLYEQ QHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSENNKVLVLGSENQ NKGNQTPYEYENGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGEK ERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRK VRAENDRHHALDAVVVACSTVAMQQKITREVRYKEMNAFDGKTIDKETGEV LHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSS RPEAVHEYVTPLEVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQL KLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQ QVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQV AKGILPDRAVVQGKDEEDWQLIDDSFNEKESLHPNDLVEVITKKARMFGYF ASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIR PCRLKKRPPVRSGKRTADGSEFESPKKKRKVE G390 single mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmAm 269 guide RNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targeting mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the rat TTR gene trRNA AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU 270 GGCACCGAGUCGGUGCUUUUUUU Not Used 271 G534 single mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAm 272 guide RNA UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA targeting mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU the rat TTR gene G000395 5' mG*mC*mA*AUGGUGUAGCGGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmG 273 truncated mCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmG inactive GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sgRNA modified sequence SV40 NLS PKKKRKV 274 Alternate PKKKRRV 275 SV40 NLS Nucleoplasmin KRPAATKKAGQAKKKK 276 NLS Exemplary gccRccAUGG 277 Kozak sequence Exemplary gccgccRccAUGG 278 Kozak sequence * = PS linkage; 'm' = 2T-O-Me nucleotide

[0658] The following sequence table provides a listing of sequences disclosed herein. It is understood that if a DNA sequence (comprising Ts) is referenced with respect to an RNA, then Ts should be replaced with Us (which may be modified or unmodified depending on the context), and vice versa.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 278 <210> SEQ ID NO 1 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 1 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 2 <211> LENGTH: 4403 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript comprising Cas9 ORF corresponding to SEQ ID NO: 205 using codons with generally high expression in humans <400> SEQUENCE: 2 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc atgcctaaga aaaagcggaa ggtcgacggg gataagaagt actcaatcgg 120 gctggatatc ggaactaatt ccgtgggttg ggcagtgatc acggatgaat acaaagtgcc 180 gtccaagaag ttcaaggtcc tggggaacac cgatagacac agcatcaaga aaaatctcat 240 cggagccctg ctgtttgact ccggcgaaac cgcagaagcg acccggctca aacgtaccgc 300 gaggcgacgc tacacccggc ggaagaatcg catctgctat ctgcaagaga tcttttcgaa 360 cgaaatggca aaggtcgacg acagcttctt ccaccgcctg gaagaatctt tcctggtgga 420 ggaggacaag aagcatgaac ggcatcctat ctttggaaac atcgtcgacg aagtggcgta 480 ccacgaaaag tacccgacca tctaccatct gcggaagaag ttggttgact caactgacaa 540 ggccgacctc agattgatct acttggccct cgcccatatg atcaaattcc gcggacactt 600 cctgatcgaa ggcgatctga accctgataa ctccgacgtg gataagcttt tcattcaact 660 ggtgcagacc tacaaccaac tgttcgaaga aaacccaatc aatgctagcg gcgtcgatgc 720 caaggccatc ctgtccgccc ggctgtcgaa gtcgcggcgc ctcgaaaacc tgatcgcaca 780 gctgccggga gagaaaaaga acggactttt cggcaacttg atcgctctct cactgggact 840 cactcccaat ttcaagtcca attttgacct ggccgaggac gcgaagctgc aactctcaaa 900 ggacacctac gacgacgact tggacaattt gctggcacaa attggcgatc agtacgcgga 960 tctgttcctt gccgctaaga acctttcgga cgcaatcttg ctgtccgata tcctgcgcgt 1020 gaacaccgaa ataaccaaag cgccgcttag cgcctcgatg attaagcggt acgacgagca 1080 tcaccaggat ctcacgctgc tcaaagcgct cgtgagacag caactgcctg aaaagtacaa 1140 ggagatcttc ttcgaccagt ccaagaatgg gtacgcaggg tacatcgatg gaggcgctag 1200 ccaggaagag ttctataagt tcatcaagcc aatcctggaa aagatggacg gaaccgaaga 1260 actgctggtc aagctgaaca gggaggatct gctccggaaa cagagaacct ttgacaacgg 1320 atccattccc caccagatcc atctgggtga gctgcacgcc atcttgcggc gccaggagga 1380 cttttaccca ttcctcaagg acaaccggga aaagatcgag aaaattctga cgttccgcat 1440 cccgtattac gtgggcccac tggcgcgcgg caattcgcgc ttcgcgtgga tgactagaaa 1500 atcagaggaa accatcactc cttggaattt cgaggaagtt gtggataagg gagcttcggc 1560 acaaagcttc atcgaacgaa tgaccaactt cgacaagaat ctcccaaacg agaaggtgct 1620 tcctaagcac agcctccttt acgaatactt cactgtctac aacgaactga ctaaagtgaa 1680 atacgttact gaaggaatga ggaagccggc ctttctgtcc ggagaacaga agaaagcaat 1740 tgtcgatctg ctgttcaaga ccaaccgcaa ggtgaccgtc aagcagctta aagaggacta 1800 cttcaagaag atcgagtgtt tcgactcagt ggaaatcagc ggggtggagg acagattcaa 1860 cgcttcgctg ggaacctatc atgatctcct gaagatcatc aaggacaagg acttccttga 1920 caacgaggag aacgaggaca tcctggaaga tatcgtcctg accttgaccc ttttcgagga 1980 tcgcgagatg atcgaggaga ggcttaagac ctacgctcat ctcttcgacg ataaggtcat 2040 gaaacaactc aagcgccgcc ggtacactgg ttggggccgc ctctcccgca agctgatcaa 2100 cggtattcgc gataaacaga gcggtaaaac tatcctggat ttcctcaaat cggatggctt 2160 cgctaatcgt aacttcatgc aattgatcca cgacgacagc ctgaccttta aggaggacat 2220 ccaaaaagca caagtgtccg gacagggaga ctcactccat gaacacatcg cgaatctggc 2280 cggttcgccg gcgattaaga agggaattct gcaaactgtg aaggtggtcg acgagctggt 2340 gaaggtcatg ggacggcaca aaccggagaa tatcgtgatt gaaatggccc gagaaaacca 2400 gactacccag aagggccaga aaaactcccg cgaaaggatg aagcggatcg aagaaggaat 2460 caaggagctg ggcagccaga tcctgaaaga gcacccggtg gaaaacacgc agctgcagaa 2520 cgagaagctc tacctgtact atttgcaaaa tggacgggac atgtacgtgg accaagagct 2580 ggacatcaat cggttgtctg attacgacgt ggaccacatc gttccacagt cctttctgaa 2640 ggatgactcg atcgataaca aggtgttgac tcgcagcgac aagaacagag ggaagtcaga 2700 taatgtgcca tcggaggagg tcgtgaagaa gatgaagaat tactggcggc agctcctgaa 2760 tgcgaagctg attacccaga gaaagtttga caatctcact aaagccgagc gcggcggact 2820 ctcagagctg gataaggctg gattcatcaa acggcagctg gtcgagactc ggcagattac 2880 caagcacgtg gcgcagatct tggactcccg catgaacact aaatacgacg agaacgataa 2940 gctcatccgg gaagtgaagg tgattaccct gaaaagcaaa cttgtgtcgg actttcggaa 3000 ggactttcag ttttacaaag tgagagaaat caacaactac catcacgcgc atgacgcata 3060 cctcaacgct gtggtcggta ccgccctgat caaaaagtac cctaaacttg aatcggagtt 3120 tgtgtacgga gactacaagg tctacgacgt gaggaagatg atagccaagt ccgaacagga 3180 aatcgggaaa gcaactgcga aatacttctt ttactcaaac atcatgaact ttttcaagac 3240 tgaaattacg ctggccaatg gagaaatcag gaagaggcca ctgatcgaaa ctaacggaga 3300 aacgggcgaa atcgtgtggg acaagggcag ggacttcgca actgttcgca aagtgctctc 3360 tatgccgcaa gtcaatattg tgaagaaaac cgaagtgcaa accggcggat tttcaaagga 3420 atcgatcctc ccaaagagaa atagcgacaa gctcattgca cgcaagaaag actgggaccc 3480 gaagaagtac ggaggattcg attcgccgac tgtcgcatac tccgtcctcg tggtggccaa 3540 ggtggagaag ggaaagagca aaaagctcaa atccgtcaaa gagctgctgg ggattaccat 3600 catggaacga tcctcgttcg agaagaaccc gattgatttc ctcgaggcga agggttacaa 3660 ggaggtgaag aaggatctga tcatcaaact ccccaagtac tcactgttcg aactggaaaa 3720 tggtcggaag cgcatgctgg cttcggccgg agaactccaa aaaggaaatg agctggcctt 3780 gcctagcaag tacgtcaact tcctctatct tgcttcgcac tacgaaaaac tcaaagggtc 3840 accggaagat aacgaacaga agcagctttt cgtggagcag cacaagcatt atctggatga 3900 aatcatcgaa caaatctccg agttttcaaa gcgcgtgatc ctcgccgacg ccaacctcga 3960 caaagtcctg tcggcctaca ataagcatag agataagccg atcagagaac aggccgagaa 4020 cattatccac ttgttcaccc tgactaacct gggagcccca gccgccttca agtacttcga 4080 tactactatc gatcgcaaaa gatacacgtc caccaaggaa gttctggacg cgaccctgat 4140 ccaccaaagc atcactggac tctacgaaac taggatcgat ctgtcgcagc tgggtggcga 4200 ttgatagtct agccatcaca tttaaaagca tctcagccta ccatgagaat aagagaaaga 4260 aaatgaagat caatagctta ttcatctctt tttctttttc gttggtgtaa agccaacacc 4320 ctgtctaaaa aacataaatt tctttaatca ttttgcctct tttctctgtg cttcaattaa 4380 taaaaaatgg aaagaacctc gag 4403 <210> SEQ ID NO 3 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: modified sgRNA sequence <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(20) <223> OTHER INFORMATION: n is a, c, g, or u <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 3 nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 4 <211> LENGTH: 105 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: 30/30/39 poly-A sequence <400> SEQUENCE: 4 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcgaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaccgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 105 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003335 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 5 cugcuccucc ucugccuugc 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003336 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 6 ccuccucugc cuugcuggac 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003337 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 7 ccaguccagc aaggcagagg 20 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003338 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 8 auaccagucc agcaaggcag 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003339 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 9 acacaaauac caguccagca 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003340 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 10 uggacuggua uuugugucug 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003341 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 11 cugguauuug ugucugaggc 20 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003342 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 12 cuucucuaca cccagggcac 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003343 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 13 cagaggacac uuggauucac 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003344 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 14 uuugaccauc agaggacacu 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003345 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 15 ucuagaacuu ugaccaucag 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003346 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 16 aaaguucuag augcuguccg 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003347 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 17 cauugauggc aggacugccu 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003348 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 18 aggcaguccu gccaucaaug 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003349 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 19 ugcacggcca cauugauggc 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003350 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 20 cacaugcacg gccacauuga 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003351 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 21 agccuuucug aacacaugca 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003352 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 22 gaaaggcugc ugaugacacc 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003353 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 23 aaaggcugcu gaugacaccu 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003354 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 24 accugggagc cauuugccuc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003355 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 25 cccagaggca aauggcuccc 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003356 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 26 gcaacuuacc cagaggcaaa 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003357 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 27 uucuuuggca acuuacccag 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003358 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 28 augcagcucu ccagacucac 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003359 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 29 agugagucug gagagcugca 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003360 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 30 gugagucugg agagcugcau 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003361 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 31 gcugcauggg cucacaacug 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003362 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 32 gcaugggcuc acaacugagg 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003363 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 33 acugaggagg aauuuguaga 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003364 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 34 cugaggagga auuuguagaa 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003365 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 35 uguagaaggg auauacaaag 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003366 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 36 aaauagacac caaaucuuac 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003367 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 37 agacaccaaa ucuuacugga 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003368 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 38 aagugccuuc caguaagauu 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003369 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 39 cucugcaugc ucauggaaug 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003370 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 40 ccucugcaug cucauggaau 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003371 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 41 accucugcau gcucauggaa 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003372 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 42 uacucaccuc ugcaugcuca 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003373 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 43 guauucacag ccaacgacuc 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003374 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 44 gcggcggggg ccggagucgu 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003375 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 45 aaugguguag cggcgggggc 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003376 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 46 cggcaauggu guagcggcgg 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003377 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 47 gcggcaaugg uguagcggcg 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003378 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 48 ggcggcaaug guguagcggc 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003379 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 49 gggcggcaau gguguagcgg 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003380 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 50 gcagggcggc aaugguguag 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003381 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 51 ggggcucagc agggcggcaa 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003382 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 52 ggaguagggg cucagcaggg 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003383 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 53 auaggaguag gggcucagca 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003384 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 54 aauaggagua ggggcucagc 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003385 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 55 ccccuacucc uauuccacca 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003386 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 56 ccguggugga auaggaguag 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003387 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 57 gccguggugg aauaggagua 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003388 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 58 gacgacagcc gugguggaau 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003389 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 59 auuggugacg acagccgugg 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003390 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 60 gggauuggug acgacagccg 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003391 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 61 ggcugucguc accaauccca 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003392 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 62 agucccucau uccuugggau 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005298 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 63 uccacucauu cuuggcagga 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005299 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 64 agccguggug gaauaggagu 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005300 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 65 ucacagaaac acucaccgua 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005301 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 66 gucacagaaa cacucaccgu 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005302 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 67 acgugucuuc ucuacaccca 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005303 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 68 ugaauccaag uguccucuga 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005304 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 69 ggccgugcau guguucagaa 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005305 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 70 uauaggaaaa ccagugaguc 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005306 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 71 aaaucuuacu ggaaggcacu 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005307 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 72 ugucugucuu cucucauagg 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR000689 gRNA targeting Cyno TTR <400> SEQUENCE: 73 acacaaauac caguccagcg 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005364 gRNA targeting Cyno TTR <400> SEQUENCE: 74 aaaggcugcu gaugagaccu 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005365 gRNA targeting Cyno TTR <400> SEQUENCE: 75 cauugacagc aggacugccu 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005366 gRNA targeting Cyno TTR <400> SEQUENCE: 76 auaccagucc agcgaggcag 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005367 gRNA targeting Cyno TTR <400> SEQUENCE: 77 ccaguccagc gaggcagagg 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005368 gRNA targeting Cyno TTR <400> SEQUENCE: 78 ccuccucugc cucgcuggac 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005369 gRNA targeting Cyno TTR <400> SEQUENCE: 79 aaaguucuag augccguccg 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005370 gRNA targeting Cyno TTR <400> SEQUENCE: 80 acuugucuuc ucuauaccca 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005371 gRNA targeting Cyno TTR <400> SEQUENCE: 81 aagugacuuc caguaagauu 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005372 gRNA targeting Cyno TTR <400> SEQUENCE: 82 aaaaggcugc ugaugagacc 20 <210> SEQ ID NO 83 <400> SEQUENCE: 83 000 <210> SEQ ID NO 84 <400> SEQUENCE: 84 000 <210> SEQ ID NO 85 <400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <400> SEQUENCE: 86 000 <210> SEQ ID NO 87 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000480 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 87 aaaggcugcu gaugacaccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 88 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000481 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 88 ucuagaacuu ugaccaucag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 89 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000482 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 89 uguagaaggg auauacaaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 90 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000483 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 90 uccacucauu cuuggcagga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 91 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000484 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 91 agacaccaaa ucuuacugga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 92 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000485 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 92 ccuccucugc cuugcuggac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 93 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000486 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 93 acacaaauac caguccagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 94 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000487 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 94 uucuuuggca acuuacccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 95 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000488 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 95 aaaguucuag augcuguccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 96 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000489 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 96 uuugaccauc agaggacacu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 97 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000490 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 97 aaauagacac caaaucuuac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 98 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000491 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 98 auaccagucc agcaaggcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 99 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000492 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 99 cuucucuaca cccagggcac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 100 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000493 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 100 aagugccuuc caguaagauu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 101 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000494 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 101 gugagucugg agagcugcau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 102 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000495 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 102 cagaggacac uuggauucac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 103 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000496 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 103 ggccgugcau guguucagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 104 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000497 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 104 cugcuccucc ucugccuugc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 105 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000498 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 105 agugagucug gagagcugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 106 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000499 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 106 ugaauccaag uguccucuga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 107 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000500 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 107 ccaguccagc aaggcagagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 108 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000501 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 108 ucacagaaac acucaccgua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 109 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000567 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 109 gaaaggcugc ugaugacacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 110 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000568 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 110 ggcugucguc accaauccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 111 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000570 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 111 cauugauggc aggacugccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 112 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000571 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 112 gucacagaaa cacucaccgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 113 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000572 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 113 ccccuacucc uauuccacca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 114 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000502 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 114 acacaaauac caguccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 115 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000503 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 115 aaaaggcugc ugaugagacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 116 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000504 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 116 aaaggcugcu gaugagaccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 117 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000505 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 117 cauugacagc aggacugccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 118 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000506 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 118 auaccagucc agcgaggcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 119 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000507 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 119 ccaguccagc gaggcagagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 120 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000508 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 120 ccuccucugc cucgcuggac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 121 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000509 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 121 aaaguucuag augccguccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 122 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000510 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 122 acuugucuuc ucuauaccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 123 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000511 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 123 aagugacuuc caguaagauu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 124 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000282 sgRNA modified sequence targeting Mouse TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 124 uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 125 <400> SEQUENCE: 125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000 <210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO 129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000 <210> SEQ ID NO 131 <400> SEQUENCE: 131 000 <210> SEQ ID NO 132 <400> SEQUENCE: 132 000 <210> SEQ ID NO 133 <400> SEQUENCE: 133 000 <210> SEQ ID NO 134 <400> SEQUENCE: 134 000 <210> SEQ ID NO 135 <400> SEQUENCE: 135 000 <210> SEQ ID NO 136 <400> SEQUENCE: 136 000 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000 <210> SEQ ID NO 138 <400> SEQUENCE: 138 000 <210> SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE: 140 000 <210> SEQ ID NO 141 <400> SEQUENCE: 141 000 <210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210> SEQ ID NO 143 <400> SEQUENCE: 143 000 <210> SEQ ID NO 144 <400> SEQUENCE: 144 000 <210> SEQ ID NO 145 <400> SEQUENCE: 145 000 <210> SEQ ID NO 146 <400> SEQUENCE: 146 000 <210> SEQ ID NO 147 <400> SEQUENCE: 147 000 <210> SEQ ID NO 148 <400> SEQUENCE: 148 000 <210> SEQ ID NO 149 <400> SEQUENCE: 149 000 <210> SEQ ID NO 150 <400> SEQUENCE: 150 000 <210> SEQ ID NO 151 <400> SEQUENCE: 151 000 <210> SEQ ID NO 152 <400> SEQUENCE: 152 000 <210> SEQ ID NO 153 <400> SEQUENCE: 153 000 <210> SEQ ID NO 154 <400> SEQUENCE: 154 000 <210> SEQ ID NO 155 <400> SEQUENCE: 155 000 <210> SEQ ID NO 156 <400> SEQUENCE: 156 000 <210> SEQ ID NO 157 <400> SEQUENCE: 157 000 <210> SEQ ID NO 158 <400> SEQUENCE: 158 000 <210> SEQ ID NO 159 <400> SEQUENCE: 159 000 <210> SEQ ID NO 160 <400> SEQUENCE: 160 000 <210> SEQ ID NO 161 <400> SEQUENCE: 161 000 <210> SEQ ID NO 162 <400> SEQUENCE: 162 000 <210> SEQ ID NO 163 <400> SEQUENCE: 163 000 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174 <400> SEQUENCE: 174 000 <210> SEQ ID NO 175 <400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000 <210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400> SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE: 198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000 <210> SEQ ID NO 200 <400> SEQUENCE: 200 000 <210> SEQ ID NO 201 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: DNA coding sequence of Cas9 using the thymidine analog of the minimal uridine codons listed in Table 3, with start and stop codons <400> SEQUENCE: 201 atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg atgggcagtc 60 atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa cacagacaga 120 cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga aacagcagaa 180 gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa cagaatctgc 240 tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt cttccacaga 300 ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc actggcacac 480 atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga caacagcgac 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact gttcggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc ggcattcctg 1620 agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag aaaggtcaca 1680 gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag cgtcgaaatc 1740 agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac aggatgggga 1980 agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa gacaatcctg 2040 gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg agacagcctg 2160 cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga aaacatcgtc 2280 atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca gaacggaaga 2460 gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagatgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt cgacaacctg 2700 acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat caagagacag 2760 ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac actgaagagc 2880 aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga aatcaacaac 2940 taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact gatcaagaag 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa gacagaagtc 3300 cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga caagctgatc 3360 gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc gacagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa cccgatcgac 3540 ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc aggagaactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag caagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac aagcacaaag 4020 gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag aaaggtctag 4140 <210> SEQ ID NO 202 <211> LENGTH: 4143 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: DNA coding sequence of Cas9 using codons with generally high expression in humans <400> SEQUENCE: 202 atggataaga agtactcaat cgggctggat atcggaacta attccgtggg ttgggcagtg 60 atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa caccgataga 120 cacagcatca agaaaaatct catcggagcc ctgctgtttg actccggcga aaccgcagaa 180 gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag agatcttttc gaacgaaatg gcaaaggtcg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtcg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcaactga caaggccgac ctcagattga tctacttggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga taactccgac 540 gtggataagc ttttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcta gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaaa agaacggact tttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatc ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc tagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga tctgctccgg 1200 aaacagagaa cctttgacaa cggatccatt ccccaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa tttcgaggaa 1440 gttgtggata agggagcttc ggcacaaagc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620 tccggagaac agaagaaagc aattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc agtggaaatc 1740 agcggggtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcaattgat ccacgacgac 2100 agcctgacct ttaaggagga catccaaaaa gcacaagtgt ccggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat tctgcaaact 2220 gtgaaggtgg tcgacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaaaaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tcgatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcttggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gtaccgccct gatcaaaaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga actttttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa aaccgaagtg 3300 caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaaaaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctcgagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actccccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 caaaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta tcttgcttcg 3720 cactacgaaa aactcaaagg gtcaccggaa gataacgaac agaagcagct tttcgtggag 3780 cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc aaagcgcgtg 3840 atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca tagagataag 3900 ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa cctgggagcc 3960 ccagccgcct tcaagtactt cgatactact atcgatcgca aaagatacac gtccaccaag 4020 gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga aactaggatc 4080 gatctgtcgc agctgggtgg cgatggcggt ggatctccga aaaagaagag aaaggtgtaa 4140 tga 4143 <210> SEQ ID NO 203 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 with one nuclear localization signal (1xNLS) as the C-terminal 7 amino acids <400> SEQUENCE: 203 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 204 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF using minimal uridine codons, with start and stop codons <400> SEQUENCE: 204 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 205 <211> LENGTH: 4143 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF using codons with generally high expression in humans, with start and stop codons <400> SEQUENCE: 205 auggauaaga aguacucaau cgggcuggau aucggaacua auuccguggg uugggcagug 60 aucacggaug aauacaaagu gccguccaag aaguucaagg uccuggggaa caccgauaga 120 cacagcauca agaaaaaucu caucggagcc cugcuguuug acuccggcga aaccgcagaa 180 gcgacccggc ucaaacguac cgcgaggcga cgcuacaccc ggcggaagaa ucgcaucugc 240 uaucugcaag agaucuuuuc gaacgaaaug gcaaaggucg acgacagcuu cuuccaccgc 300 cuggaagaau cuuuccuggu ggaggaggac aagaagcaug aacggcaucc uaucuuugga 360 aacaucgucg acgaaguggc guaccacgaa aaguacccga ccaucuacca ucugcggaag 420 aaguugguug acucaacuga caaggccgac cucagauuga ucuacuuggc ccucgcccau 480 augaucaaau uccgcggaca cuuccugauc gaaggcgauc ugaacccuga uaacuccgac 540 guggauaagc uuuucauuca acuggugcag accuacaacc aacuguucga agaaaaccca 600 aucaaugcua gcggcgucga ugccaaggcc auccuguccg cccggcuguc gaagucgcgg 660 cgccucgaaa accugaucgc acagcugccg ggagagaaaa agaacggacu uuucggcaac 720 uugaucgcuc ucucacuggg acucacuccc aauuucaagu ccaauuuuga ccuggccgag 780 gacgcgaagc ugcaacucuc aaaggacacc uacgacgacg acuuggacaa uuugcuggca 840 caaauuggcg aucaguacgc ggaucuguuc cuugccgcua agaaccuuuc ggacgcaauc 900 uugcuguccg auauccugcg cgugaacacc gaaauaacca aagcgccgcu uagcgccucg 960 augauuaagc gguacgacga gcaucaccag gaucucacgc ugcucaaagc gcucgugaga 1020 cagcaacugc cugaaaagua caaggagauc uucuucgacc aguccaagaa uggguacgca 1080 ggguacaucg auggaggcgc uagccaggaa gaguucuaua aguucaucaa gccaauccug 1140 gaaaagaugg acggaaccga agaacugcug gucaagcuga acagggagga ucugcuccgg 1200 aaacagagaa ccuuugacaa cggauccauu ccccaccaga uccaucuggg ugagcugcac 1260 gccaucuugc ggcgccagga ggacuuuuac ccauuccuca aggacaaccg ggaaaagauc 1320 gagaaaauuc ugacguuccg caucccguau uacgugggcc cacuggcgcg cggcaauucg 1380 cgcuucgcgu ggaugacuag aaaaucagag gaaaccauca cuccuuggaa uuucgaggaa 1440 guuguggaua agggagcuuc ggcacaaagc uucaucgaac gaaugaccaa cuucgacaag 1500 aaucucccaa acgagaaggu gcuuccuaag cacagccucc uuuacgaaua cuucacuguc 1560 uacaacgaac ugacuaaagu gaaauacguu acugaaggaa ugaggaagcc ggccuuucug 1620 uccggagaac agaagaaagc aauugucgau cugcuguuca agaccaaccg caaggugacc 1680 gucaagcagc uuaaagagga cuacuucaag aagaucgagu guuucgacuc aguggaaauc 1740 agcggggugg aggacagauu caacgcuucg cugggaaccu aucaugaucu ccugaagauc 1800 aucaaggaca aggacuuccu ugacaacgag gagaacgagg acauccugga agauaucguc 1860 cugaccuuga cccuuuucga ggaucgcgag augaucgagg agaggcuuaa gaccuacgcu 1920 caucucuucg acgauaaggu caugaaacaa cucaagcgcc gccgguacac ugguuggggc 1980 cgccucuccc gcaagcugau caacgguauu cgcgauaaac agagcgguaa aacuauccug 2040 gauuuccuca aaucggaugg cuucgcuaau cguaacuuca ugcaauugau ccacgacgac 2100 agccugaccu uuaaggagga cauccaaaaa gcacaagugu ccggacaggg agacucacuc 2160 caugaacaca ucgcgaaucu ggccgguucg ccggcgauua agaagggaau ucugcaaacu 2220 gugaaggugg ucgacgagcu ggugaagguc augggacggc acaaaccgga gaauaucgug 2280 auugaaaugg cccgagaaaa ccagacuacc cagaagggcc agaaaaacuc ccgcgaaagg 2340 augaagcgga ucgaagaagg aaucaaggag cugggcagcc agauccugaa agagcacccg 2400 guggaaaaca cgcagcugca gaacgagaag cucuaccugu acuauuugca aaauggacgg 2460 gacauguacg uggaccaaga gcuggacauc aaucgguugu cugauuacga cguggaccac 2520 aucguuccac aguccuuucu gaaggaugac ucgaucgaua acaagguguu gacucgcagc 2580 gacaagaaca gagggaaguc agauaaugug ccaucggagg aggucgugaa gaagaugaag 2640 aauuacuggc ggcagcuccu gaaugcgaag cugauuaccc agagaaaguu ugacaaucuc 2700 acuaaagccg agcgcggcgg acucucagag cuggauaagg cuggauucau caaacggcag 2760 cuggucgaga cucggcagau uaccaagcac guggcgcaga ucuuggacuc ccgcaugaac 2820 acuaaauacg acgagaacga uaagcucauc cgggaaguga aggugauuac ccugaaaagc 2880 aaacuugugu cggacuuucg gaaggacuuu caguuuuaca aagugagaga aaucaacaac 2940 uaccaucacg cgcaugacgc auaccucaac gcuguggucg guaccgcccu gaucaaaaag 3000 uacccuaaac uugaaucgga guuuguguac ggagacuaca aggucuacga cgugaggaag 3060 augauagcca aguccgaaca ggaaaucggg aaagcaacug cgaaauacuu cuuuuacuca 3120 aacaucauga acuuuuucaa gacugaaauu acgcuggcca auggagaaau caggaagagg 3180 ccacugaucg aaacuaacgg agaaacgggc gaaaucgugu gggacaaggg cagggacuuc 3240 gcaacuguuc gcaaagugcu cucuaugccg caagucaaua uugugaagaa aaccgaagug 3300 caaaccggcg gauuuucaaa ggaaucgauc cucccaaaga gaaauagcga caagcucauu 3360 gcacgcaaga aagacuggga cccgaagaag uacggaggau ucgauucgcc gacugucgca 3420 uacuccgucc ucgugguggc caagguggag aagggaaaga gcaaaaagcu caaauccguc 3480 aaagagcugc uggggauuac caucauggaa cgauccucgu ucgagaagaa cccgauugau 3540 uuccucgagg cgaaggguua caaggaggug aagaaggauc ugaucaucaa acuccccaag 3600 uacucacugu ucgaacugga aaauggucgg aagcgcaugc uggcuucggc cggagaacuc 3660 caaaaaggaa augagcuggc cuugccuagc aaguacguca acuuccucua ucuugcuucg 3720 cacuacgaaa aacucaaagg gucaccggaa gauaacgaac agaagcagcu uuucguggag 3780 cagcacaagc auuaucugga ugaaaucauc gaacaaaucu ccgaguuuuc aaagcgcgug 3840 auccucgccg acgccaaccu cgacaaaguc cugucggccu acaauaagca uagagauaag 3900 ccgaucagag aacaggccga gaacauuauc cacuuguuca cccugacuaa ccugggagcc 3960 ccagccgccu ucaaguacuu cgauacuacu aucgaucgca aaagauacac guccaccaag 4020 gaaguucugg acgcgacccu gauccaccaa agcaucacug gacucuacga aacuaggauc 4080 gaucugucgc agcugggugg cgauggcggu ggaucuccga aaaagaagag aaagguguaa 4140 uga 4143 <210> SEQ ID NO 206 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase (D10A) amino acid sequence <400> SEQUENCE: 206 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 207 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase (D10A) mRNA ORF <400> SEQUENCE: 207 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 208 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 (D10A H840A) amino acid sequence <400> SEQUENCE: 208 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 209 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 (D10A H840A) mRNA ORF <400> SEQUENCE: 209 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 210 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA coding sequence using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 210 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 211 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase bare coding sequence <400> SEQUENCE: 211 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 212 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 bare coding sequence <400> SEQUENCE: 212 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 213 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 (without NLS) <400> SEQUENCE: 213 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 214 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF encoding SEQ ID NO: 213 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 214 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 215 <211> LENGTH: 4101 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 coding sequence encoding SEQ ID NO: 213 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 215 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga c 4101 <210> SEQ ID NO 216 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 nickase (without NLS) <400> SEQUENCE: 216 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 217 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase mRNA ORF encoding SEQ ID NO: 216 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 217 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 218 <211> LENGTH: 4101 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase coding sequence encoding SEQ ID NO: 216 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 218 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga c 4101 <210> SEQ ID NO 219 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of dCas9 (without NLS) <400> SEQUENCE: 219 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 220 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 mRNA ORF encoding SEQ ID NO: 219 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 220 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 221 <211> LENGTH: 4113 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 coding sequence encoding SEQ ID NO: 219 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 221 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agc 4113 <210> SEQ ID NO 222 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 with two nuclear localization signals (2xNLS) as the C-terminal amino acids <400> SEQUENCE: 222 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 223 <211> LENGTH: 4233 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF encoding SEQ ID NO: 222 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 223 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucccg 4140 aagaagaaga gaaaggucgg aagcggaagc ccgaagaaga agagaaaggu cgacggaagc 4200 ccgaagaaga agagaaaggu cgacagcgga uag 4233 <210> SEQ ID NO 224 <211> LENGTH: 4227 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 coding sequence encoding SEQ ID NO: 222 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 224 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa ggucccgaag 4140 aagaagagaa aggucggaag cggaagcccg aagaagaaga gaaaggucga cggaagcccg 4200 aagaagaaga gaaaggucga cagcgga 4227 <210> SEQ ID NO 225 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 nickase with two nuclear localization signals as the C-terminal amino acids <400> SEQUENCE: 225 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 226 <211> LENGTH: 4179 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase mRNA ORF encoding SEQ ID NO: 25 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 226 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggaagc ggaagcccga agaagaagag aaaggucgac 4140 ggaagcccga agaagaagag aaaggucgac agcggauag 4179 <210> SEQ ID NO 227 <211> LENGTH: 4173 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase coding sequence encoding SEQ ID NO: 25 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 227 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaagcgga agcccgaaga agaagagaaa ggucgacgga 4140 agcccgaaga agaagagaaa ggucgacagc gga 4173 <210> SEQ ID NO 228 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of dCas9 with two nuclear localization signals as the C-terminal amino acids <400> SEQUENCE: 228 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 229 <211> LENGTH: 4179 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 mRNA ORF encoding SEQ ID NO: 228 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 229 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggaagc ggaagcccga agaagaagag aaaggucgac 4140 ggaagcccga agaagaagag aaaggucgac agcggauag 4179 <210> SEQ ID NO 230 <211> LENGTH: 4173 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 coding sequence encoding SEQ ID NO: 228 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 230 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaagcgga agcccgaaga agaagagaaa ggucgacgga 4140 agcccgaaga agaagagaaa ggucgacagc gga 4173 <210> SEQ ID NO 231 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: T7 Promoter <400> SEQUENCE: 231 taatacgact cactata 17 <210> SEQ ID NO 232 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(50) <223> OTHER INFORMATION: Human beta-globin 5 UTR <400> SEQUENCE: 232 acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc 50 <210> SEQ ID NO 233 <211> LENGTH: 132 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(132) <223> OTHER INFORMATION: Human beta-globin 3 UTR <400> SEQUENCE: 233 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60 taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120 tattttcatt gc 132 <210> SEQ ID NO 234 <211> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(66) <223> OTHER INFORMATION: Human alpha-globin 5 UTR <400> SEQUENCE: 234 cataaaccct ggcgcgctcg cggcccggca ctcttctggt ccccacagac tcagagagaa 60 cccacc 66 <210> SEQ ID NO 235 <211> LENGTH: 110 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(110) <223> OTHER INFORMATION: Human alpha-globin 3 UTR <400> SEQUENCE: 235 gctggagcct cggtggccat gcttcttgcc ccttgggcct ccccccagcc cctcctcccc 60 ttcctgcacc cgtacccccg tggtctttga ataaagtctg agtgggcggc 110 <210> SEQ ID NO 236 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Xenopus laevis <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(29) <223> OTHER INFORMATION: Xenopus laevis beta-globin 5 UTR <400> SEQUENCE: 236 aagctcagaa taaacgctca actttggcc 29 <210> SEQ ID NO 237 <211> LENGTH: 130 <212> TYPE: DNA <213> ORGANISM: Xenopus laevis <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(130) <223> OTHER INFORMATION: Xenopus laevis beta-globin 3 UTR <400> SEQUENCE: 237 accagcctca agaacacccg aatggagtct ctaagctaca taataccaac ttacacttta 60 caaaatgttg tcccccaaaa tgtagccatt cgtatctgct cctaataaaa agaaagtttc 120 ttcacattct 130 <210> SEQ ID NO 238 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Bos taurus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(27) <223> OTHER INFORMATION: Bovine Growth Hormone 5 UTR <400> SEQUENCE: 238 cagggtcctg tggacagctc accagct 27 <210> SEQ ID NO 239 <211> LENGTH: 102 <212> TYPE: DNA <213> ORGANISM: Bos taurus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(102) <223> OTHER INFORMATION: Bovine Growth Hormone 3 UTR <400> SEQUENCE: 239 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 60 tcccactgtc ctttcctaat aaaatgagga aattgcatcg ca 102 <210> SEQ ID NO 240 <211> LENGTH: 93 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(93) <223> OTHER INFORMATION: Mus musculus hemoglobin alpha, adult chain 1 (Hba-a1), 3UTR <400> SEQUENCE: 240 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 93 <210> SEQ ID NO 241 <211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: HSD17B4 5 UTR <400> SEQUENCE: 241 tcccgcagtc ggcgtccagc ggctctgctt gttcgtgtgt gtgtcgttgc aggccttatt 60 c 61 <210> SEQ ID NO 242 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G282 single guide RNA targeting the mouse TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 242 uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 243 <400> SEQUENCE: 243 000 <210> SEQ ID NO 244 <211> LENGTH: 4405 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, and 3 UTR of ALB <400> SEQUENCE: 244 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg 120 atgggcagtc atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa 180 cacagacaga cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga 240 aacagcagaa gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa 300 cagaatctgc tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt 360 cttccacaga ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc 420 gatcttcgga aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca 480 cctgagaaag aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc 540 actggcacac atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga 600 caacagcgac gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga 660 agaaaacccg atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag 720 caagagcaga agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact 780 gttcggaaac ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga 840 cctggcagaa gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa 900 cctgctggca cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag 960 cgacgcaatc ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct 1020 gagcgcaagc atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc 1080 actggtcaga cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa 1140 cggatacgca ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa 1200 gccgatcctg gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga 1260 cctgctgaga aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg 1320 agaactgcac gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag 1380 agaaaagatc gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag 1440 aggaaacagc agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa 1500 cttcgaagaa gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa 1560 cttcgacaag aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata 1620 cttcacagtc tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc 1680 ggcattcctg agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag 1740 aaaggtcaca gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag 1800 cgtcgaaatc agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct 1860 gctgaagatc atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga 1920 agacatcgtc ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa 1980 gacatacgca cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac 2040 aggatgggga agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa 2100 gacaatcctg gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat 2160 ccacgacgac agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg 2220 agacagcctg cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat 2280 cctgcagaca gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga 2340 aaacatcgtc atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag 2400 cagagaaaga atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa 2460 ggaacacccg gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca 2520 gaacggaaga gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga 2580 cgtcgaccac atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct 2640 gacaagaagc gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa 2700 gaagatgaag aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt 2760 cgacaacctg acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat 2820 caagagacag ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag 2880 cagaatgaac acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac 2940 actgaagagc aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga 3000 aatcaacaac taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact 3060 gatcaagaag tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga 3120 cgtcagaaag atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt 3180 cttctacagc aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat 3240 cagaaagaga ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg 3300 aagagacttc gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa 3360 gacagaagtc cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga 3420 caagctgatc gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc 3480 gacagtcgca tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct 3540 gaagagcgtc aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa 3600 cccgatcgac ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa 3660 gctgccgaag tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc 3720 aggagaactg cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta 3780 cctggcaagc cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct 3840 gttcgtcgaa cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag 3900 caagagagtc atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca 3960 cagagacaag ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa 4020 cctgggagca ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac 4080 aagcacaaag gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga 4140 aacaagaatc gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag 4200 aaaggtctag ctagccatca catttaaaag catctcagcc taccatgaga ataagagaaa 4260 gaaaatgaag atcaatagct tattcatctc tttttctttt tcgttggtgt aaagccaaca 4320 ccctgtctaa aaaacataaa tttctttaat cattttgcct cttttctctg tgcttcaatt 4380 aataaaaaat ggaaagaacc tcgag 4405 <210> SEQ ID NO 245 <211> LENGTH: 4188 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Alternative Cas9 ORF with 19.36% U content <400> SEQUENCE: 245 atggataaga agtactcgat cgggctggat atcggaacta attccgtggg ttgggcagtg 60 atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa caccgataga 120 cacagcatca agaagaatct catcggagcc ctgctgtttg actccggcga aaccgcagaa 180 gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag aaatcttttc gaacgaaatg gcaaaggtgg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtgg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcaactga caaggccgac ctcagattga tctacttggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga taactccgac 540 gtggataagc tgttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcca gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaga agaacggact tttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatt ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc cagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga tctgctccgc 1200 aaacagagaa cctttgacaa cggaagcatt ccacaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa tttcgaggaa 1440 gttgtggata agggagcttc ggcacaatcc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620 agcggagaac agaagaaagc gattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc agtggaaatc 1740 agcggagtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcagttgat ccacgacgac 2100 agcctgacct ttaaggagga catccagaaa gcacaagtga gcggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat cctgcaaact 2220 gtgaaggtgg tggacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaagaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tccatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcctggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gcaccgccct gatcaagaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga acttcttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa aaccgaagtg 3300 caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaagaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctggagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actgcccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 cagaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta tcttgcttcg 3720 cactacgaga aactcaaagg gtcaccggaa gataacgaac agaagcagct tttcgtggag 3780 cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc aaagcgcgtg 3840 atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca tagagataag 3900 ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa cctgggagct 3960 ccagccgcct tcaagtactt cgatactact atcgaccgca aaagatacac gtccaccaag 4020 gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga aactaggatc 4080 gatctgtcgc agctgggtgg cgatggtggc ggtggatcct acccatacga cgtgcctgac 4140 tacgcctccg gaggtggtgg ccccaagaag aaacggaagg tgtgatag 4188 <210> SEQ ID NO 246 <211> LENGTH: 4459 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 245, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 246 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatct gccaccatgg ataagaagta ctcgatcggg ctggatatcg gaactaattc 120 cgtgggttgg gcagtgatca cggatgaata caaagtgccg tccaagaagt tcaaggtcct 180 ggggaacacc gatagacaca gcatcaagaa gaatctcatc ggagccctgc tgtttgactc 240 cggcgaaacc gcagaagcga cccggctcaa acgtaccgcg aggcgacgct acacccggcg 300 gaagaatcgc atctgctatc tgcaagaaat cttttcgaac gaaatggcaa aggtggacga 360 cagcttcttc caccgcctgg aagaatcttt cctggtggag gaggacaaga agcatgaacg 420 gcatcctatc tttggaaaca tcgtggacga agtggcgtac cacgaaaagt acccgaccat 480 ctaccatctg cggaagaagt tggttgactc aactgacaag gccgacctca gattgatcta 540 cttggccctc gcccatatga tcaaattccg cggacacttc ctgatcgaag gcgatctgaa 600 ccctgataac tccgacgtgg ataagctgtt cattcaactg gtgcagacct acaaccaact 660 gttcgaagaa aacccaatca atgccagcgg cgtcgatgcc aaggccatcc tgtccgcccg 720 gctgtcgaag tcgcggcgcc tcgaaaacct gatcgcacag ctgccgggag agaagaagaa 780 cggacttttc ggcaacttga tcgctctctc actgggactc actcccaatt tcaagtccaa 840 ttttgacctg gccgaggacg cgaagctgca actctcaaag gacacctacg acgacgactt 900 ggacaatttg ctggcacaaa ttggcgatca gtacgcggat ctgttccttg ccgctaagaa 960 cctttcggac gcaatcttgc tgtccgatat cctgcgcgtg aacaccgaaa taaccaaagc 1020 gccgcttagc gcctcgatga ttaagcggta cgacgagcat caccaggatc tcacgctgct 1080 caaagcgctc gtgagacagc aactgcctga aaagtacaag gagattttct tcgaccagtc 1140 caagaatggg tacgcagggt acatcgatgg aggcgccagc caggaagagt tctataagtt 1200 catcaagcca atcctggaaa agatggacgg aaccgaagaa ctgctggtca agctgaacag 1260 ggaggatctg ctccgcaaac agagaacctt tgacaacgga agcattccac accagatcca 1320 tctgggtgag ctgcacgcca tcttgcggcg ccaggaggac ttttacccat tcctcaagga 1380 caaccgggaa aagatcgaga aaattctgac gttccgcatc ccgtattacg tgggcccact 1440 ggcgcgcggc aattcgcgct tcgcgtggat gactagaaaa tcagaggaaa ccatcactcc 1500 ttggaatttc gaggaagttg tggataaggg agcttcggca caatccttca tcgaacgaat 1560 gaccaacttc gacaagaatc tcccaaacga gaaggtgctt cctaagcaca gcctccttta 1620 cgaatacttc actgtctaca acgaactgac taaagtgaaa tacgttactg aaggaatgag 1680 gaagccggcc tttctgagcg gagaacagaa gaaagcgatt gtcgatctgc tgttcaagac 1740 caaccgcaag gtgaccgtca agcagcttaa agaggactac ttcaagaaga tcgagtgttt 1800 cgactcagtg gaaatcagcg gagtggagga cagattcaac gcttcgctgg gaacctatca 1860 tgatctcctg aagatcatca aggacaagga cttccttgac aacgaggaga acgaggacat 1920 cctggaagat atcgtcctga ccttgaccct tttcgaggat cgcgagatga tcgaggagag 1980 gcttaagacc tacgctcatc tcttcgacga taaggtcatg aaacaactca agcgccgccg 2040 gtacactggt tggggccgcc tctcccgcaa gctgatcaac ggtattcgcg ataaacagag 2100 cggtaaaact atcctggatt tcctcaaatc ggatggcttc gctaatcgta acttcatgca 2160 gttgatccac gacgacagcc tgacctttaa ggaggacatc cagaaagcac aagtgagcgg 2220 acagggagac tcactccatg aacacatcgc gaatctggcc ggttcgccgg cgattaagaa 2280 gggaatcctg caaactgtga aggtggtgga cgagctggtg aaggtcatgg gacggcacaa 2340 accggagaat atcgtgattg aaatggcccg agaaaaccag actacccaga agggccagaa 2400 gaactcccgc gaaaggatga agcggatcga agaaggaatc aaggagctgg gcagccagat 2460 cctgaaagag cacccggtgg aaaacacgca gctgcagaac gagaagctct acctgtacta 2520 tttgcaaaat ggacgggaca tgtacgtgga ccaagagctg gacatcaatc ggttgtctga 2580 ttacgacgtg gaccacatcg ttccacagtc ctttctgaag gatgactcca tcgataacaa 2640 ggtgttgact cgcagcgaca agaacagagg gaagtcagat aatgtgccat cggaggaggt 2700 cgtgaagaag atgaagaatt actggcggca gctcctgaat gcgaagctga ttacccagag 2760 aaagtttgac aatctcacta aagccgagcg cggcggactc tcagagctgg ataaggctgg 2820 attcatcaaa cggcagctgg tcgagactcg gcagattacc aagcacgtgg cgcagatcct 2880 ggactcccgc atgaacacta aatacgacga gaacgataag ctcatccggg aagtgaaggt 2940 gattaccctg aaaagcaaac ttgtgtcgga ctttcggaag gactttcagt tttacaaagt 3000 gagagaaatc aacaactacc atcacgcgca tgacgcatac ctcaacgctg tggtcggcac 3060 cgccctgatc aagaagtacc ctaaacttga atcggagttt gtgtacggag actacaaggt 3120 ctacgacgtg aggaagatga tagccaagtc cgaacaggaa atcgggaaag caactgcgaa 3180 atacttcttt tactcaaaca tcatgaactt cttcaagact gaaattacgc tggccaatgg 3240 agaaatcagg aagaggccac tgatcgaaac taacggagaa acgggcgaaa tcgtgtggga 3300 caagggcagg gacttcgcaa ctgttcgcaa agtgctctct atgccgcaag tcaatattgt 3360 gaagaaaacc gaagtgcaaa ccggcggatt ttcaaaggaa tcgatcctcc caaagagaaa 3420 tagcgacaag ctcattgcac gcaagaaaga ctgggacccg aagaagtacg gaggattcga 3480 ttcgccgact gtcgcatact ccgtcctcgt ggtggccaag gtggagaagg gaaagagcaa 3540 gaagctcaaa tccgtcaaag agctgctggg gattaccatc atggaacgat cctcgttcga 3600 gaagaacccg attgatttcc tggaggcgaa gggttacaag gaggtgaaga aggatctgat 3660 catcaaactg cccaagtact cactgttcga actggaaaat ggtcggaagc gcatgctggc 3720 ttcggccgga gaactccaga aaggaaatga gctggccttg cctagcaagt acgtcaactt 3780 cctctatctt gcttcgcact acgagaaact caaagggtca ccggaagata acgaacagaa 3840 gcagcttttc gtggagcagc acaagcatta tctggatgaa atcatcgaac aaatctccga 3900 gttttcaaag cgcgtgatcc tcgccgacgc caacctcgac aaagtcctgt cggcctacaa 3960 taagcataga gataagccga tcagagaaca ggccgagaac attatccact tgttcaccct 4020 gactaacctg ggagctccag ccgccttcaa gtacttcgat actactatcg accgcaaaag 4080 atacacgtcc accaaggaag ttctggacgc gaccctgatc caccaaagca tcactggact 4140 ctacgaaact aggatcgatc tgtcgcagct gggtggcgat ggtggcggtg gatcctaccc 4200 atacgacgtg cctgactacg cctccggagg tggtggcccc aagaagaaac ggaaggtgtg 4260 atagctagcc atcacattta aaagcatctc agcctaccat gagaataaga gaaagaaaat 4320 gaagatcaat agcttattca tctctttttc tttttcgttg gtgtaaagcc aacaccctgt 4380 ctaaaaaaca taaatttctt taatcatttt gcctcttttc tctgtgcttc aattaataaa 4440 aaatggaaag aacctcgag 4459 <210> SEQ ID NO 247 <211> LENGTH: 4453 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 245, and 3 UTR of ALB <400> SEQUENCE: 247 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatct atggataaga agtactcgat cgggctggat atcggaacta attccgtggg 120 ttgggcagtg atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa 180 caccgataga cacagcatca agaagaatct catcggagcc ctgctgtttg actccggcga 240 aaccgcagaa gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa 300 tcgcatctgc tatctgcaag aaatcttttc gaacgaaatg gcaaaggtgg acgacagctt 360 cttccaccgc ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc 420 tatctttgga aacatcgtgg acgaagtggc gtaccacgaa aagtacccga ccatctacca 480 tctgcggaag aagttggttg actcaactga caaggccgac ctcagattga tctacttggc 540 cctcgcccat atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga 600 taactccgac gtggataagc tgttcattca actggtgcag acctacaacc aactgttcga 660 agaaaaccca atcaatgcca gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc 720 gaagtcgcgg cgcctcgaaa acctgatcgc acagctgccg ggagagaaga agaacggact 780 tttcggcaac ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga 840 cctggccgag gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa 900 tttgctggca caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc 960 ggacgcaatc ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct 1020 tagcgcctcg atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc 1080 gctcgtgaga cagcaactgc ctgaaaagta caaggagatt ttcttcgacc agtccaagaa 1140 tgggtacgca gggtacatcg atggaggcgc cagccaggaa gagttctata agttcatcaa 1200 gccaatcctg gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga 1260 tctgctccgc aaacagagaa cctttgacaa cggaagcatt ccacaccaga tccatctggg 1320 tgagctgcac gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg 1380 ggaaaagatc gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg 1440 cggcaattcg cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa 1500 tttcgaggaa gttgtggata agggagcttc ggcacaatcc ttcatcgaac gaatgaccaa 1560 cttcgacaag aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata 1620 cttcactgtc tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc 1680 ggcctttctg agcggagaac agaagaaagc gattgtcgat ctgctgttca agaccaaccg 1740 caaggtgacc gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc 1800 agtggaaatc agcggagtgg aggacagatt caacgcttcg ctgggaacct atcatgatct 1860 cctgaagatc atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga 1920 agatatcgtc ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa 1980 gacctacgct catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac 2040 tggttggggc cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa 2100 aactatcctg gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcagttgat 2160 ccacgacgac agcctgacct ttaaggagga catccagaaa gcacaagtga gcggacaggg 2220 agactcactc catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat 2280 cctgcaaact gtgaaggtgg tggacgagct ggtgaaggtc atgggacggc acaaaccgga 2340 gaatatcgtg attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaagaactc 2400 ccgcgaaagg atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa 2460 agagcacccg gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca 2520 aaatggacgg gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga 2580 cgtggaccac atcgttccac agtcctttct gaaggatgac tccatcgata acaaggtgtt 2640 gactcgcagc gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa 2700 gaagatgaag aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt 2760 tgacaatctc actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat 2820 caaacggcag ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcctggactc 2880 ccgcatgaac actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac 2940 cctgaaaagc aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga 3000 aatcaacaac taccatcacg cgcatgacgc atacctcaac gctgtggtcg gcaccgccct 3060 gatcaagaag taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga 3120 cgtgaggaag atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt 3180 cttttactca aacatcatga acttcttcaa gactgaaatt acgctggcca atggagaaat 3240 caggaagagg ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg 3300 cagggacttc gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa 3360 aaccgaagtg caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga 3420 caagctcatt gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc 3480 gactgtcgca tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaagaagct 3540 caaatccgtc aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa 3600 cccgattgat ttcctggagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa 3660 actgcccaag tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc 3720 cggagaactc cagaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta 3780 tcttgcttcg cactacgaga aactcaaagg gtcaccggaa gataacgaac agaagcagct 3840 tttcgtggag cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc 3900 aaagcgcgtg atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca 3960 tagagataag ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa 4020 cctgggagct ccagccgcct tcaagtactt cgatactact atcgaccgca aaagatacac 4080 gtccaccaag gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga 4140 aactaggatc gatctgtcgc agctgggtgg cgatggtggc ggtggatcct acccatacga 4200 cgtgcctgac tacgcctccg gaggtggtgg ccccaagaag aaacggaagg tgtgatagct 4260 agccatcaca tttaaaagca tctcagccta ccatgagaat aagagaaaga aaatgaagat 4320 caatagctta ttcatctctt tttctttttc gttggtgtaa agccaacacc ctgtctaaaa 4380 aacataaatt tctttaatca ttttgcctct tttctctgtg cttcaattaa taaaaaatgg 4440 aaagaacctc gag 4453 <210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210> SEQ ID NO 249 <211> LENGTH: 4409 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript comprising Kozak sequence with Cas9 ORF using codons with generally high expression in humans <400> SEQUENCE: 249 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgc ctaagaaaaa gcggaaggtc gacggggata agaagtactc 120 aatcgggctg gatatcggaa ctaattccgt gggttgggca gtgatcacgg atgaatacaa 180 agtgccgtcc aagaagttca aggtcctggg gaacaccgat agacacagca tcaagaaaaa 240 tctcatcgga gccctgctgt ttgactccgg cgaaaccgca gaagcgaccc ggctcaaacg 300 taccgcgagg cgacgctaca cccggcggaa gaatcgcatc tgctatctgc aagagatctt 360 ttcgaacgaa atggcaaagg tcgacgacag cttcttccac cgcctggaag aatctttcct 420 ggtggaggag gacaagaagc atgaacggca tcctatcttt ggaaacatcg tcgacgaagt 480 ggcgtaccac gaaaagtacc cgaccatcta ccatctgcgg aagaagttgg ttgactcaac 540 tgacaaggcc gacctcagat tgatctactt ggccctcgcc catatgatca aattccgcgg 600 acacttcctg atcgaaggcg atctgaaccc tgataactcc gacgtggata agcttttcat 660 tcaactggtg cagacctaca accaactgtt cgaagaaaac ccaatcaatg ctagcggcgt 720 cgatgccaag gccatcctgt ccgcccggct gtcgaagtcg cggcgcctcg aaaacctgat 780 cgcacagctg ccgggagaga aaaagaacgg acttttcggc aacttgatcg ctctctcact 840 gggactcact cccaatttca agtccaattt tgacctggcc gaggacgcga agctgcaact 900 ctcaaaggac acctacgacg acgacttgga caatttgctg gcacaaattg gcgatcagta 960 cgcggatctg ttccttgccg ctaagaacct ttcggacgca atcttgctgt ccgatatcct 1020 gcgcgtgaac accgaaataa ccaaagcgcc gcttagcgcc tcgatgatta agcggtacga 1080 cgagcatcac caggatctca cgctgctcaa agcgctcgtg agacagcaac tgcctgaaaa 1140 gtacaaggag atcttcttcg accagtccaa gaatgggtac gcagggtaca tcgatggagg 1200 cgctagccag gaagagttct ataagttcat caagccaatc ctggaaaaga tggacggaac 1260 cgaagaactg ctggtcaagc tgaacaggga ggatctgctc cggaaacaga gaacctttga 1320 caacggatcc attccccacc agatccatct gggtgagctg cacgccatct tgcggcgcca 1380 ggaggacttt tacccattcc tcaaggacaa ccgggaaaag atcgagaaaa ttctgacgtt 1440 ccgcatcccg tattacgtgg gcccactggc gcgcggcaat tcgcgcttcg cgtggatgac 1500 tagaaaatca gaggaaacca tcactccttg gaatttcgag gaagttgtgg ataagggagc 1560 ttcggcacaa agcttcatcg aacgaatgac caacttcgac aagaatctcc caaacgagaa 1620 ggtgcttcct aagcacagcc tcctttacga atacttcact gtctacaacg aactgactaa 1680 agtgaaatac gttactgaag gaatgaggaa gccggccttt ctgtccggag aacagaagaa 1740 agcaattgtc gatctgctgt tcaagaccaa ccgcaaggtg accgtcaagc agcttaaaga 1800 ggactacttc aagaagatcg agtgtttcga ctcagtggaa atcagcgggg tggaggacag 1860 attcaacgct tcgctgggaa cctatcatga tctcctgaag atcatcaagg acaaggactt 1920 ccttgacaac gaggagaacg aggacatcct ggaagatatc gtcctgacct tgaccctttt 1980 cgaggatcgc gagatgatcg aggagaggct taagacctac gctcatctct tcgacgataa 2040 ggtcatgaaa caactcaagc gccgccggta cactggttgg ggccgcctct cccgcaagct 2100 gatcaacggt attcgcgata aacagagcgg taaaactatc ctggatttcc tcaaatcgga 2160 tggcttcgct aatcgtaact tcatgcaatt gatccacgac gacagcctga cctttaagga 2220 ggacatccaa aaagcacaag tgtccggaca gggagactca ctccatgaac acatcgcgaa 2280 tctggccggt tcgccggcga ttaagaaggg aattctgcaa actgtgaagg tggtcgacga 2340 gctggtgaag gtcatgggac ggcacaaacc ggagaatatc gtgattgaaa tggcccgaga 2400 aaaccagact acccagaagg gccagaaaaa ctcccgcgaa aggatgaagc ggatcgaaga 2460 aggaatcaag gagctgggca gccagatcct gaaagagcac ccggtggaaa acacgcagct 2520 gcagaacgag aagctctacc tgtactattt gcaaaatgga cgggacatgt acgtggacca 2580 agagctggac atcaatcggt tgtctgatta cgacgtggac cacatcgttc cacagtcctt 2640 tctgaaggat gactcgatcg ataacaaggt gttgactcgc agcgacaaga acagagggaa 2700 gtcagataat gtgccatcgg aggaggtcgt gaagaagatg aagaattact ggcggcagct 2760 cctgaatgcg aagctgatta cccagagaaa gtttgacaat ctcactaaag ccgagcgcgg 2820 cggactctca gagctggata aggctggatt catcaaacgg cagctggtcg agactcggca 2880 gattaccaag cacgtggcgc agatcttgga ctcccgcatg aacactaaat acgacgagaa 2940 cgataagctc atccgggaag tgaaggtgat taccctgaaa agcaaacttg tgtcggactt 3000 tcggaaggac tttcagtttt acaaagtgag agaaatcaac aactaccatc acgcgcatga 3060 cgcatacctc aacgctgtgg tcggtaccgc cctgatcaaa aagtacccta aacttgaatc 3120 ggagtttgtg tacggagact acaaggtcta cgacgtgagg aagatgatag ccaagtccga 3180 acaggaaatc gggaaagcaa ctgcgaaata cttcttttac tcaaacatca tgaacttttt 3240 caagactgaa attacgctgg ccaatggaga aatcaggaag aggccactga tcgaaactaa 3300 cggagaaacg ggcgaaatcg tgtgggacaa gggcagggac ttcgcaactg ttcgcaaagt 3360 gctctctatg ccgcaagtca atattgtgaa gaaaaccgaa gtgcaaaccg gcggattttc 3420 aaaggaatcg atcctcccaa agagaaatag cgacaagctc attgcacgca agaaagactg 3480 ggacccgaag aagtacggag gattcgattc gccgactgtc gcatactccg tcctcgtggt 3540 ggccaaggtg gagaagggaa agagcaaaaa gctcaaatcc gtcaaagagc tgctggggat 3600 taccatcatg gaacgatcct cgttcgagaa gaacccgatt gatttcctcg aggcgaaggg 3660 ttacaaggag gtgaagaagg atctgatcat caaactcccc aagtactcac tgttcgaact 3720 ggaaaatggt cggaagcgca tgctggcttc ggccggagaa ctccaaaaag gaaatgagct 3780 ggccttgcct agcaagtacg tcaacttcct ctatcttgct tcgcactacg aaaaactcaa 3840 agggtcaccg gaagataacg aacagaagca gcttttcgtg gagcagcaca agcattatct 3900 ggatgaaatc atcgaacaaa tctccgagtt ttcaaagcgc gtgatcctcg ccgacgccaa 3960 cctcgacaaa gtcctgtcgg cctacaataa gcatagagat aagccgatca gagaacaggc 4020 cgagaacatt atccacttgt tcaccctgac taacctggga gccccagccg ccttcaagta 4080 cttcgatact actatcgatc gcaaaagata cacgtccacc aaggaagttc tggacgcgac 4140 cctgatccac caaagcatca ctggactcta cgaaactagg atcgatctgt cgcagctggg 4200 tggcgattga tagtctagcc atcacattta aaagcatctc agcctaccat gagaataaga 4260 gaaagaaaat gaagatcaat agcttattca tctctttttc tttttcgttg gtgtaaagcc 4320 aacaccctgt ctaaaaaaca taaatttctt taatcatttt gcctcttttc tctgtgcttc 4380 aattaataaa aaatggaaag aacctcgag 4409 <210> SEQ ID NO 250 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with splice junctions removed; 12.75% U content <400> SEQUENCE: 250 atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg atgggcagtc 60 atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa cacagacaga 120 cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga aacagcagaa 180 gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa cagaatctgc 240 tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt cttccaccgg 300 ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc actggcacac 480 atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga caacagcgac 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact gttcggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc ggcattcctg 1620 agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag aaaggtcaca 1680 gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag cgtcgaaatc 1740 agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac aggatgggga 1980 agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa gacaatcctg 2040 gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg agacagcctg 2160 cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga aaacatcgtc 2280 atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca aaacggaaga 2460 gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagatgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt cgacaacctg 2700 acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat caagagacag 2760 ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac actgaagagc 2880 aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga aatcaacaac 2940 taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact gatcaagaag 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa gacagaagtc 3300 cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga caagctgatc 3360 gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc gacagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa cccgatcgac 3540 ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc aggagaactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag caagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac aagcacaaag 4020 gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag aaaggtctag 4140 <210> SEQ ID NO 251 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 250, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 251 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc caccggctgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcaaaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 252 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with minimal uridine codons frequently used in humans in general; 12.75% U content <400> SEQUENCE: 252 atggacaaga agtacagcat cggcctggac atcggcacca acagcgtggg ctgggccgtg 60 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgacaga 120 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag 180 gccaccagac tgaagagaac cgccagaaga agatacacca gaagaaagaa cagaatctgc 240 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 300 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agagacaccc catcttcggc 360 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 420 aagctggtgg acagcaccga caaggccgac ctgagactga tctacctggc cctggcccac 480 atgatcaagt tcagaggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg ccagactgag caagagcaga 660 agactggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc 900 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgccagc 960 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgaga 1020 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc 1080 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg 1140 gagaagatgg acggcaccga ggagctgctg gtgaagctga acagagagga cctgctgaga 1200 aagcagagaa ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac 1260 gccatcctga gaagacagga ggacttctac cccttcctga aggacaacag agagaagatc 1320 gagaagatcc tgaccttcag aatcccctac tacgtgggcc ccctggccag aggcaacagc 1380 agattcgcct ggatgaccag aaagagcgag gagaccatca ccccctggaa cttcgaggag 1440 gtggtggaca agggcgccag cgcccagagc ttcatcgaga gaatgaccaa cttcgacaag 1500 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgagaaagcc cgccttcctg 1620 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaacag aaaggtgacc 1680 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc 1740 agcggcgtgg aggacagatt caacgccagc ctgggcacct accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860 ctgaccctga ccctgttcga ggacagagag atgatcgagg agagactgaa gacctacgcc 1920 cacctgttcg acgacaaggt gatgaagcag ctgaagagaa gaagatacac cggctggggc 1980 agactgagca gaaagctgat caacggcatc agagacaagc agagcggcaa gaccatcctg 2040 gacttcctga agagcgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg 2160 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc 2220 gtgaaggtgg tggacgagct ggtgaaggtg atgggcagac acaagcccga gaacatcgtg 2280 atcgagatgg ccagagagaa ccagaccacc cagaagggcc agaagaacag cagagagaga 2340 atgaagagaa tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc 2400 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggcaga 2460 gacatgtacg tggaccagga gctggacatc aacagactga gcgactacga cgtggaccac 2520 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gaccagaagc 2580 gacaagaaca gaggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag 2640 aactactgga gacagctgct gaacgccaag ctgatcaccc agagaaagtt cgacaacctg 2700 accaaggccg agagaggcgg cctgagcgag ctggacaagg ccggcttcat caagagacag 2760 ctggtggaga ccagacagat caccaagcac gtggcccaga tcctggacag cagaatgaac 2820 accaagtacg acgagaacga caagctgatc agagaggtga aggtgatcac cctgaagagc 2880 aagctggtga gcgacttcag aaaggacttc cagttctaca aggtgagaga gatcaacaac 2940 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgagaaag 3060 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc 3120 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat cagaaagaga 3180 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg cagagacttc 3240 gccaccgtga gaaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300 cagaccggcg gcttcagcaa ggagagcatc ctgcccaaga gaaacagcga caagctgatc 3360 gccagaaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc 3420 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg 3480 aaggagctgc tgggcatcac catcatggag agaagcagct tcgagaagaa ccccatcgac 3540 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600 tacagcctgt tcgagctgga gaacggcaga aagagaatgc tggccagcgc cggcgagctg 3660 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc 3720 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag 3780 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagagagtg 3840 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca cagagacaag 3900 cccatcagag agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960 cccgccgcct tcaagtactt cgacaccacc atcgacagaa agagatacac cagcaccaag 4020 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gaccagaatc 4080 gacctgagcc agctgggcgg cgacggcggc ggcagcccca agaagaagag aaaggtgtga 4140 <210> SEQ ID NO 253 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 252, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 253 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaacag 120 cgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaagt tcaaggtgct 180 gggcaacacc gacagacaca gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag 240 cggcgagacc gccgaggcca ccagactgaa gagaaccgcc agaagaagat acaccagaag 300 aaagaacaga atctgctacc tgcaggagat cttcagcaac gagatggcca aggtggacga 360 cagcttcttc cacagactgg aggagagctt cctggtggag gaggacaaga agcacgagag 420 acaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 480 ctaccacctg agaaagaagc tggtggacag caccgacaag gccgacctga gactgatcta 540 cctggccctg gcccacatga tcaagttcag aggccacttc ctgatcgagg gcgacctgaa 600 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 660 gttcgaggag aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgagcgccag 720 actgagcaag agcagaagac tggagaacct gatcgcccag ctgcccggcg agaagaagaa 780 cggcctgttc ggcaacctga tcgccctgag cctgggcctg acccccaact tcaagagcaa 840 cttcgacctg gccgaggacg ccaagctgca gctgagcaag gacacctacg acgacgacct 900 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgttcctgg ccgccaagaa 960 cctgagcgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 1020 ccccctgagc gccagcatga tcaagagata cgacgagcac caccaggacc tgaccctgct 1080 gaaggccctg gtgagacagc agctgcccga gaagtacaag gagatcttct tcgaccagag 1140 caagaacggc tacgccggct acatcgacgg cggcgccagc caggaggagt tctacaagtt 1200 catcaagccc atcctggaga agatggacgg caccgaggag ctgctggtga agctgaacag 1260 agaggacctg ctgagaaagc agagaacctt cgacaacggc agcatccccc accagatcca 1320 cctgggcgag ctgcacgcca tcctgagaag acaggaggac ttctacccct tcctgaagga 1380 caacagagag aagatcgaga agatcctgac cttcagaatc ccctactacg tgggccccct 1440 ggccagaggc aacagcagat tcgcctggat gaccagaaag agcgaggaga ccatcacccc 1500 ctggaacttc gaggaggtgg tggacaaggg cgccagcgcc cagagcttca tcgagagaat 1560 gaccaacttc gacaagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 1620 cgagtacttc accgtgtaca acgagctgac caaggtgaag tacgtgaccg agggcatgag 1680 aaagcccgcc ttcctgagcg gcgagcagaa gaaggccatc gtggacctgc tgttcaagac 1740 caacagaaag gtgaccgtga agcagctgaa ggaggactac ttcaagaaga tcgagtgctt 1800 cgacagcgtg gagatcagcg gcgtggagga cagattcaac gccagcctgg gcacctacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaggaga acgaggacat 1920 cctggaggac atcgtgctga ccctgaccct gttcgaggac agagagatga tcgaggagag 1980 actgaagacc tacgcccacc tgttcgacga caaggtgatg aagcagctga agagaagaag 2040 atacaccggc tggggcagac tgagcagaaa gctgatcaac ggcatcagag acaagcagag 2100 cggcaagacc atcctggact tcctgaagag cgacggcttc gccaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgaccttcaa ggaggacatc cagaaggccc aggtgagcgg 2220 ccagggcgac agcctgcacg agcacatcgc caacctggcc ggcagccccg ccatcaagaa 2280 gggcatcctg cagaccgtga aggtggtgga cgagctggtg aaggtgatgg gcagacacaa 2340 gcccgagaac atcgtgatcg agatggccag agagaaccag accacccaga agggccagaa 2400 gaacagcaga gagagaatga agagaatcga ggagggcatc aaggagctgg gcagccagat 2460 cctgaaggag caccccgtgg agaacaccca gctgcagaac gagaagctgt acctgtacta 2520 cctgcagaac ggcagagaca tgtacgtgga ccaggagctg gacatcaaca gactgagcga 2580 ctacgacgtg gaccacatcg tgccccagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtgctgacc agaagcgaca agaacagagg caagagcgac aacgtgccca gcgaggaggt 2700 ggtgaagaag atgaagaact actggagaca gctgctgaac gccaagctga tcacccagag 2760 aaagttcgac aacctgacca aggccgagag aggcggcctg agcgagctgg acaaggccgg 2820 cttcatcaag agacagctgg tggagaccag acagatcacc aagcacgtgg cccagatcct 2880 ggacagcaga atgaacacca agtacgacga gaacgacaag ctgatcagag aggtgaaggt 2940 gatcaccctg aagagcaagc tggtgagcga cttcagaaag gacttccagt tctacaaggt 3000 gagagagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tggtgggcac 3060 cgccctgatc aagaagtacc ccaagctgga gagcgagttc gtgtacggcg actacaaggt 3120 gtacgacgtg agaaagatga tcgccaagag cgagcaggag atcggcaagg ccaccgccaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagacc gagatcaccc tggccaacgg 3240 cgagatcaga aagagacccc tgatcgagac caacggcgag accggcgaga tcgtgtggga 3300 caagggcaga gacttcgcca ccgtgagaaa ggtgctgagc atgccccagg tgaacatcgt 3360 gaagaagacc gaggtgcaga ccggcggctt cagcaaggag agcatcctgc ccaagagaaa 3420 cagcgacaag ctgatcgcca gaaagaagga ctgggacccc aagaagtacg gcggcttcga 3480 cagccccacc gtggcctaca gcgtgctggt ggtggccaag gtggagaagg gcaagagcaa 3540 gaagctgaag agcgtgaagg agctgctggg catcaccatc atggagagaa gcagcttcga 3600 gaagaacccc atcgacttcc tggaggccaa gggctacaag gaggtgaaga aggacctgat 3660 catcaagctg cccaagtaca gcctgttcga gctggagaac ggcagaaaga gaatgctggc 3720 cagcgccggc gagctgcaga agggcaacga gctggccctg cccagcaagt acgtgaactt 3780 cctgtacctg gccagccact acgagaagct gaagggcagc cccgaggaca acgagcagaa 3840 gcagctgttc gtggagcagc acaagcacta cctggacgag atcatcgagc agatcagcga 3900 gttcagcaag agagtgatcc tggccgacgc caacctggac aaggtgctga gcgcctacaa 3960 caagcacaga gacaagccca tcagagagca ggccgagaac atcatccacc tgttcaccct 4020 gaccaacctg ggcgcccccg ccgccttcaa gtacttcgac accaccatcg acagaaagag 4080 atacaccagc accaaggagg tgctggacgc caccctgatc caccagagca tcaccggcct 4140 gtacgagacc agaatcgacc tgagccagct gggcggcgac ggcggcggca gccccaagaa 4200 gaagagaaag gtgtgactag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 254 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with minimal uridine codons infrequently used in humans in general; 12.75% U content <400> SEQUENCE: 254 atggacaaaa aatacagcat agggctagac atagggacga acagcgtagg gtgggcggta 60 ataacggacg aatacaaagt accgagcaaa aaattcaaag tactagggaa cacggaccga 120 cacagcataa aaaaaaacct aataggggcg ctactattcg acagcgggga aacggcggaa 180 gcgacgcgac taaaacgaac ggcgcgacga cgatacacgc gacgaaaaaa ccgaatatgc 240 tacctacaag aaatattcag caacgaaatg gcgaaagtag acgacagctt cttccaccga 300 ctagaagaaa gcttcctagt agaagaagac aaaaaacacg aacgacaccc gatattcggg 360 aacatagtag acgaagtagc gtaccacgaa aaatacccga cgatatacca cctacgaaaa 420 aaactagtag acagcacgga caaagcggac ctacgactaa tatacctagc gctagcgcac 480 atgataaaat tccgagggca cttcctaata gaaggggacc taaacccgga caacagcgac 540 gtagacaaac tattcataca actagtacaa acgtacaacc aactattcga agaaaacccg 600 ataaacgcga gcggggtaga cgcgaaagcg atactaagcg cgcgactaag caaaagccga 660 cgactagaaa acctaatagc gcaactaccg ggggaaaaaa aaaacgggct attcgggaac 720 ctaatagcgc taagcctagg gctaacgccg aacttcaaaa gcaacttcga cctagcggaa 780 gacgcgaaac tacaactaag caaagacacg tacgacgacg acctagacaa cctactagcg 840 caaatagggg accaatacgc ggacctattc ctagcggcga aaaacctaag cgacgcgata 900 ctactaagcg acatactacg agtaaacacg gaaataacga aagcgccgct aagcgcgagc 960 atgataaaac gatacgacga acaccaccaa gacctaacgc tactaaaagc gctagtacga 1020 caacaactac cggaaaaata caaagaaata ttcttcgacc aaagcaaaaa cgggtacgcg 1080 gggtacatag acgggggggc gagccaagaa gaattctaca aattcataaa accgatacta 1140 gaaaaaatgg acgggacgga agaactacta gtaaaactaa accgagaaga cctactacga 1200 aaacaacgaa cgttcgacaa cgggagcata ccgcaccaaa tacacctagg ggaactacac 1260 gcgatactac gacgacaaga agacttctac ccgttcctaa aagacaaccg agaaaaaata 1320 gaaaaaatac taacgttccg aataccgtac tacgtagggc cgctagcgcg agggaacagc 1380 cgattcgcgt ggatgacgcg aaaaagcgaa gaaacgataa cgccgtggaa cttcgaagaa 1440 gtagtagaca aaggggcgag cgcgcaaagc ttcatagaac gaatgacgaa cttcgacaaa 1500 aacctaccga acgaaaaagt actaccgaaa cacagcctac tatacgaata cttcacggta 1560 tacaacgaac taacgaaagt aaaatacgta acggaaggga tgcgaaaacc ggcgttccta 1620 agcggggaac aaaaaaaagc gatagtagac ctactattca aaacgaaccg aaaagtaacg 1680 gtaaaacaac taaaagaaga ctacttcaaa aaaatagaat gcttcgacag cgtagaaata 1740 agcggggtag aagaccgatt caacgcgagc ctagggacgt accacgacct actaaaaata 1800 ataaaagaca aagacttcct agacaacgaa gaaaacgaag acatactaga agacatagta 1860 ctaacgctaa cgctattcga agaccgagaa atgatagaag aacgactaaa aacgtacgcg 1920 cacctattcg acgacaaagt aatgaaacaa ctaaaacgac gacgatacac ggggtggggg 1980 cgactaagcc gaaaactaat aaacgggata cgagacaaac aaagcgggaa aacgatacta 2040 gacttcctaa aaagcgacgg gttcgcgaac cgaaacttca tgcaactaat acacgacgac 2100 agcctaacgt tcaaagaaga catacaaaaa gcgcaagtaa gcgggcaagg ggacagccta 2160 cacgaacaca tagcgaacct agcggggagc ccggcgataa aaaaagggat actacaaacg 2220 gtaaaagtag tagacgaact agtaaaagta atggggcgac acaaaccgga aaacatagta 2280 atagaaatgg cgcgagaaaa ccaaacgacg caaaaagggc aaaaaaacag ccgagaacga 2340 atgaaacgaa tagaagaagg gataaaagaa ctagggagcc aaatactaaa agaacacccg 2400 gtagaaaaca cgcaactaca aaacgaaaaa ctatacctat actacctaca aaacgggcga 2460 gacatgtacg tagaccaaga actagacata aaccgactaa gcgactacga cgtagaccac 2520 atagtaccgc aaagcttcct aaaagacgac agcatagaca acaaagtact aacgcgaagc 2580 gacaaaaacc gagggaaaag cgacaacgta ccgagcgaag aagtagtaaa aaaaatgaaa 2640 aactactggc gacaactact aaacgcgaaa ctaataacgc aacgaaaatt cgacaaccta 2700 acgaaagcgg aacgaggggg gctaagcgaa ctagacaaag cggggttcat aaaacgacaa 2760 ctagtagaaa cgcgacaaat aacgaaacac gtagcgcaaa tactagacag ccgaatgaac 2820 acgaaatacg acgaaaacga caaactaata cgagaagtaa aagtaataac gctaaaaagc 2880 aaactagtaa gcgacttccg aaaagacttc caattctaca aagtacgaga aataaacaac 2940 taccaccacg cgcacgacgc gtacctaaac gcggtagtag ggacggcgct aataaaaaaa 3000 tacccgaaac tagaaagcga attcgtatac ggggactaca aagtatacga cgtacgaaaa 3060 atgatagcga aaagcgaaca agaaataggg aaagcgacgg cgaaatactt cttctacagc 3120 aacataatga acttcttcaa aacggaaata acgctagcga acggggaaat acgaaaacga 3180 ccgctaatag aaacgaacgg ggaaacgggg gaaatagtat gggacaaagg gcgagacttc 3240 gcgacggtac gaaaagtact aagcatgccg caagtaaaca tagtaaaaaa aacggaagta 3300 caaacggggg ggttcagcaa agaaagcata ctaccgaaac gaaacagcga caaactaata 3360 gcgcgaaaaa aagactggga cccgaaaaaa tacggggggt tcgacagccc gacggtagcg 3420 tacagcgtac tagtagtagc gaaagtagaa aaagggaaaa gcaaaaaact aaaaagcgta 3480 aaagaactac tagggataac gataatggaa cgaagcagct tcgaaaaaaa cccgatagac 3540 ttcctagaag cgaaagggta caaagaagta aaaaaagacc taataataaa actaccgaaa 3600 tacagcctat tcgaactaga aaacgggcga aaacgaatgc tagcgagcgc gggggaacta 3660 caaaaaggga acgaactagc gctaccgagc aaatacgtaa acttcctata cctagcgagc 3720 cactacgaaa aactaaaagg gagcccggaa gacaacgaac aaaaacaact attcgtagaa 3780 caacacaaac actacctaga cgaaataata gaacaaataa gcgaattcag caaacgagta 3840 atactagcgg acgcgaacct agacaaagta ctaagcgcgt acaacaaaca ccgagacaaa 3900 ccgatacgag aacaagcgga aaacataata cacctattca cgctaacgaa cctaggggcg 3960 ccggcggcgt tcaaatactt cgacacgacg atagaccgaa aacgatacac gagcacgaaa 4020 gaagtactag acgcgacgct aatacaccaa agcataacgg ggctatacga aacgcgaata 4080 gacctaagcc aactaggggg ggacgggggg gggagcccga aaaaaaaacg aaaagtatga 4140 <210> SEQ ID NO 255 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 254, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 255 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaaaaaata cagcataggg ctagacatag ggacgaacag 120 cgtagggtgg gcggtaataa cggacgaata caaagtaccg agcaaaaaat tcaaagtact 180 agggaacacg gaccgacaca gcataaaaaa aaacctaata ggggcgctac tattcgacag 240 cggggaaacg gcggaagcga cgcgactaaa acgaacggcg cgacgacgat acacgcgacg 300 aaaaaaccga atatgctacc tacaagaaat attcagcaac gaaatggcga aagtagacga 360 cagcttcttc caccgactag aagaaagctt cctagtagaa gaagacaaaa aacacgaacg 420 acacccgata ttcgggaaca tagtagacga agtagcgtac cacgaaaaat acccgacgat 480 ataccaccta cgaaaaaaac tagtagacag cacggacaaa gcggacctac gactaatata 540 cctagcgcta gcgcacatga taaaattccg agggcacttc ctaatagaag gggacctaaa 600 cccggacaac agcgacgtag acaaactatt catacaacta gtacaaacgt acaaccaact 660 attcgaagaa aacccgataa acgcgagcgg ggtagacgcg aaagcgatac taagcgcgcg 720 actaagcaaa agccgacgac tagaaaacct aatagcgcaa ctaccggggg aaaaaaaaaa 780 cgggctattc gggaacctaa tagcgctaag cctagggcta acgccgaact tcaaaagcaa 840 cttcgaccta gcggaagacg cgaaactaca actaagcaaa gacacgtacg acgacgacct 900 agacaaccta ctagcgcaaa taggggacca atacgcggac ctattcctag cggcgaaaaa 960 cctaagcgac gcgatactac taagcgacat actacgagta aacacggaaa taacgaaagc 1020 gccgctaagc gcgagcatga taaaacgata cgacgaacac caccaagacc taacgctact 1080 aaaagcgcta gtacgacaac aactaccgga aaaatacaaa gaaatattct tcgaccaaag 1140 caaaaacggg tacgcggggt acatagacgg gggggcgagc caagaagaat tctacaaatt 1200 cataaaaccg atactagaaa aaatggacgg gacggaagaa ctactagtaa aactaaaccg 1260 agaagaccta ctacgaaaac aacgaacgtt cgacaacggg agcataccgc accaaataca 1320 cctaggggaa ctacacgcga tactacgacg acaagaagac ttctacccgt tcctaaaaga 1380 caaccgagaa aaaatagaaa aaatactaac gttccgaata ccgtactacg tagggccgct 1440 agcgcgaggg aacagccgat tcgcgtggat gacgcgaaaa agcgaagaaa cgataacgcc 1500 gtggaacttc gaagaagtag tagacaaagg ggcgagcgcg caaagcttca tagaacgaat 1560 gacgaacttc gacaaaaacc taccgaacga aaaagtacta ccgaaacaca gcctactata 1620 cgaatacttc acggtataca acgaactaac gaaagtaaaa tacgtaacgg aagggatgcg 1680 aaaaccggcg ttcctaagcg gggaacaaaa aaaagcgata gtagacctac tattcaaaac 1740 gaaccgaaaa gtaacggtaa aacaactaaa agaagactac ttcaaaaaaa tagaatgctt 1800 cgacagcgta gaaataagcg gggtagaaga ccgattcaac gcgagcctag ggacgtacca 1860 cgacctacta aaaataataa aagacaaaga cttcctagac aacgaagaaa acgaagacat 1920 actagaagac atagtactaa cgctaacgct attcgaagac cgagaaatga tagaagaacg 1980 actaaaaacg tacgcgcacc tattcgacga caaagtaatg aaacaactaa aacgacgacg 2040 atacacgggg tgggggcgac taagccgaaa actaataaac gggatacgag acaaacaaag 2100 cgggaaaacg atactagact tcctaaaaag cgacgggttc gcgaaccgaa acttcatgca 2160 actaatacac gacgacagcc taacgttcaa agaagacata caaaaagcgc aagtaagcgg 2220 gcaaggggac agcctacacg aacacatagc gaacctagcg gggagcccgg cgataaaaaa 2280 agggatacta caaacggtaa aagtagtaga cgaactagta aaagtaatgg ggcgacacaa 2340 accggaaaac atagtaatag aaatggcgcg agaaaaccaa acgacgcaaa aagggcaaaa 2400 aaacagccga gaacgaatga aacgaataga agaagggata aaagaactag ggagccaaat 2460 actaaaagaa cacccggtag aaaacacgca actacaaaac gaaaaactat acctatacta 2520 cctacaaaac gggcgagaca tgtacgtaga ccaagaacta gacataaacc gactaagcga 2580 ctacgacgta gaccacatag taccgcaaag cttcctaaaa gacgacagca tagacaacaa 2640 agtactaacg cgaagcgaca aaaaccgagg gaaaagcgac aacgtaccga gcgaagaagt 2700 agtaaaaaaa atgaaaaact actggcgaca actactaaac gcgaaactaa taacgcaacg 2760 aaaattcgac aacctaacga aagcggaacg aggggggcta agcgaactag acaaagcggg 2820 gttcataaaa cgacaactag tagaaacgcg acaaataacg aaacacgtag cgcaaatact 2880 agacagccga atgaacacga aatacgacga aaacgacaaa ctaatacgag aagtaaaagt 2940 aataacgcta aaaagcaaac tagtaagcga cttccgaaaa gacttccaat tctacaaagt 3000 acgagaaata aacaactacc accacgcgca cgacgcgtac ctaaacgcgg tagtagggac 3060 ggcgctaata aaaaaatacc cgaaactaga aagcgaattc gtatacgggg actacaaagt 3120 atacgacgta cgaaaaatga tagcgaaaag cgaacaagaa atagggaaag cgacggcgaa 3180 atacttcttc tacagcaaca taatgaactt cttcaaaacg gaaataacgc tagcgaacgg 3240 ggaaatacga aaacgaccgc taatagaaac gaacggggaa acgggggaaa tagtatggga 3300 caaagggcga gacttcgcga cggtacgaaa agtactaagc atgccgcaag taaacatagt 3360 aaaaaaaacg gaagtacaaa cgggggggtt cagcaaagaa agcatactac cgaaacgaaa 3420 cagcgacaaa ctaatagcgc gaaaaaaaga ctgggacccg aaaaaatacg gggggttcga 3480 cagcccgacg gtagcgtaca gcgtactagt agtagcgaaa gtagaaaaag ggaaaagcaa 3540 aaaactaaaa agcgtaaaag aactactagg gataacgata atggaacgaa gcagcttcga 3600 aaaaaacccg atagacttcc tagaagcgaa agggtacaaa gaagtaaaaa aagacctaat 3660 aataaaacta ccgaaataca gcctattcga actagaaaac gggcgaaaac gaatgctagc 3720 gagcgcgggg gaactacaaa aagggaacga actagcgcta ccgagcaaat acgtaaactt 3780 cctataccta gcgagccact acgaaaaact aaaagggagc ccggaagaca acgaacaaaa 3840 acaactattc gtagaacaac acaaacacta cctagacgaa ataatagaac aaataagcga 3900 attcagcaaa cgagtaatac tagcggacgc gaacctagac aaagtactaa gcgcgtacaa 3960 caaacaccga gacaaaccga tacgagaaca agcggaaaac ataatacacc tattcacgct 4020 aacgaaccta ggggcgccgg cggcgttcaa atacttcgac acgacgatag accgaaaacg 4080 atacacgagc acgaaagaag tactagacgc gacgctaata caccaaagca taacggggct 4140 atacgaaacg cgaatagacc taagccaact agggggggac ggggggggga gcccgaaaaa 4200 aaaacgaaaa gtatgactag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 256 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 256 aggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 257 <211> LENGTH: 4481 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from CMV, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 257 gggcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac 60 cgatccagcc tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt 120 gactcaccgt ccttgacacg gccaccatgg acaagaagta cagcatcgga ctggacatcg 180 gaacaaacag cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt 240 tcaaggtcct gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc 300 tgttcgacag cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat 360 acacaagaag aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa 420 aggtcgacga cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga 480 agcacgaaag acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt 540 acccgacaat ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga 600 gactgatcta cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag 660 gagacctgaa cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat 720 acaaccagct gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc 780 tgagcgcaag actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag 840 aaaagaagaa cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact 900 tcaagagcaa cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg 960 acgacgacct ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg 1020 cagcaaagaa cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa 1080 tcacaaaggc accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc 1140 tgacactgct gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct 1200 tcgaccagag caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat 1260 tctacaagtt catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca 1320 agctgaacag agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc 1380 accagatcca cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt 1440 tcctgaagga caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg 1500 tcggaccgct ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa 1560 caatcacacc gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca 1620 tcgaaagaat gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca 1680 gcctgctgta cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag 1740 aaggaatgag aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc 1800 tgttcaagac aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga 1860 tcgaatgctt cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg 1920 gaacatacca cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa 1980 acgaagacat cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga 2040 tcgaagaaag actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga 2100 agagaagaag atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag 2160 acaagcagag cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa 2220 acttcatgca gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac 2280 aggtcagcgg acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg 2340 caatcaagaa gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg 2400 gaagacacaa gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga 2460 agggacagaa gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg 2520 gaagccagat cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt 2580 acctgtacta cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca 2640 gactgagcga ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca 2700 tcgacaacaa ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga 2760 gcgaagaagt cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga 2820 tcacacagag aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg 2880 acaaggcagg attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg 2940 cacagatcct ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag 3000 aagtcaaggt catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt 3060 tctacaaggt cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag 3120 tcgtcggaac agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag 3180 actacaaggt ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg 3240 caacagcaaa gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac 3300 tggcaaacgg agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa 3360 tcgtctggga caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg 3420 tcaacatcgt caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc 3480 cgaagagaaa cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg 3540 gaggattcga cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg 3600 gaaagagcaa gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa 3660 gcagcttcga aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga 3720 aggacctgat catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga 3780 gaatgctggc aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt 3840 acgtcaactt cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca 3900 acgaacagaa gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac 3960 agatcagcga attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga 4020 gcgcatacaa caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc 4080 tgttcacact gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg 4140 acagaaagag atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca 4200 tcacaggact gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa 4260 gcccgaagaa gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc 4320 atgagaataa gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt 4380 tggtgtaaag ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt 4440 tctctgtgct tcaattaata aaaaatggaa agaacctcga g 4481 <210> SEQ ID NO 258 <211> LENGTH: 4348 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from HBB, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of HBB <400> SEQUENCE: 258 gggacatttg cttctgacac aactgtgttc actagcaacc tcaaacagac accggatctg 60 ccaccatgga caagaagtac agcatcggac tggacatcgg aacaaacagc gtcggatggg 120 cagtcatcac agacgaatac aaggtcccga gcaagaagtt caaggtcctg ggaaacacag 180 acagacacag catcaagaag aacctgatcg gagcactgct gttcgacagc ggagaaacag 240 cagaagcaac aagactgaag agaacagcaa gaagaagata cacaagaaga aagaacagaa 300 tctgctacct gcaggaaatc ttcagcaacg aaatggcaaa ggtcgacgac agcttcttcc 360 acagactgga agaaagcttc ctggtcgaag aagacaagaa gcacgaaaga cacccgatct 420 tcggaaacat cgtcgacgaa gtcgcatacc acgaaaagta cccgacaatc taccacctga 480 gaaagaagct ggtcgacagc acagacaagg cagacctgag actgatctac ctggcactgg 540 cacacatgat caagttcaga ggacacttcc tgatcgaagg agacctgaac ccggacaaca 600 gcgacgtcga caagctgttc atccagctgg tccagacata caaccagctg ttcgaagaaa 660 acccgatcaa cgcaagcgga gtcgacgcaa aggcaatcct gagcgcaaga ctgagcaaga 720 gcagaagact ggaaaacctg atcgcacagc tgccgggaga aaagaagaac ggactgttcg 780 gaaacctgat cgcactgagc ctgggactga caccgaactt caagagcaac ttcgacctgg 840 cagaagacgc aaagctgcag ctgagcaagg acacatacga cgacgacctg gacaacctgc 900 tggcacagat cggagaccag tacgcagacc tgttcctggc agcaaagaac ctgagcgacg 960 caatcctgct gagcgacatc ctgagagtca acacagaaat cacaaaggca ccgctgagcg 1020 caagcatgat caagagatac gacgaacacc accaggacct gacactgctg aaggcactgg 1080 tcagacagca gctgccggaa aagtacaagg aaatcttctt cgaccagagc aagaacggat 1140 acgcaggata catcgacgga ggagcaagcc aggaagaatt ctacaagttc atcaagccga 1200 tcctggaaaa gatggacgga acagaagaac tgctggtcaa gctgaacaga gaagacctgc 1260 tgagaaagca gagaacattc gacaacggaa gcatcccgca ccagatccac ctgggagaac 1320 tgcacgcaat cctgagaaga caggaagact tctacccgtt cctgaaggac aacagagaaa 1380 agatcgaaaa gatcctgaca ttcagaatcc cgtactacgt cggaccgctg gcaagaggaa 1440 acagcagatt cgcatggatg acaagaaaga gcgaagaaac aatcacaccg tggaacttcg 1500 aagaagtcgt cgacaaggga gcaagcgcac agagcttcat cgaaagaatg acaaacttcg 1560 acaagaacct gccgaacgaa aaggtcctgc cgaagcacag cctgctgtac gaatacttca 1620 cagtctacaa cgaactgaca aaggtcaagt acgtcacaga aggaatgaga aagccggcat 1680 tcctgagcgg agaacagaag aaggcaatcg tcgacctgct gttcaagaca aacagaaagg 1740 tcacagtcaa gcagctgaag gaagactact tcaagaagat cgaatgcttc gacagcgtcg 1800 aaatcagcgg agtcgaagac agattcaacg caagcctggg aacataccac gacctgctga 1860 agatcatcaa ggacaaggac ttcctggaca acgaagaaaa cgaagacatc ctggaagaca 1920 tcgtcctgac actgacactg ttcgaagaca gagaaatgat cgaagaaaga ctgaagacat 1980 acgcacacct gttcgacgac aaggtcatga agcagctgaa gagaagaaga tacacaggat 2040 ggggaagact gagcagaaag ctgatcaacg gaatcagaga caagcagagc ggaaagacaa 2100 tcctggactt cctgaagagc gacggattcg caaacagaaa cttcatgcag ctgatccacg 2160 acgacagcct gacattcaag gaagacatcc agaaggcaca ggtcagcgga cagggagaca 2220 gcctgcacga acacatcgca aacctggcag gaagcccggc aatcaagaag ggaatcctgc 2280 agacagtcaa ggtcgtcgac gaactggtca aggtcatggg aagacacaag ccggaaaaca 2340 tcgtcatcga aatggcaaga gaaaaccaga caacacagaa gggacagaag aacagcagag 2400 aaagaatgaa gagaatcgaa gaaggaatca aggaactggg aagccagatc ctgaaggaac 2460 acccggtcga aaacacacag ctgcagaacg aaaagctgta cctgtactac ctgcagaacg 2520 gaagagacat gtacgtcgac caggaactgg acatcaacag actgagcgac tacgacgtcg 2580 accacatcgt cccgcagagc ttcctgaagg acgacagcat cgacaacaag gtcctgacaa 2640 gaagcgacaa gaacagagga aagagcgaca acgtcccgag cgaagaagtc gtcaagaaga 2700 tgaagaacta ctggagacag ctgctgaacg caaagctgat cacacagaga aagttcgaca 2760 acctgacaaa ggcagagaga ggaggactga gcgaactgga caaggcagga ttcatcaaga 2820 gacagctggt cgaaacaaga cagatcacaa agcacgtcgc acagatcctg gacagcagaa 2880 tgaacacaaa gtacgacgaa aacgacaagc tgatcagaga agtcaaggtc atcacactga 2940 agagcaagct ggtcagcgac ttcagaaagg acttccagtt ctacaaggtc agagaaatca 3000 acaactacca ccacgcacac gacgcatacc tgaacgcagt cgtcggaaca gcactgatca 3060 agaagtaccc gaagctggaa agcgaattcg tctacggaga ctacaaggtc tacgacgtca 3120 gaaagatgat cgcaaagagc gaacaggaaa tcggaaaggc aacagcaaag tacttcttct 3180 acagcaacat catgaacttc ttcaagacag aaatcacact ggcaaacgga gaaatcagaa 3240 agagaccgct gatcgaaaca aacggagaaa caggagaaat cgtctgggac aagggaagag 3300 acttcgcaac agtcagaaag gtcctgagca tgccgcaggt caacatcgtc aagaagacag 3360 aagtccagac aggaggattc agcaaggaaa gcatcctgcc gaagagaaac agcgacaagc 3420 tgatcgcaag aaagaaggac tgggacccga agaagtacgg aggattcgac agcccgacag 3480 tcgcatacag cgtcctggtc gtcgcaaagg tcgaaaaggg aaagagcaag aagctgaaga 3540 gcgtcaagga actgctggga atcacaatca tggaaagaag cagcttcgaa aagaacccga 3600 tcgacttcct ggaagcaaag ggatacaagg aagtcaagaa ggacctgatc atcaagctgc 3660 cgaagtacag cctgttcgaa ctggaaaacg gaagaaagag aatgctggca agcgcaggag 3720 aactgcagaa gggaaacgaa ctggcactgc cgagcaagta cgtcaacttc ctgtacctgg 3780 caagccacta cgaaaagctg aagggaagcc cggaagacaa cgaacagaag cagctgttcg 3840 tcgaacagca caagcactac ctggacgaaa tcatcgaaca gatcagcgaa ttcagcaaga 3900 gagtcatcct ggcagacgca aacctggaca aggtcctgag cgcatacaac aagcacagag 3960 acaagccgat cagagaacag gcagaaaaca tcatccacct gttcacactg acaaacctgg 4020 gagcaccggc agcattcaag tacttcgaca caacaatcga cagaaagaga tacacaagca 4080 caaaggaagt cctggacgca acactgatcc accagagcat cacaggactg tacgaaacaa 4140 gaatcgacct gagccagctg ggaggagacg gaggaggaag cccgaagaag aagagaaagg 4200 tctagctagc gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa 4260 gtccaactac taaactgggg gatattatga agggccttga gcatctggat tctgcctaat 4320 aaaaaacatt tattttcatt gcctcgag 4348 <210> SEQ ID NO 259 <211> LENGTH: 4325 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from XBG, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of XBG <400> SEQUENCE: 259 gggaagctca gaataaacgc tcaactttgg ccggatctgc caccatggac aagaagtaca 60 gcatcggact ggacatcgga acaaacagcg tcggatgggc agtcatcaca gacgaataca 120 aggtcccgag caagaagttc aaggtcctgg gaaacacaga cagacacagc atcaagaaga 180 acctgatcgg agcactgctg ttcgacagcg gagaaacagc agaagcaaca agactgaaga 240 gaacagcaag aagaagatac acaagaagaa agaacagaat ctgctacctg caggaaatct 300 tcagcaacga aatggcaaag gtcgacgaca gcttcttcca cagactggaa gaaagcttcc 360 tggtcgaaga agacaagaag cacgaaagac acccgatctt cggaaacatc gtcgacgaag 420 tcgcatacca cgaaaagtac ccgacaatct accacctgag aaagaagctg gtcgacagca 480 cagacaaggc agacctgaga ctgatctacc tggcactggc acacatgatc aagttcagag 540 gacacttcct gatcgaagga gacctgaacc cggacaacag cgacgtcgac aagctgttca 600 tccagctggt ccagacatac aaccagctgt tcgaagaaaa cccgatcaac gcaagcggag 660 tcgacgcaaa ggcaatcctg agcgcaagac tgagcaagag cagaagactg gaaaacctga 720 tcgcacagct gccgggagaa aagaagaacg gactgttcgg aaacctgatc gcactgagcc 780 tgggactgac accgaacttc aagagcaact tcgacctggc agaagacgca aagctgcagc 840 tgagcaagga cacatacgac gacgacctgg acaacctgct ggcacagatc ggagaccagt 900 acgcagacct gttcctggca gcaaagaacc tgagcgacgc aatcctgctg agcgacatcc 960 tgagagtcaa cacagaaatc acaaaggcac cgctgagcgc aagcatgatc aagagatacg 1020 acgaacacca ccaggacctg acactgctga aggcactggt cagacagcag ctgccggaaa 1080 agtacaagga aatcttcttc gaccagagca agaacggata cgcaggatac atcgacggag 1140 gagcaagcca ggaagaattc tacaagttca tcaagccgat cctggaaaag atggacggaa 1200 cagaagaact gctggtcaag ctgaacagag aagacctgct gagaaagcag agaacattcg 1260 acaacggaag catcccgcac cagatccacc tgggagaact gcacgcaatc ctgagaagac 1320 aggaagactt ctacccgttc ctgaaggaca acagagaaaa gatcgaaaag atcctgacat 1380 tcagaatccc gtactacgtc ggaccgctgg caagaggaaa cagcagattc gcatggatga 1440 caagaaagag cgaagaaaca atcacaccgt ggaacttcga agaagtcgtc gacaagggag 1500 caagcgcaca gagcttcatc gaaagaatga caaacttcga caagaacctg ccgaacgaaa 1560 aggtcctgcc gaagcacagc ctgctgtacg aatacttcac agtctacaac gaactgacaa 1620 aggtcaagta cgtcacagaa ggaatgagaa agccggcatt cctgagcgga gaacagaaga 1680 aggcaatcgt cgacctgctg ttcaagacaa acagaaaggt cacagtcaag cagctgaagg 1740 aagactactt caagaagatc gaatgcttcg acagcgtcga aatcagcgga gtcgaagaca 1800 gattcaacgc aagcctggga acataccacg acctgctgaa gatcatcaag gacaaggact 1860 tcctggacaa cgaagaaaac gaagacatcc tggaagacat cgtcctgaca ctgacactgt 1920 tcgaagacag agaaatgatc gaagaaagac tgaagacata cgcacacctg ttcgacgaca 1980 aggtcatgaa gcagctgaag agaagaagat acacaggatg gggaagactg agcagaaagc 2040 tgatcaacgg aatcagagac aagcagagcg gaaagacaat cctggacttc ctgaagagcg 2100 acggattcgc aaacagaaac ttcatgcagc tgatccacga cgacagcctg acattcaagg 2160 aagacatcca gaaggcacag gtcagcggac agggagacag cctgcacgaa cacatcgcaa 2220 acctggcagg aagcccggca atcaagaagg gaatcctgca gacagtcaag gtcgtcgacg 2280 aactggtcaa ggtcatggga agacacaagc cggaaaacat cgtcatcgaa atggcaagag 2340 aaaaccagac aacacagaag ggacagaaga acagcagaga aagaatgaag agaatcgaag 2400 aaggaatcaa ggaactggga agccagatcc tgaaggaaca cccggtcgaa aacacacagc 2460 tgcagaacga aaagctgtac ctgtactacc tgcagaacgg aagagacatg tacgtcgacc 2520 aggaactgga catcaacaga ctgagcgact acgacgtcga ccacatcgtc ccgcagagct 2580 tcctgaagga cgacagcatc gacaacaagg tcctgacaag aagcgacaag aacagaggaa 2640 agagcgacaa cgtcccgagc gaagaagtcg tcaagaagat gaagaactac tggagacagc 2700 tgctgaacgc aaagctgatc acacagagaa agttcgacaa cctgacaaag gcagagagag 2760 gaggactgag cgaactggac aaggcaggat tcatcaagag acagctggtc gaaacaagac 2820 agatcacaaa gcacgtcgca cagatcctgg acagcagaat gaacacaaag tacgacgaaa 2880 acgacaagct gatcagagaa gtcaaggtca tcacactgaa gagcaagctg gtcagcgact 2940 tcagaaagga cttccagttc tacaaggtca gagaaatcaa caactaccac cacgcacacg 3000 acgcatacct gaacgcagtc gtcggaacag cactgatcaa gaagtacccg aagctggaaa 3060 gcgaattcgt ctacggagac tacaaggtct acgacgtcag aaagatgatc gcaaagagcg 3120 aacaggaaat cggaaaggca acagcaaagt acttcttcta cagcaacatc atgaacttct 3180 tcaagacaga aatcacactg gcaaacggag aaatcagaaa gagaccgctg atcgaaacaa 3240 acggagaaac aggagaaatc gtctgggaca agggaagaga cttcgcaaca gtcagaaagg 3300 tcctgagcat gccgcaggtc aacatcgtca agaagacaga agtccagaca ggaggattca 3360 gcaaggaaag catcctgccg aagagaaaca gcgacaagct gatcgcaaga aagaaggact 3420 gggacccgaa gaagtacgga ggattcgaca gcccgacagt cgcatacagc gtcctggtcg 3480 tcgcaaaggt cgaaaaggga aagagcaaga agctgaagag cgtcaaggaa ctgctgggaa 3540 tcacaatcat ggaaagaagc agcttcgaaa agaacccgat cgacttcctg gaagcaaagg 3600 gatacaagga agtcaagaag gacctgatca tcaagctgcc gaagtacagc ctgttcgaac 3660 tggaaaacgg aagaaagaga atgctggcaa gcgcaggaga actgcagaag ggaaacgaac 3720 tggcactgcc gagcaagtac gtcaacttcc tgtacctggc aagccactac gaaaagctga 3780 agggaagccc ggaagacaac gaacagaagc agctgttcgt cgaacagcac aagcactacc 3840 tggacgaaat catcgaacag atcagcgaat tcagcaagag agtcatcctg gcagacgcaa 3900 acctggacaa ggtcctgagc gcatacaaca agcacagaga caagccgatc agagaacagg 3960 cagaaaacat catccacctg ttcacactga caaacctggg agcaccggca gcattcaagt 4020 acttcgacac aacaatcgac agaaagagat acacaagcac aaaggaagtc ctggacgcaa 4080 cactgatcca ccagagcatc acaggactgt acgaaacaag aatcgacctg agccagctgg 4140 gaggagacgg aggaggaagc ccgaagaaga agagaaaggt ctagctagca ccagcctcaa 4200 gaacacccga atggagtctc taagctacat aataccaact tacactttac aaaatgttgt 4260 cccccaaaat gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacattctc 4320 tcgag 4325 <210> SEQ ID NO 260 <211> LENGTH: 4325 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR from XBG, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of XBG <400> SEQUENCE: 260 aggaagctca gaataaacgc tcaactttgg ccggatctgc caccatggac aagaagtaca 60 gcatcggact ggacatcgga acaaacagcg tcggatgggc agtcatcaca gacgaataca 120 aggtcccgag caagaagttc aaggtcctgg gaaacacaga cagacacagc atcaagaaga 180 acctgatcgg agcactgctg ttcgacagcg gagaaacagc agaagcaaca agactgaaga 240 gaacagcaag aagaagatac acaagaagaa agaacagaat ctgctacctg caggaaatct 300 tcagcaacga aatggcaaag gtcgacgaca gcttcttcca cagactggaa gaaagcttcc 360 tggtcgaaga agacaagaag cacgaaagac acccgatctt cggaaacatc gtcgacgaag 420 tcgcatacca cgaaaagtac ccgacaatct accacctgag aaagaagctg gtcgacagca 480 cagacaaggc agacctgaga ctgatctacc tggcactggc acacatgatc aagttcagag 540 gacacttcct gatcgaagga gacctgaacc cggacaacag cgacgtcgac aagctgttca 600 tccagctggt ccagacatac aaccagctgt tcgaagaaaa cccgatcaac gcaagcggag 660 tcgacgcaaa ggcaatcctg agcgcaagac tgagcaagag cagaagactg gaaaacctga 720 tcgcacagct gccgggagaa aagaagaacg gactgttcgg aaacctgatc gcactgagcc 780 tgggactgac accgaacttc aagagcaact tcgacctggc agaagacgca aagctgcagc 840 tgagcaagga cacatacgac gacgacctgg acaacctgct ggcacagatc ggagaccagt 900 acgcagacct gttcctggca gcaaagaacc tgagcgacgc aatcctgctg agcgacatcc 960 tgagagtcaa cacagaaatc acaaaggcac cgctgagcgc aagcatgatc aagagatacg 1020 acgaacacca ccaggacctg acactgctga aggcactggt cagacagcag ctgccggaaa 1080 agtacaagga aatcttcttc gaccagagca agaacggata cgcaggatac atcgacggag 1140 gagcaagcca ggaagaattc tacaagttca tcaagccgat cctggaaaag atggacggaa 1200 cagaagaact gctggtcaag ctgaacagag aagacctgct gagaaagcag agaacattcg 1260 acaacggaag catcccgcac cagatccacc tgggagaact gcacgcaatc ctgagaagac 1320 aggaagactt ctacccgttc ctgaaggaca acagagaaaa gatcgaaaag atcctgacat 1380 tcagaatccc gtactacgtc ggaccgctgg caagaggaaa cagcagattc gcatggatga 1440 caagaaagag cgaagaaaca atcacaccgt ggaacttcga agaagtcgtc gacaagggag 1500 caagcgcaca gagcttcatc gaaagaatga caaacttcga caagaacctg ccgaacgaaa 1560 aggtcctgcc gaagcacagc ctgctgtacg aatacttcac agtctacaac gaactgacaa 1620 aggtcaagta cgtcacagaa ggaatgagaa agccggcatt cctgagcgga gaacagaaga 1680 aggcaatcgt cgacctgctg ttcaagacaa acagaaaggt cacagtcaag cagctgaagg 1740 aagactactt caagaagatc gaatgcttcg acagcgtcga aatcagcgga gtcgaagaca 1800 gattcaacgc aagcctggga acataccacg acctgctgaa gatcatcaag gacaaggact 1860 tcctggacaa cgaagaaaac gaagacatcc tggaagacat cgtcctgaca ctgacactgt 1920 tcgaagacag agaaatgatc gaagaaagac tgaagacata cgcacacctg ttcgacgaca 1980 aggtcatgaa gcagctgaag agaagaagat acacaggatg gggaagactg agcagaaagc 2040 tgatcaacgg aatcagagac aagcagagcg gaaagacaat cctggacttc ctgaagagcg 2100 acggattcgc aaacagaaac ttcatgcagc tgatccacga cgacagcctg acattcaagg 2160 aagacatcca gaaggcacag gtcagcggac agggagacag cctgcacgaa cacatcgcaa 2220 acctggcagg aagcccggca atcaagaagg gaatcctgca gacagtcaag gtcgtcgacg 2280 aactggtcaa ggtcatggga agacacaagc cggaaaacat cgtcatcgaa atggcaagag 2340 aaaaccagac aacacagaag ggacagaaga acagcagaga aagaatgaag agaatcgaag 2400 aaggaatcaa ggaactggga agccagatcc tgaaggaaca cccggtcgaa aacacacagc 2460 tgcagaacga aaagctgtac ctgtactacc tgcagaacgg aagagacatg tacgtcgacc 2520 aggaactgga catcaacaga ctgagcgact acgacgtcga ccacatcgtc ccgcagagct 2580 tcctgaagga cgacagcatc gacaacaagg tcctgacaag aagcgacaag aacagaggaa 2640 agagcgacaa cgtcccgagc gaagaagtcg tcaagaagat gaagaactac tggagacagc 2700 tgctgaacgc aaagctgatc acacagagaa agttcgacaa cctgacaaag gcagagagag 2760 gaggactgag cgaactggac aaggcaggat tcatcaagag acagctggtc gaaacaagac 2820 agatcacaaa gcacgtcgca cagatcctgg acagcagaat gaacacaaag tacgacgaaa 2880 acgacaagct gatcagagaa gtcaaggtca tcacactgaa gagcaagctg gtcagcgact 2940 tcagaaagga cttccagttc tacaaggtca gagaaatcaa caactaccac cacgcacacg 3000 acgcatacct gaacgcagtc gtcggaacag cactgatcaa gaagtacccg aagctggaaa 3060 gcgaattcgt ctacggagac tacaaggtct acgacgtcag aaagatgatc gcaaagagcg 3120 aacaggaaat cggaaaggca acagcaaagt acttcttcta cagcaacatc atgaacttct 3180 tcaagacaga aatcacactg gcaaacggag aaatcagaaa gagaccgctg atcgaaacaa 3240 acggagaaac aggagaaatc gtctgggaca agggaagaga cttcgcaaca gtcagaaagg 3300 tcctgagcat gccgcaggtc aacatcgtca agaagacaga agtccagaca ggaggattca 3360 gcaaggaaag catcctgccg aagagaaaca gcgacaagct gatcgcaaga aagaaggact 3420 gggacccgaa gaagtacgga ggattcgaca gcccgacagt cgcatacagc gtcctggtcg 3480 tcgcaaaggt cgaaaaggga aagagcaaga agctgaagag cgtcaaggaa ctgctgggaa 3540 tcacaatcat ggaaagaagc agcttcgaaa agaacccgat cgacttcctg gaagcaaagg 3600 gatacaagga agtcaagaag gacctgatca tcaagctgcc gaagtacagc ctgttcgaac 3660 tggaaaacgg aagaaagaga atgctggcaa gcgcaggaga actgcagaag ggaaacgaac 3720 tggcactgcc gagcaagtac gtcaacttcc tgtacctggc aagccactac gaaaagctga 3780 agggaagccc ggaagacaac gaacagaagc agctgttcgt cgaacagcac aagcactacc 3840 tggacgaaat catcgaacag atcagcgaat tcagcaagag agtcatcctg gcagacgcaa 3900 acctggacaa ggtcctgagc gcatacaaca agcacagaga caagccgatc agagaacagg 3960 cagaaaacat catccacctg ttcacactga caaacctggg agcaccggca gcattcaagt 4020 acttcgacac aacaatcgac agaaagagat acacaagcac aaaggaagtc ctggacgcaa 4080 cactgatcca ccagagcatc acaggactgt acgaaacaag aatcgacctg agccagctgg 4140 gaggagacgg aggaggaagc ccgaagaaga agagaaaggt ctagctagca ccagcctcaa 4200 gaacacccga atggagtctc taagctacat aataccaact tacactttac aaaatgttgt 4260 cccccaaaat gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacattctc 4320 tcgag 4325 <210> SEQ ID NO 261 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR from HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 261 aggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 262 <400> SEQUENCE: 262 000 <210> SEQ ID NO 263 <211> LENGTH: 93 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: poly-A 100 sequence <400> SEQUENCE: 263 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 93 <210> SEQ ID NO 264 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G209 single guide RNA targeting the mouse TTR gene <400> SEQUENCE: 264 aaataagaga gaaaagaaga gtaagaagaa atataagagc cacc 44 <210> SEQ ID NO 265 <211> LENGTH: 3312 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: ORF encoding Neisseria meningitidis Cas9 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 265 atggcagcat tcaagccgaa ctcgatcaac tacatcctgg gactggacat cggaatcgca 60 tcggtcggat gggcaatggt cgaaatcgac gaagaagaaa acccgatcag actgatcgac 120 ctgggagtca gagtcttcga aagagcagaa gtcccgaaga caggagactc gctggcaatg 180 gcaagaagac tggcaagatc ggtcagaaga ctgacaagaa gaagagcaca cagactgctg 240 agaacaagaa gactgctgaa gagagaagga gtcctgcagg cagcaaactt cgacgaaaac 300 ggactgatca agtcgctgcc gaacacaccg tggcagctga gagcagcagc actggacaga 360 aagctgacac cgctggaatg gtcggcagtc ctgctgcacc tgatcaagca cagaggatac 420 ctgtcgcaga gaaagaacga aggagaaaca gcagacaagg aactgggagc actgctgaag 480 ggagtcgcag gaaacgcaca cgcactgcag acaggagact tcagaacacc ggcagaactg 540 gcactgaaca agttcgaaaa ggaatcggga cacatcagaa accagagatc ggactactcg 600 cacacattct cgagaaagga cctgcaggca gaactgatcc tgctgttcga aaagcagaag 660 gaattcggaa acccgcacgt ctcgggagga ctgaaggaag gaatcgaaac actgctgatg 720 acacagagac cggcactgtc gggagacgca gtccagaaga tgctgggaca ctgcacattc 780 gaaccggcag aaccgaaggc agcaaagaac acatacacag cagaaagatt catctggctg 840 acaaagctga acaacctgag aatcctggaa cagggatcgg aaagaccgct gacagacaca 900 gaaagagcaa cactgatgga cgaaccgtac agaaagtcga agctgacata cgcacaggca 960 agaaagctgc tgggactgga agacacagca ttcttcaagg gactgagata cggaaaggac 1020 aacgcagaag catcgacact gatggaaatg aaggcatacc acgcaatctc gagagcactg 1080 gaaaaggaag gactgaagga caagaagtcg ccgctgaacc tgtcgccgga actgcaggac 1140 gaaatcggaa cagcattctc gctgttcaag acagacgaag acatcacagg aagactgaag 1200 gacagaatcc agccggaaat cctggaagca ctgctgaagc acatctcgtt cgacaagttc 1260 gtccagatct cgctgaaggc actgagaaga atcgtcccgc tgatggaaca gggaaagaga 1320 tacgacgaag catgcgcaga aatctacgga gaccactacg gaaagaagaa cacagaagaa 1380 aagatctacc tgccgccgat cccggcagac gaaatcagaa acccggtcgt cctgagagca 1440 ctgtcgcagg caagaaaggt catcaacgga gtcgtcagaa gatacggatc gccggcaaga 1500 atccacatcg aaacagcaag agaagtcgga aagtcgttca aggacagaaa ggaaatcgaa 1560 aagagacagg aagaaaacag aaaggacaga gaaaaggcag cagcaaagtt cagagaatac 1620 ttcccgaact tcgtcggaga accgaagtcg aaggacatcc tgaagctgag actgtacgaa 1680 cagcagcacg gaaagtgcct gtactcggga aaggaaatca acctgggaag actgaacgaa 1740 aagggatacg tcgaaatcga ccacgcactg ccgttctcga gaacatggga cgactcgttc 1800 aacaacaagg tcctggtcct gggatcggaa aaccagaaca agggaaacca gacaccgtac 1860 gaatacttca acggaaagga caactcgaga gaatggcagg aattcaaggc aagagtcgaa 1920 acatcgagat tcccgagatc gaagaagcag agaatcctgc tgcagaagtt cgacgaagac 1980 ggattcaagg aaagaaacct gaacgacaca agatacgtca acagattcct gtgccagttc 2040 gtcgcagaca gaatgagact gacaggaaag ggaaagaaga gagtcttcgc atcgaacgga 2100 cagatcacaa acctgctgag aggattctgg ggactgagaa aggtcagagc agaaaacgac 2160 agacaccacg cactggacgc agtcgtcgtc gcatgctcga cagtcgcaat gcagcagaag 2220 atcacaagat tcgtcagata caaggaaatg aacgcattcg acggaaagac aatcgacaag 2280 gaaacaggag aagtcctgca ccagaagaca cacttcccgc agccgtggga attcttcgca 2340 caggaagtca tgatcagagt cttcggaaag ccggacggaa agccggaatt cgaagaagca 2400 gacacactgg aaaagctgag aacactgctg gcagaaaagc tgtcgtcgag accggaagca 2460 gtccacgaat acgtcacacc gctgttcgtc tcgagagcac cgaacagaaa gatgtcggga 2520 cagggacaca tggaaacagt caagtcggca aagagactgg acgaaggagt ctcggtcctg 2580 agagtcccgc tgacacagct gaagctgaag gacctggaaa agatggtcaa cagagaaaga 2640 gaaccgaagc tgtacgaagc actgaaggca agactggaag cacacaagga cgacccggca 2700 aaggcattcg cagaaccgtt ctacaagtac gacaaggcag gaaacagaac acagcaggtc 2760 aaggcagtca gagtcgaaca ggtccagaag acaggagtct gggtcagaaa ccacaacgga 2820 atcgcagaca acgcaacaat ggtcagagta gacgtcttcg aaaagggaga caagtactac 2880 ctggtcccga tctactcgtg gcaggtcgca aagggaatcc tgccggacag agcagtcgtc 2940 cagggaaagg acgaagaaga ctggcagctg atcgacgact cgttcaactt caagttctcg 3000 ctgcacccga acgacctggt cgaagtcatc acaaagaagg caagaatgtt cggatacttc 3060 gcatcgtgcc acagaggaac aggaaacatc aacatcagaa tccacgacct ggaccacaag 3120 atcggaaaga acggaatcct ggaaggaatc ggagtcaaga cagcactgtc gttccagaag 3180 taccagatcg acgaactggg aaaggaaatc agaccgtgca gactgaagaa gagaccgccg 3240 gtcagatccg gaaagagaac agcagacgga tcggaattcg aatcgccgaa gaagaagaga 3300 aaggtcgaat ga 3312 <210> SEQ ID NO 266 <211> LENGTH: 3306 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: ORF encoding Neisseria meningitidis Cas9 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 266 gcagcattca agccgaactc gatcaactac atcctgggac tggacatcgg aatcgcatcg 60 gtcggatggg caatggtcga aatcgacgaa gaagaaaacc cgatcagact gatcgacctg 120 ggagtcagag tcttcgaaag agcagaagtc ccgaagacag gagactcgct ggcaatggca 180 agaagactgg caagatcggt cagaagactg acaagaagaa gagcacacag actgctgaga 240 acaagaagac tgctgaagag agaaggagtc ctgcaggcag caaacttcga cgaaaacgga 300 ctgatcaagt cgctgccgaa cacaccgtgg cagctgagag cagcagcact ggacagaaag 360 ctgacaccgc tggaatggtc ggcagtcctg ctgcacctga tcaagcacag aggatacctg 420 tcgcagagaa agaacgaagg agaaacagca gacaaggaac tgggagcact gctgaaggga 480 gtcgcaggaa acgcacacgc actgcagaca ggagacttca gaacaccggc agaactggca 540 ctgaacaagt tcgaaaagga atcgggacac atcagaaacc agagatcgga ctactcgcac 600 acattctcga gaaaggacct gcaggcagaa ctgatcctgc tgttcgaaaa gcagaaggaa 660 ttcggaaacc cgcacgtctc gggaggactg aaggaaggaa tcgaaacact gctgatgaca 720 cagagaccgg cactgtcggg agacgcagtc cagaagatgc tgggacactg cacattcgaa 780 ccggcagaac cgaaggcagc aaagaacaca tacacagcag aaagattcat ctggctgaca 840 aagctgaaca acctgagaat cctggaacag ggatcggaaa gaccgctgac agacacagaa 900 agagcaacac tgatggacga accgtacaga aagtcgaagc tgacatacgc acaggcaaga 960 aagctgctgg gactggaaga cacagcattc ttcaagggac tgagatacgg aaaggacaac 1020 gcagaagcat cgacactgat ggaaatgaag gcataccacg caatctcgag agcactggaa 1080 aaggaaggac tgaaggacaa gaagtcgccg ctgaacctgt cgccggaact gcaggacgaa 1140 atcggaacag cattctcgct gttcaagaca gacgaagaca tcacaggaag actgaaggac 1200 agaatccagc cggaaatcct ggaagcactg ctgaagcaca tctcgttcga caagttcgtc 1260 cagatctcgc tgaaggcact gagaagaatc gtcccgctga tggaacaggg aaagagatac 1320 gacgaagcat gcgcagaaat ctacggagac cactacggaa agaagaacac agaagaaaag 1380 atctacctgc cgccgatccc ggcagacgaa atcagaaacc cggtcgtcct gagagcactg 1440 tcgcaggcaa gaaaggtcat caacggagtc gtcagaagat acggatcgcc ggcaagaatc 1500 cacatcgaaa cagcaagaga agtcggaaag tcgttcaagg acagaaagga aatcgaaaag 1560 agacaggaag aaaacagaaa ggacagagaa aaggcagcag caaagttcag agaatacttc 1620 ccgaacttcg tcggagaacc gaagtcgaag gacatcctga agctgagact gtacgaacag 1680 cagcacggaa agtgcctgta ctcgggaaag gaaatcaacc tgggaagact gaacgaaaag 1740 ggatacgtcg aaatcgacca cgcactgccg ttctcgagaa catgggacga ctcgttcaac 1800 aacaaggtcc tggtcctggg atcggaaaac cagaacaagg gaaaccagac accgtacgaa 1860 tacttcaacg gaaaggacaa ctcgagagaa tggcaggaat tcaaggcaag agtcgaaaca 1920 tcgagattcc cgagatcgaa gaagcagaga atcctgctgc agaagttcga cgaagacgga 1980 ttcaaggaaa gaaacctgaa cgacacaaga tacgtcaaca gattcctgtg ccagttcgtc 2040 gcagacagaa tgagactgac aggaaaggga aagaagagag tcttcgcatc gaacggacag 2100 atcacaaacc tgctgagagg attctgggga ctgagaaagg tcagagcaga aaacgacaga 2160 caccacgcac tggacgcagt cgtcgtcgca tgctcgacag tcgcaatgca gcagaagatc 2220 acaagattcg tcagatacaa ggaaatgaac gcattcgacg gaaagacaat cgacaaggaa 2280 acaggagaag tcctgcacca gaagacacac ttcccgcagc cgtgggaatt cttcgcacag 2340 gaagtcatga tcagagtctt cggaaagccg gacggaaagc cggaattcga agaagcagac 2400 acactggaaa agctgagaac actgctggca gaaaagctgt cgtcgagacc ggaagcagtc 2460 cacgaatacg tcacaccgct gttcgtctcg agagcaccga acagaaagat gtcgggacag 2520 ggacacatgg aaacagtcaa gtcggcaaag agactggacg aaggagtctc ggtcctgaga 2580 gtcccgctga cacagctgaa gctgaaggac ctggaaaaga tggtcaacag agaaagagaa 2640 ccgaagctgt acgaagcact gaaggcaaga ctggaagcac acaaggacga cccggcaaag 2700 gcattcgcag aaccgttcta caagtacgac aaggcaggaa acagaacaca gcaggtcaag 2760 gcagtcagag tcgaacaggt ccagaagaca ggagtctggg tcagaaacca caacggaatc 2820 gcagacaacg caacaatggt cagagtagac gtcttcgaaa agggagacaa gtactacctg 2880 gtcccgatct actcgtggca ggtcgcaaag ggaatcctgc cggacagagc agtcgtccag 2940 ggaaaggacg aagaagactg gcagctgatc gacgactcgt tcaacttcaa gttctcgctg 3000 cacccgaacg acctggtcga agtcatcaca aagaaggcaa gaatgttcgg atacttcgca 3060 tcgtgccaca gaggaacagg aaacatcaac atcagaatcc acgacctgga ccacaagatc 3120 ggaaagaacg gaatcctgga aggaatcgga gtcaagacag cactgtcgtt ccagaagtac 3180 cagatcgacg aactgggaaa ggaaatcaga ccgtgcagac tgaagaagag accgccggtc 3240 agatccggaa agagaacagc agacggatcg gaattcgaat cgccgaagaa gaagagaaag 3300 gtcgaa 3306 <210> SEQ ID NO 267 <211> LENGTH: 3636 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Transcript comprising SEQ ID NO: 265 (encoding Neisseria meningitidis Cas9) <400> SEQUENCE: 267 gggagaccca agctggctag cgtttaaact taagcttgga tccgccacca tggcagcatt 60 caagccgaac tcgatcaact acatcctggg actggacatc ggaatcgcat cggtcggatg 120 ggcaatggtc gaaatcgacg aagaagaaaa cccgatcaga ctgatcgacc tgggagtcag 180 agtcttcgaa agagcagaag tcccgaagac aggagactcg ctggcaatgg caagaagact 240 ggcaagatcg gtcagaagac tgacaagaag aagagcacac agactgctga gaacaagaag 300 actgctgaag agagaaggag tcctgcaggc agcaaacttc gacgaaaacg gactgatcaa 360 gtcgctgccg aacacaccgt ggcagctgag agcagcagca ctggacagaa agctgacacc 420 gctggaatgg tcggcagtcc tgctgcacct gatcaagcac agaggatacc tgtcgcagag 480 aaagaacgaa ggagaaacag cagacaagga actgggagca ctgctgaagg gagtcgcagg 540 aaacgcacac gcactgcaga caggagactt cagaacaccg gcagaactgg cactgaacaa 600 gttcgaaaag gaatcgggac acatcagaaa ccagagatcg gactactcgc acacattctc 660 gagaaaggac ctgcaggcag aactgatcct gctgttcgaa aagcagaagg aattcggaaa 720 cccgcacgtc tcgggaggac tgaaggaagg aatcgaaaca ctgctgatga cacagagacc 780 ggcactgtcg ggagacgcag tccagaagat gctgggacac tgcacattcg aaccggcaga 840 accgaaggca gcaaagaaca catacacagc agaaagattc atctggctga caaagctgaa 900 caacctgaga atcctggaac agggatcgga aagaccgctg acagacacag aaagagcaac 960 actgatggac gaaccgtaca gaaagtcgaa gctgacatac gcacaggcaa gaaagctgct 1020 gggactggaa gacacagcat tcttcaaggg actgagatac ggaaaggaca acgcagaagc 1080 atcgacactg atggaaatga aggcatacca cgcaatctcg agagcactgg aaaaggaagg 1140 actgaaggac aagaagtcgc cgctgaacct gtcgccggaa ctgcaggacg aaatcggaac 1200 agcattctcg ctgttcaaga cagacgaaga catcacagga agactgaagg acagaatcca 1260 gccggaaatc ctggaagcac tgctgaagca catctcgttc gacaagttcg tccagatctc 1320 gctgaaggca ctgagaagaa tcgtcccgct gatggaacag ggaaagagat acgacgaagc 1380 atgcgcagaa atctacggag accactacgg aaagaagaac acagaagaaa agatctacct 1440 gccgccgatc ccggcagacg aaatcagaaa cccggtcgtc ctgagagcac tgtcgcaggc 1500 aagaaaggtc atcaacggag tcgtcagaag atacggatcg ccggcaagaa tccacatcga 1560 aacagcaaga gaagtcggaa agtcgttcaa ggacagaaag gaaatcgaaa agagacagga 1620 agaaaacaga aaggacagag aaaaggcagc agcaaagttc agagaatact tcccgaactt 1680 cgtcggagaa ccgaagtcga aggacatcct gaagctgaga ctgtacgaac agcagcacgg 1740 aaagtgcctg tactcgggaa aggaaatcaa cctgggaaga ctgaacgaaa agggatacgt 1800 cgaaatcgac cacgcactgc cgttctcgag aacatgggac gactcgttca acaacaaggt 1860 cctggtcctg ggatcggaaa accagaacaa gggaaaccag acaccgtacg aatacttcaa 1920 cggaaaggac aactcgagag aatggcagga attcaaggca agagtcgaaa catcgagatt 1980 cccgagatcg aagaagcaga gaatcctgct gcagaagttc gacgaagacg gattcaagga 2040 aagaaacctg aacgacacaa gatacgtcaa cagattcctg tgccagttcg tcgcagacag 2100 aatgagactg acaggaaagg gaaagaagag agtcttcgca tcgaacggac agatcacaaa 2160 cctgctgaga ggattctggg gactgagaaa ggtcagagca gaaaacgaca gacaccacgc 2220 actggacgca gtcgtcgtcg catgctcgac agtcgcaatg cagcagaaga tcacaagatt 2280 cgtcagatac aaggaaatga acgcattcga cggaaagaca atcgacaagg aaacaggaga 2340 agtcctgcac cagaagacac acttcccgca gccgtgggaa ttcttcgcac aggaagtcat 2400 gatcagagtc ttcggaaagc cggacggaaa gccggaattc gaagaagcag acacactgga 2460 aaagctgaga acactgctgg cagaaaagct gtcgtcgaga ccggaagcag tccacgaata 2520 cgtcacaccg ctgttcgtct cgagagcacc gaacagaaag atgtcgggac agggacacat 2580 ggaaacagtc aagtcggcaa agagactgga cgaaggagtc tcggtcctga gagtcccgct 2640 gacacagctg aagctgaagg acctggaaaa gatggtcaac agagaaagag aaccgaagct 2700 gtacgaagca ctgaaggcaa gactggaagc acacaaggac gacccggcaa aggcattcgc 2760 agaaccgttc tacaagtacg acaaggcagg aaacagaaca cagcaggtca aggcagtcag 2820 agtcgaacag gtccagaaga caggagtctg ggtcagaaac cacaacggaa tcgcagacaa 2880 cgcaacaatg gtcagagtag acgtcttcga aaagggagac aagtactacc tggtcccgat 2940 ctactcgtgg caggtcgcaa agggaatcct gccggacaga gcagtcgtcc agggaaagga 3000 cgaagaagac tggcagctga tcgacgactc gttcaacttc aagttctcgc tgcacccgaa 3060 cgacctggtc gaagtcatca caaagaaggc aagaatgttc ggatacttcg catcgtgcca 3120 cagaggaaca ggaaacatca acatcagaat ccacgacctg gaccacaaga tcggaaagaa 3180 cggaatcctg gaaggaatcg gagtcaagac agcactgtcg ttccagaagt accagatcga 3240 cgaactggga aaggaaatca gaccgtgcag actgaagaag agaccgccgg tcagatccgg 3300 aaagagaaca gcagacggat cggaattcga atcgccgaag aagaagagaa aggtcgaatg 3360 atagctagct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 3420 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 3480 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 3540 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 3600 atagcaggca tgctggggat gcggtgggct ctatgg 3636 <210> SEQ ID NO 268 <211> LENGTH: 1103 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Neisseria meningitidis Cas9 <400> SEQUENCE: 268 Met Ala Ala Phe Lys Pro Asn Ser Ile Asn Tyr Ile Leu Gly Leu Asp 1 5 10 15 Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Glu 20 25 30 Glu Asn Pro Ile Arg Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg 35 40 45 Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu 50 55 60 Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu 65 70 75 80 Arg Thr Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asn 85 90 95 Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln 100 105 110 Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser 115 120 125 Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg 130 135 140 Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys 145 150 155 160 Gly Val Ala Gly Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr 165 170 175 Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile 180 185 190 Arg Asn Gln Arg Ser Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu 195 200 205 Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn 210 215 220 Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met 225 230 235 240 Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly 245 250 255 His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr 260 265 270 Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile 275 280 285 Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr 290 295 300 Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala 305 310 315 320 Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg 325 330 335 Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala 340 345 350 Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys 355 360 365 Lys Ser Pro Leu Asn Leu Ser Pro Glu Leu Gln Asp Glu Ile Gly Thr 370 375 380 Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys 385 390 395 400 Asp Arg Ile Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser 405 410 415 Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val 420 425 430 Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile 435 440 445 Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu 450 455 460 Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala 465 470 475 480 Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly 485 490 495 Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser 500 505 510 Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys 515 520 525 Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe 530 535 540 Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu 545 550 555 560 Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Gly 565 570 575 Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe 580 585 590 Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly 595 600 605 Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn 610 615 620 Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu 625 630 635 640 Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys 645 650 655 Phe Asp Glu Asp Gly Phe Lys Glu Arg Asn Leu Asn Asp Thr Arg Tyr 660 665 670 Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp Arg Met Arg Leu Thr 675 680 685 Gly Lys Gly Lys Lys Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn 690 695 700 Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp 705 710 715 720 Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala 725 730 735 Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala 740 745 750 Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Glu Val Leu His Gln 755 760 765 Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met 770 775 780 Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala 785 790 795 800 Asp Thr Leu Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser 805 810 815 Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg 820 825 830 Ala Pro Asn Arg Lys Met Ser Gly Gln Gly His Met Glu Thr Val Lys 835 840 845 Ser Ala Lys Arg Leu Asp Glu Gly Val Ser Val Leu Arg Val Pro Leu 850 855 860 Thr Gln Leu Lys Leu Lys Asp Leu Glu Lys Met Val Asn Arg Glu Arg 865 870 875 880 Glu Pro Lys Leu Tyr Glu Ala Leu Lys Ala Arg Leu Glu Ala His Lys 885 890 895 Asp Asp Pro Ala Lys Ala Phe Ala Glu Pro Phe Tyr Lys Tyr Asp Lys 900 905 910 Ala Gly Asn Arg Thr Gln Gln Val Lys Ala Val Arg Val Glu Gln Val 915 920 925 Gln Lys Thr Gly Val Trp Val Arg Asn His Asn Gly Ile Ala Asp Asn 930 935 940 Ala Thr Met Val Arg Val Asp Val Phe Glu Lys Gly Asp Lys Tyr Tyr 945 950 955 960 Leu Val Pro Ile Tyr Ser Trp Gln Val Ala Lys Gly Ile Leu Pro Asp 965 970 975 Arg Ala Val Val Gln Gly Lys Asp Glu Glu Asp Trp Gln Leu Ile Asp 980 985 990 Asp Ser Phe Asn Phe Lys Phe Ser Leu His Pro Asn Asp Leu Val Glu 995 1000 1005 Val Ile Thr Lys Lys Ala Arg Met Phe Gly Tyr Phe Ala Ser Cys 1010 1015 1020 His Arg Gly Thr Gly Asn Ile Asn Ile Arg Ile His Asp Leu Asp 1025 1030 1035 His Lys Ile Gly Lys Asn Gly Ile Leu Glu Gly Ile Gly Val Lys 1040 1045 1050 Thr Ala Leu Ser Phe Gln Lys Tyr Gln Ile Asp Glu Leu Gly Lys 1055 1060 1065 Glu Ile Arg Pro Cys Arg Leu Lys Lys Arg Pro Pro Val Arg Ser 1070 1075 1080 Gly Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys 1085 1090 1095 Lys Arg Lys Val Glu 1100 <210> SEQ ID NO 269 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G390 single guide RNA targeting the rat TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 269 gccgagucug gagagcugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 270 <211> LENGTH: 74 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: trRNA <400> SEQUENCE: 270 aacagcauag caaguuaaaa uaaggcuagu ccguuaucaa cuugaaaaag uggcaccgag 60 ucggugcuuu uuuu 74 <210> SEQ ID NO 271 <400> SEQUENCE: 271 000 <210> SEQ ID NO 272 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G534 single guide RNA targeting the rat TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 272 acgcaaauau caguccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 273 <211> LENGTH: 95 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000395 5 truncated inactive sgRNA modified sequence <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(35) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (64)..(91) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (92)..(95) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 273 gcaauggugu agcggguuuu agagcuagaa auagcaaguu aaaauaaggc uaguccguua 60 ucaacuugaa aaaguggcac cgagucggug cuuuu 95 <210> SEQ ID NO 274 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: SV40 NLS <400> SEQUENCE: 274 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 275 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Alternate SV40 NLS <400> SEQUENCE: 275 Pro Lys Lys Lys Arg Arg Val 1 5 <210> SEQ ID NO 276 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Nucleoplasmin NLS <400> SEQUENCE: 276 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> SEQ ID NO 277 <211> LENGTH: 10 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Exemplary Kozak sequence <400> SEQUENCE: 277 gccrccaugg 10 <210> SEQ ID NO 278 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Exemplary Kozak sequence <400> SEQUENCE: 278 gccgccrcca ugg 13

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 278 <210> SEQ ID NO 1 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 1 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 2 <211> LENGTH: 4403 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript comprising Cas9 ORF corresponding to SEQ ID NO: 205 using codons with generally high expression in humans <400> SEQUENCE: 2 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc atgcctaaga aaaagcggaa ggtcgacggg gataagaagt actcaatcgg 120 gctggatatc ggaactaatt ccgtgggttg ggcagtgatc acggatgaat acaaagtgcc 180 gtccaagaag ttcaaggtcc tggggaacac cgatagacac agcatcaaga aaaatctcat 240 cggagccctg ctgtttgact ccggcgaaac cgcagaagcg acccggctca aacgtaccgc 300 gaggcgacgc tacacccggc ggaagaatcg catctgctat ctgcaagaga tcttttcgaa 360 cgaaatggca aaggtcgacg acagcttctt ccaccgcctg gaagaatctt tcctggtgga 420 ggaggacaag aagcatgaac ggcatcctat ctttggaaac atcgtcgacg aagtggcgta 480 ccacgaaaag tacccgacca tctaccatct gcggaagaag ttggttgact caactgacaa 540 ggccgacctc agattgatct acttggccct cgcccatatg atcaaattcc gcggacactt 600 cctgatcgaa ggcgatctga accctgataa ctccgacgtg gataagcttt tcattcaact 660 ggtgcagacc tacaaccaac tgttcgaaga aaacccaatc aatgctagcg gcgtcgatgc 720 caaggccatc ctgtccgccc ggctgtcgaa gtcgcggcgc ctcgaaaacc tgatcgcaca 780 gctgccggga gagaaaaaga acggactttt cggcaacttg atcgctctct cactgggact 840 cactcccaat ttcaagtcca attttgacct ggccgaggac gcgaagctgc aactctcaaa 900 ggacacctac gacgacgact tggacaattt gctggcacaa attggcgatc agtacgcgga 960 tctgttcctt gccgctaaga acctttcgga cgcaatcttg ctgtccgata tcctgcgcgt 1020 gaacaccgaa ataaccaaag cgccgcttag cgcctcgatg attaagcggt acgacgagca 1080 tcaccaggat ctcacgctgc tcaaagcgct cgtgagacag caactgcctg aaaagtacaa 1140 ggagatcttc ttcgaccagt ccaagaatgg gtacgcaggg tacatcgatg gaggcgctag 1200 ccaggaagag ttctataagt tcatcaagcc aatcctggaa aagatggacg gaaccgaaga 1260 actgctggtc aagctgaaca gggaggatct gctccggaaa cagagaacct ttgacaacgg 1320 atccattccc caccagatcc atctgggtga gctgcacgcc atcttgcggc gccaggagga 1380 cttttaccca ttcctcaagg acaaccggga aaagatcgag aaaattctga cgttccgcat 1440 cccgtattac gtgggcccac tggcgcgcgg caattcgcgc ttcgcgtgga tgactagaaa 1500 atcagaggaa accatcactc cttggaattt cgaggaagtt gtggataagg gagcttcggc 1560 acaaagcttc atcgaacgaa tgaccaactt cgacaagaat ctcccaaacg agaaggtgct 1620 tcctaagcac agcctccttt acgaatactt cactgtctac aacgaactga ctaaagtgaa 1680 atacgttact gaaggaatga ggaagccggc ctttctgtcc ggagaacaga agaaagcaat 1740 tgtcgatctg ctgttcaaga ccaaccgcaa ggtgaccgtc aagcagctta aagaggacta 1800 cttcaagaag atcgagtgtt tcgactcagt ggaaatcagc ggggtggagg acagattcaa 1860 cgcttcgctg ggaacctatc atgatctcct gaagatcatc aaggacaagg acttccttga 1920 caacgaggag aacgaggaca tcctggaaga tatcgtcctg accttgaccc ttttcgagga 1980 tcgcgagatg atcgaggaga ggcttaagac ctacgctcat ctcttcgacg ataaggtcat 2040 gaaacaactc aagcgccgcc ggtacactgg ttggggccgc ctctcccgca agctgatcaa 2100 cggtattcgc gataaacaga gcggtaaaac tatcctggat ttcctcaaat cggatggctt 2160

cgctaatcgt aacttcatgc aattgatcca cgacgacagc ctgaccttta aggaggacat 2220 ccaaaaagca caagtgtccg gacagggaga ctcactccat gaacacatcg cgaatctggc 2280 cggttcgccg gcgattaaga agggaattct gcaaactgtg aaggtggtcg acgagctggt 2340 gaaggtcatg ggacggcaca aaccggagaa tatcgtgatt gaaatggccc gagaaaacca 2400 gactacccag aagggccaga aaaactcccg cgaaaggatg aagcggatcg aagaaggaat 2460 caaggagctg ggcagccaga tcctgaaaga gcacccggtg gaaaacacgc agctgcagaa 2520 cgagaagctc tacctgtact atttgcaaaa tggacgggac atgtacgtgg accaagagct 2580 ggacatcaat cggttgtctg attacgacgt ggaccacatc gttccacagt cctttctgaa 2640 ggatgactcg atcgataaca aggtgttgac tcgcagcgac aagaacagag ggaagtcaga 2700 taatgtgcca tcggaggagg tcgtgaagaa gatgaagaat tactggcggc agctcctgaa 2760 tgcgaagctg attacccaga gaaagtttga caatctcact aaagccgagc gcggcggact 2820 ctcagagctg gataaggctg gattcatcaa acggcagctg gtcgagactc ggcagattac 2880 caagcacgtg gcgcagatct tggactcccg catgaacact aaatacgacg agaacgataa 2940 gctcatccgg gaagtgaagg tgattaccct gaaaagcaaa cttgtgtcgg actttcggaa 3000 ggactttcag ttttacaaag tgagagaaat caacaactac catcacgcgc atgacgcata 3060 cctcaacgct gtggtcggta ccgccctgat caaaaagtac cctaaacttg aatcggagtt 3120 tgtgtacgga gactacaagg tctacgacgt gaggaagatg atagccaagt ccgaacagga 3180 aatcgggaaa gcaactgcga aatacttctt ttactcaaac atcatgaact ttttcaagac 3240 tgaaattacg ctggccaatg gagaaatcag gaagaggcca ctgatcgaaa ctaacggaga 3300 aacgggcgaa atcgtgtggg acaagggcag ggacttcgca actgttcgca aagtgctctc 3360 tatgccgcaa gtcaatattg tgaagaaaac cgaagtgcaa accggcggat tttcaaagga 3420 atcgatcctc ccaaagagaa atagcgacaa gctcattgca cgcaagaaag actgggaccc 3480 gaagaagtac ggaggattcg attcgccgac tgtcgcatac tccgtcctcg tggtggccaa 3540 ggtggagaag ggaaagagca aaaagctcaa atccgtcaaa gagctgctgg ggattaccat 3600 catggaacga tcctcgttcg agaagaaccc gattgatttc ctcgaggcga agggttacaa 3660 ggaggtgaag aaggatctga tcatcaaact ccccaagtac tcactgttcg aactggaaaa 3720 tggtcggaag cgcatgctgg cttcggccgg agaactccaa aaaggaaatg agctggcctt 3780 gcctagcaag tacgtcaact tcctctatct tgcttcgcac tacgaaaaac tcaaagggtc 3840 accggaagat aacgaacaga agcagctttt cgtggagcag cacaagcatt atctggatga 3900 aatcatcgaa caaatctccg agttttcaaa gcgcgtgatc ctcgccgacg ccaacctcga 3960 caaagtcctg tcggcctaca ataagcatag agataagccg atcagagaac aggccgagaa 4020 cattatccac ttgttcaccc tgactaacct gggagcccca gccgccttca agtacttcga 4080 tactactatc gatcgcaaaa gatacacgtc caccaaggaa gttctggacg cgaccctgat 4140 ccaccaaagc atcactggac tctacgaaac taggatcgat ctgtcgcagc tgggtggcga 4200 ttgatagtct agccatcaca tttaaaagca tctcagccta ccatgagaat aagagaaaga 4260 aaatgaagat caatagctta ttcatctctt tttctttttc gttggtgtaa agccaacacc 4320 ctgtctaaaa aacataaatt tctttaatca ttttgcctct tttctctgtg cttcaattaa 4380 taaaaaatgg aaagaacctc gag 4403 <210> SEQ ID NO 3 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: modified sgRNA sequence <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(20) <223> OTHER INFORMATION: n is a, c, g, or u <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 3 nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 4 <211> LENGTH: 105 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: 30/30/39 poly-A sequence <400> SEQUENCE: 4 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcgaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaccgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 105 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003335 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 5 cugcuccucc ucugccuugc 20 <210> SEQ ID NO 6 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003336 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 6 ccuccucugc cuugcuggac 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003337 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 7 ccaguccagc aaggcagagg 20 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003338 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 8 auaccagucc agcaaggcag 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003339 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 9 acacaaauac caguccagca 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003340 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 10 uggacuggua uuugugucug 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003341 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 11 cugguauuug ugucugaggc 20 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003342 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 12 cuucucuaca cccagggcac 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003343 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 13 cagaggacac uuggauucac 20

<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003344 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 14 uuugaccauc agaggacacu 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003345 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 15 ucuagaacuu ugaccaucag 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003346 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 16 aaaguucuag augcuguccg 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003347 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 17 cauugauggc aggacugccu 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003348 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 18 aggcaguccu gccaucaaug 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003349 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 19 ugcacggcca cauugauggc 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003350 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 20 cacaugcacg gccacauuga 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003351 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 21 agccuuucug aacacaugca 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003352 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 22 gaaaggcugc ugaugacacc 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003353 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 23 aaaggcugcu gaugacaccu 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003354 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 24 accugggagc cauuugccuc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003355 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 25 cccagaggca aauggcuccc 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003356 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 26 gcaacuuacc cagaggcaaa 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003357 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 27 uucuuuggca acuuacccag 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003358 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 28 augcagcucu ccagacucac 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003359 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 29 agugagucug gagagcugca 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003360 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 30 gugagucugg agagcugcau 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003361 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 31 gcugcauggg cucacaacug 20

<210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003362 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 32 gcaugggcuc acaacugagg 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003363 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 33 acugaggagg aauuuguaga 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003364 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 34 cugaggagga auuuguagaa 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003365 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 35 uguagaaggg auauacaaag 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003366 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 36 aaauagacac caaaucuuac 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003367 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 37 agacaccaaa ucuuacugga 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003368 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 38 aagugccuuc caguaagauu 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003369 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 39 cucugcaugc ucauggaaug 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003370 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 40 ccucugcaug cucauggaau 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003371 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 41 accucugcau gcucauggaa 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003372 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 42 uacucaccuc ugcaugcuca 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003373 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 43 guauucacag ccaacgacuc 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003374 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 44 gcggcggggg ccggagucgu 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003375 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 45 aaugguguag cggcgggggc 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003376 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 46 cggcaauggu guagcggcgg 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003377 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 47 gcggcaaugg uguagcggcg 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003378 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 48 ggcggcaaug guguagcggc 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003379 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 49

gggcggcaau gguguagcgg 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003380 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 50 gcagggcggc aaugguguag 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003381 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 51 ggggcucagc agggcggcaa 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003382 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 52 ggaguagggg cucagcaggg 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003383 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 53 auaggaguag gggcucagca 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003384 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 54 aauaggagua ggggcucagc 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003385 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 55 ccccuacucc uauuccacca 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003386 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 56 ccguggugga auaggaguag 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003387 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 57 gccguggugg aauaggagua 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003388 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 58 gacgacagcc gugguggaau 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003389 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 59 auuggugacg acagccgugg 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003390 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 60 gggauuggug acgacagccg 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003391 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 61 ggcugucguc accaauccca 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR003392 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 62 agucccucau uccuugggau 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005298 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 63 uccacucauu cuuggcagga 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005299 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 64 agccguggug gaauaggagu 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005300 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 65 ucacagaaac acucaccgua 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005301 gRNA targeting Human TTR (Exon 1) <400> SEQUENCE: 66 gucacagaaa cacucaccgu 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005302 gRNA targeting Human TTR (Exon 2)

<400> SEQUENCE: 67 acgugucuuc ucuacaccca 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005303 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 68 ugaauccaag uguccucuga 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005304 gRNA targeting Human TTR (Exon 2) <400> SEQUENCE: 69 ggccgugcau guguucagaa 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005305 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 70 uauaggaaaa ccagugaguc 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005306 gRNA targeting Human TTR (Exon 3) <400> SEQUENCE: 71 aaaucuuacu ggaaggcacu 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005307 gRNA targeting Human TTR (Exon 4) <400> SEQUENCE: 72 ugucugucuu cucucauagg 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR000689 gRNA targeting Cyno TTR <400> SEQUENCE: 73 acacaaauac caguccagcg 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005364 gRNA targeting Cyno TTR <400> SEQUENCE: 74 aaaggcugcu gaugagaccu 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005365 gRNA targeting Cyno TTR <400> SEQUENCE: 75 cauugacagc aggacugccu 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005366 gRNA targeting Cyno TTR <400> SEQUENCE: 76 auaccagucc agcgaggcag 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005367 gRNA targeting Cyno TTR <400> SEQUENCE: 77 ccaguccagc gaggcagagg 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005368 gRNA targeting Cyno TTR <400> SEQUENCE: 78 ccuccucugc cucgcuggac 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005369 gRNA targeting Cyno TTR <400> SEQUENCE: 79 aaaguucuag augccguccg 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005370 gRNA targeting Cyno TTR <400> SEQUENCE: 80 acuugucuuc ucuauaccca 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005371 gRNA targeting Cyno TTR <400> SEQUENCE: 81 aagugacuuc caguaagauu 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: CR005372 gRNA targeting Cyno TTR <400> SEQUENCE: 82 aaaaggcugc ugaugagacc 20 <210> SEQ ID NO 83 <400> SEQUENCE: 83 000 <210> SEQ ID NO 84 <400> SEQUENCE: 84 000 <210> SEQ ID NO 85 <400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <400> SEQUENCE: 86 000 <210> SEQ ID NO 87 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000480 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96)

<223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 87 aaaggcugcu gaugacaccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 88 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000481 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 88 ucuagaacuu ugaccaucag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 89 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000482 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 89 uguagaaggg auauacaaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 90 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000483 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 90 uccacucauu cuuggcagga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 91 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000484 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 91 agacaccaaa ucuuacugga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 92 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000485 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 92 ccuccucugc cuugcuggac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 93 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000486 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 93 acacaaauac caguccagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 94 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000487 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 94 uucuuuggca acuuacccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 95 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000488 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base

<222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 95 aaaguucuag augcuguccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 96 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000489 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 96 uuugaccauc agaggacacu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 97 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000490 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 97 aaauagacac caaaucuuac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 98 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000491 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 98 auaccagucc agcaaggcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 99 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000492 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 99 cuucucuaca cccagggcac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 100 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000493 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 100 aagugccuuc caguaagauu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 101 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000494 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 101 gugagucugg agagcugcau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 102 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000495 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 102 cagaggacac uuggauucac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 103 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000496 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide

<400> SEQUENCE: 103 ggccgugcau guguucagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 104 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000497 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 104 cugcuccucc ucugccuugc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 105 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000498 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 105 agugagucug gagagcugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 106 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000499 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 106 ugaauccaag uguccucuga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 107 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000500 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 107 ccaguccagc aaggcagagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 108 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000501 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 108 ucacagaaac acucaccgua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 109 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000567 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 109 gaaaggcugc ugaugacacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 110 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000568 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 110 ggcugucguc accaauccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 111 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000570 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 111 cauugauggc aggacugccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 112 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000571 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 112 gucacagaaa cacucaccgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 113 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000572 sgRNA modified sequence targeting Human TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 113 ccccuacucc uauuccacca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 114 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000502 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 114 acacaaauac caguccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 115 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000503 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 115 aaaaggcugc ugaugagacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 116 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000504 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 116 aaaggcugcu gaugagaccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 117 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000505 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 117 cauugacagc aggacugccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 118 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000506 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 118 auaccagucc agcgaggcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 119 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000507 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 119 ccaguccagc gaggcagagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> SEQ ID NO 120 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000508 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 120 ccuccucugc cucgcuggac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 121 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000509 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 121 aaaguucuag augccguccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 122 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000510 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 122 acuugucuuc ucuauaccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 123 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000511 sgRNA modified sequence targeting Cyno TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 123 aagugacuuc caguaagauu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 124 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000282 sgRNA modified sequence targeting Mouse TTR <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 124 uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 125 <400> SEQUENCE: 125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000 <210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO 129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000 <210> SEQ ID NO 131 <400> SEQUENCE: 131 000 <210> SEQ ID NO 132 <400> SEQUENCE: 132 000 <210> SEQ ID NO 133 <400> SEQUENCE: 133 000 <210> SEQ ID NO 134 <400> SEQUENCE: 134 000 <210> SEQ ID NO 135 <400> SEQUENCE: 135 000 <210> SEQ ID NO 136 <400> SEQUENCE: 136 000 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000 <210> SEQ ID NO 138 <400> SEQUENCE: 138

000 <210> SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE: 140 000 <210> SEQ ID NO 141 <400> SEQUENCE: 141 000 <210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210> SEQ ID NO 143 <400> SEQUENCE: 143 000 <210> SEQ ID NO 144 <400> SEQUENCE: 144 000 <210> SEQ ID NO 145 <400> SEQUENCE: 145 000 <210> SEQ ID NO 146 <400> SEQUENCE: 146 000 <210> SEQ ID NO 147 <400> SEQUENCE: 147 000 <210> SEQ ID NO 148 <400> SEQUENCE: 148 000 <210> SEQ ID NO 149 <400> SEQUENCE: 149 000 <210> SEQ ID NO 150 <400> SEQUENCE: 150 000 <210> SEQ ID NO 151 <400> SEQUENCE: 151 000 <210> SEQ ID NO 152 <400> SEQUENCE: 152 000 <210> SEQ ID NO 153 <400> SEQUENCE: 153 000 <210> SEQ ID NO 154 <400> SEQUENCE: 154 000 <210> SEQ ID NO 155 <400> SEQUENCE: 155 000 <210> SEQ ID NO 156 <400> SEQUENCE: 156 000 <210> SEQ ID NO 157 <400> SEQUENCE: 157 000 <210> SEQ ID NO 158 <400> SEQUENCE: 158 000 <210> SEQ ID NO 159 <400> SEQUENCE: 159 000 <210> SEQ ID NO 160 <400> SEQUENCE: 160 000 <210> SEQ ID NO 161 <400> SEQUENCE: 161 000 <210> SEQ ID NO 162 <400> SEQUENCE: 162 000 <210> SEQ ID NO 163 <400> SEQUENCE: 163 000 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174 <400> SEQUENCE: 174

000 <210> SEQ ID NO 175 <400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000 <210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400> SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE: 198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000 <210> SEQ ID NO 200 <400> SEQUENCE: 200 000 <210> SEQ ID NO 201 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: DNA coding sequence of Cas9 using the thymidine analog of the minimal uridine codons listed in Table 3, with start and stop codons <400> SEQUENCE: 201 atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg atgggcagtc 60 atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa cacagacaga 120 cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga aacagcagaa 180 gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa cagaatctgc 240 tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt cttccacaga 300 ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc actggcacac 480 atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga caacagcgac 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact gttcggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc ggcattcctg 1620

agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag aaaggtcaca 1680 gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag cgtcgaaatc 1740 agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac aggatgggga 1980 agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa gacaatcctg 2040 gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg agacagcctg 2160 cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga aaacatcgtc 2280 atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca gaacggaaga 2460 gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagatgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt cgacaacctg 2700 acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat caagagacag 2760 ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac actgaagagc 2880 aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga aatcaacaac 2940 taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact gatcaagaag 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa gacagaagtc 3300 cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga caagctgatc 3360 gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc gacagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa cccgatcgac 3540 ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc aggagaactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag caagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac aagcacaaag 4020 gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag aaaggtctag 4140 <210> SEQ ID NO 202 <211> LENGTH: 4143 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: DNA coding sequence of Cas9 using codons with generally high expression in humans <400> SEQUENCE: 202 atggataaga agtactcaat cgggctggat atcggaacta attccgtggg ttgggcagtg 60 atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa caccgataga 120 cacagcatca agaaaaatct catcggagcc ctgctgtttg actccggcga aaccgcagaa 180 gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag agatcttttc gaacgaaatg gcaaaggtcg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtcg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcaactga caaggccgac ctcagattga tctacttggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga taactccgac 540 gtggataagc ttttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcta gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaaa agaacggact tttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatc ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc tagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga tctgctccgg 1200 aaacagagaa cctttgacaa cggatccatt ccccaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa tttcgaggaa 1440 gttgtggata agggagcttc ggcacaaagc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620 tccggagaac agaagaaagc aattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc agtggaaatc 1740 agcggggtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcaattgat ccacgacgac 2100 agcctgacct ttaaggagga catccaaaaa gcacaagtgt ccggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat tctgcaaact 2220 gtgaaggtgg tcgacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaaaaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tcgatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcttggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gtaccgccct gatcaaaaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga actttttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa aaccgaagtg 3300 caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaaaaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctcgagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actccccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 caaaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta tcttgcttcg 3720 cactacgaaa aactcaaagg gtcaccggaa gataacgaac agaagcagct tttcgtggag 3780 cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc aaagcgcgtg 3840 atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca tagagataag 3900 ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa cctgggagcc 3960 ccagccgcct tcaagtactt cgatactact atcgatcgca aaagatacac gtccaccaag 4020 gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga aactaggatc 4080 gatctgtcgc agctgggtgg cgatggcggt ggatctccga aaaagaagag aaaggtgtaa 4140 tga 4143 <210> SEQ ID NO 203 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 with one nuclear localization signal (1xNLS) as the C-terminal 7 amino acids <400> SEQUENCE: 203 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 204 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF using minimal uridine codons, with start and stop codons <400> SEQUENCE: 204 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 205 <211> LENGTH: 4143 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF using codons with generally high expression in humans, with start and stop codons <400> SEQUENCE: 205 auggauaaga aguacucaau cgggcuggau aucggaacua auuccguggg uugggcagug 60 aucacggaug aauacaaagu gccguccaag aaguucaagg uccuggggaa caccgauaga 120 cacagcauca agaaaaaucu caucggagcc cugcuguuug acuccggcga aaccgcagaa 180 gcgacccggc ucaaacguac cgcgaggcga cgcuacaccc ggcggaagaa ucgcaucugc 240 uaucugcaag agaucuuuuc gaacgaaaug gcaaaggucg acgacagcuu cuuccaccgc 300 cuggaagaau cuuuccuggu ggaggaggac aagaagcaug aacggcaucc uaucuuugga 360 aacaucgucg acgaaguggc guaccacgaa aaguacccga ccaucuacca ucugcggaag 420 aaguugguug acucaacuga caaggccgac cucagauuga ucuacuuggc ccucgcccau 480 augaucaaau uccgcggaca cuuccugauc gaaggcgauc ugaacccuga uaacuccgac 540 guggauaagc uuuucauuca acuggugcag accuacaacc aacuguucga agaaaaccca 600 aucaaugcua gcggcgucga ugccaaggcc auccuguccg cccggcuguc gaagucgcgg 660 cgccucgaaa accugaucgc acagcugccg ggagagaaaa agaacggacu uuucggcaac 720 uugaucgcuc ucucacuggg acucacuccc aauuucaagu ccaauuuuga ccuggccgag 780 gacgcgaagc ugcaacucuc aaaggacacc uacgacgacg acuuggacaa uuugcuggca 840 caaauuggcg aucaguacgc ggaucuguuc cuugccgcua agaaccuuuc ggacgcaauc 900 uugcuguccg auauccugcg cgugaacacc gaaauaacca aagcgccgcu uagcgccucg 960 augauuaagc gguacgacga gcaucaccag gaucucacgc ugcucaaagc gcucgugaga 1020 cagcaacugc cugaaaagua caaggagauc uucuucgacc aguccaagaa uggguacgca 1080 ggguacaucg auggaggcgc uagccaggaa gaguucuaua aguucaucaa gccaauccug 1140 gaaaagaugg acggaaccga agaacugcug gucaagcuga acagggagga ucugcuccgg 1200 aaacagagaa ccuuugacaa cggauccauu ccccaccaga uccaucuggg ugagcugcac 1260 gccaucuugc ggcgccagga ggacuuuuac ccauuccuca aggacaaccg ggaaaagauc 1320 gagaaaauuc ugacguuccg caucccguau uacgugggcc cacuggcgcg cggcaauucg 1380 cgcuucgcgu ggaugacuag aaaaucagag gaaaccauca cuccuuggaa uuucgaggaa 1440 guuguggaua agggagcuuc ggcacaaagc uucaucgaac gaaugaccaa cuucgacaag 1500 aaucucccaa acgagaaggu gcuuccuaag cacagccucc uuuacgaaua cuucacuguc 1560 uacaacgaac ugacuaaagu gaaauacguu acugaaggaa ugaggaagcc ggccuuucug 1620 uccggagaac agaagaaagc aauugucgau cugcuguuca agaccaaccg caaggugacc 1680 gucaagcagc uuaaagagga cuacuucaag aagaucgagu guuucgacuc aguggaaauc 1740 agcggggugg aggacagauu caacgcuucg cugggaaccu aucaugaucu ccugaagauc 1800 aucaaggaca aggacuuccu ugacaacgag gagaacgagg acauccugga agauaucguc 1860 cugaccuuga cccuuuucga ggaucgcgag augaucgagg agaggcuuaa gaccuacgcu 1920 caucucuucg acgauaaggu caugaaacaa cucaagcgcc gccgguacac ugguuggggc 1980 cgccucuccc gcaagcugau caacgguauu cgcgauaaac agagcgguaa aacuauccug 2040 gauuuccuca aaucggaugg cuucgcuaau cguaacuuca ugcaauugau ccacgacgac 2100 agccugaccu uuaaggagga cauccaaaaa gcacaagugu ccggacaggg agacucacuc 2160 caugaacaca ucgcgaaucu ggccgguucg ccggcgauua agaagggaau ucugcaaacu 2220 gugaaggugg ucgacgagcu ggugaagguc augggacggc acaaaccgga gaauaucgug 2280 auugaaaugg cccgagaaaa ccagacuacc cagaagggcc agaaaaacuc ccgcgaaagg 2340

augaagcgga ucgaagaagg aaucaaggag cugggcagcc agauccugaa agagcacccg 2400 guggaaaaca cgcagcugca gaacgagaag cucuaccugu acuauuugca aaauggacgg 2460 gacauguacg uggaccaaga gcuggacauc aaucgguugu cugauuacga cguggaccac 2520 aucguuccac aguccuuucu gaaggaugac ucgaucgaua acaagguguu gacucgcagc 2580 gacaagaaca gagggaaguc agauaaugug ccaucggagg aggucgugaa gaagaugaag 2640 aauuacuggc ggcagcuccu gaaugcgaag cugauuaccc agagaaaguu ugacaaucuc 2700 acuaaagccg agcgcggcgg acucucagag cuggauaagg cuggauucau caaacggcag 2760 cuggucgaga cucggcagau uaccaagcac guggcgcaga ucuuggacuc ccgcaugaac 2820 acuaaauacg acgagaacga uaagcucauc cgggaaguga aggugauuac ccugaaaagc 2880 aaacuugugu cggacuuucg gaaggacuuu caguuuuaca aagugagaga aaucaacaac 2940 uaccaucacg cgcaugacgc auaccucaac gcuguggucg guaccgcccu gaucaaaaag 3000 uacccuaaac uugaaucgga guuuguguac ggagacuaca aggucuacga cgugaggaag 3060 augauagcca aguccgaaca ggaaaucggg aaagcaacug cgaaauacuu cuuuuacuca 3120 aacaucauga acuuuuucaa gacugaaauu acgcuggcca auggagaaau caggaagagg 3180 ccacugaucg aaacuaacgg agaaacgggc gaaaucgugu gggacaaggg cagggacuuc 3240 gcaacuguuc gcaaagugcu cucuaugccg caagucaaua uugugaagaa aaccgaagug 3300 caaaccggcg gauuuucaaa ggaaucgauc cucccaaaga gaaauagcga caagcucauu 3360 gcacgcaaga aagacuggga cccgaagaag uacggaggau ucgauucgcc gacugucgca 3420 uacuccgucc ucgugguggc caagguggag aagggaaaga gcaaaaagcu caaauccguc 3480 aaagagcugc uggggauuac caucauggaa cgauccucgu ucgagaagaa cccgauugau 3540 uuccucgagg cgaaggguua caaggaggug aagaaggauc ugaucaucaa acuccccaag 3600 uacucacugu ucgaacugga aaauggucgg aagcgcaugc uggcuucggc cggagaacuc 3660 caaaaaggaa augagcuggc cuugccuagc aaguacguca acuuccucua ucuugcuucg 3720 cacuacgaaa aacucaaagg gucaccggaa gauaacgaac agaagcagcu uuucguggag 3780 cagcacaagc auuaucugga ugaaaucauc gaacaaaucu ccgaguuuuc aaagcgcgug 3840 auccucgccg acgccaaccu cgacaaaguc cugucggccu acaauaagca uagagauaag 3900 ccgaucagag aacaggccga gaacauuauc cacuuguuca cccugacuaa ccugggagcc 3960 ccagccgccu ucaaguacuu cgauacuacu aucgaucgca aaagauacac guccaccaag 4020 gaaguucugg acgcgacccu gauccaccaa agcaucacug gacucuacga aacuaggauc 4080 gaucugucgc agcugggugg cgauggcggu ggaucuccga aaaagaagag aaagguguaa 4140 uga 4143 <210> SEQ ID NO 206 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase (D10A) amino acid sequence <400> SEQUENCE: 206 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 207 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase (D10A) mRNA ORF <400> SEQUENCE: 207 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 208 <211> LENGTH: 1379 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 (D10A H840A) amino acid sequence <400> SEQUENCE: 208 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> SEQ ID NO 209 <211> LENGTH: 4140 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 (D10A H840A) mRNA ORF <400> SEQUENCE: 209 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> SEQ ID NO 210 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA coding sequence using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 210 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520

gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 211 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase bare coding sequence <400> SEQUENCE: 211 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 212 <211> LENGTH: 4134 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 bare coding sequence <400> SEQUENCE: 212 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140

aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> SEQ ID NO 213 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 (without NLS) <400> SEQUENCE: 213 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 214 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF encoding SEQ ID NO: 213 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 214 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540

uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 215 <211> LENGTH: 4101 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 coding sequence encoding SEQ ID NO: 213 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 215 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga c 4101 <210> SEQ ID NO 216 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 nickase (without NLS) <400> SEQUENCE: 216 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 217 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase mRNA ORF encoding SEQ ID NO: 216 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 217 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380

agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 218 <211> LENGTH: 4101 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase coding sequence encoding SEQ ID NO: 216 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 218 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga c 4101 <210> SEQ ID NO 219 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of dCas9 (without NLS)

<400> SEQUENCE: 219 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> SEQ ID NO 220 <211> LENGTH: 4107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 mRNA ORF encoding SEQ ID NO: 219 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 220 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> SEQ ID NO 221 <211> LENGTH: 4113 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 coding sequence encoding SEQ ID NO: 219 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 221 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220

aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agc 4113 <210> SEQ ID NO 222 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 with two nuclear localization signals (2xNLS) as the C-terminal amino acids <400> SEQUENCE: 222 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 223 <211> LENGTH: 4233 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 mRNA ORF encoding SEQ ID NO: 222 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 223 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucccg 4140 aagaagaaga gaaaggucgg aagcggaagc ccgaagaaga agagaaaggu cgacggaagc 4200 ccgaagaaga agagaaaggu cgacagcgga uag 4233 <210> SEQ ID NO 224 <211> LENGTH: 4227

<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 coding sequence encoding SEQ ID NO: 222 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 224 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa ggucccgaag 4140 aagaagagaa aggucggaag cggaagcccg aagaagaaga gaaaggucga cggaagcccg 4200 aagaagaaga gaaaggucga cagcgga 4227 <210> SEQ ID NO 225 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Cas9 nickase with two nuclear localization signals as the C-terminal amino acids <400> SEQUENCE: 225 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 226 <211> LENGTH: 4179 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase mRNA ORF encoding SEQ ID NO: 25 using minimal uridine codons as listed in Table 3, with start and stop codons <400> SEQUENCE: 226 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740

agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggaagc ggaagcccga agaagaagag aaaggucgac 4140 ggaagcccga agaagaagag aaaggucgac agcggauag 4179 <210> SEQ ID NO 227 <211> LENGTH: 4173 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 nickase coding sequence encoding SEQ ID NO: 25 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 227 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaagcgga agcccgaaga agaagagaaa ggucgacgga 4140 agcccgaaga agaagagaaa ggucgacagc gga 4173 <210> SEQ ID NO 228 <211> LENGTH: 1392 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of dCas9 with two nuclear localization signals as the C-terminal amino acids <400> SEQUENCE: 228 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> SEQ ID NO 229 <211> LENGTH: 4179 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 mRNA ORF encoding SEQ ID NO: 228 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 229 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggaagc ggaagcccga agaagaagag aaaggucgac 4140 ggaagcccga agaagaagag aaaggucgac agcggauag 4179 <210> SEQ ID NO 230 <211> LENGTH: 4173 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: dCas9 coding sequence encoding SEQ ID NO: 228 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 230 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220

aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaagcgga agcccgaaga agaagagaaa ggucgacgga 4140 agcccgaaga agaagagaaa ggucgacagc gga 4173 <210> SEQ ID NO 231 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: T7 Promoter <400> SEQUENCE: 231 taatacgact cactata 17 <210> SEQ ID NO 232 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(50) <223> OTHER INFORMATION: Human beta-globin 5 UTR <400> SEQUENCE: 232 acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc 50 <210> SEQ ID NO 233 <211> LENGTH: 132 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(132) <223> OTHER INFORMATION: Human beta-globin 3 UTR <400> SEQUENCE: 233 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60 taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120 tattttcatt gc 132 <210> SEQ ID NO 234 <211> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(66) <223> OTHER INFORMATION: Human alpha-globin 5 UTR <400> SEQUENCE: 234 cataaaccct ggcgcgctcg cggcccggca ctcttctggt ccccacagac tcagagagaa 60 cccacc 66 <210> SEQ ID NO 235 <211> LENGTH: 110 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(110) <223> OTHER INFORMATION: Human alpha-globin 3 UTR <400> SEQUENCE: 235 gctggagcct cggtggccat gcttcttgcc ccttgggcct ccccccagcc cctcctcccc 60 ttcctgcacc cgtacccccg tggtctttga ataaagtctg agtgggcggc 110 <210> SEQ ID NO 236 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Xenopus laevis <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(29) <223> OTHER INFORMATION: Xenopus laevis beta-globin 5 UTR <400> SEQUENCE: 236 aagctcagaa taaacgctca actttggcc 29 <210> SEQ ID NO 237 <211> LENGTH: 130 <212> TYPE: DNA <213> ORGANISM: Xenopus laevis <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(130) <223> OTHER INFORMATION: Xenopus laevis beta-globin 3 UTR <400> SEQUENCE: 237 accagcctca agaacacccg aatggagtct ctaagctaca taataccaac ttacacttta 60 caaaatgttg tcccccaaaa tgtagccatt cgtatctgct cctaataaaa agaaagtttc 120 ttcacattct 130 <210> SEQ ID NO 238 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Bos taurus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(27) <223> OTHER INFORMATION: Bovine Growth Hormone 5 UTR <400> SEQUENCE: 238 cagggtcctg tggacagctc accagct 27 <210> SEQ ID NO 239 <211> LENGTH: 102 <212> TYPE: DNA <213> ORGANISM: Bos taurus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(102) <223> OTHER INFORMATION: Bovine Growth Hormone 3 UTR <400> SEQUENCE: 239 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 60 tcccactgtc ctttcctaat aaaatgagga aattgcatcg ca 102 <210> SEQ ID NO 240 <211> LENGTH: 93 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(93) <223> OTHER INFORMATION: Mus musculus hemoglobin alpha, adult chain 1 (Hba-a1), 3UTR <400> SEQUENCE: 240 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 93 <210> SEQ ID NO 241 <211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: HSD17B4 5 UTR <400> SEQUENCE: 241 tcccgcagtc ggcgtccagc ggctctgctt gttcgtgtgt gtgtcgttgc aggccttatt 60 c 61 <210> SEQ ID NO 242 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G282 single guide RNA targeting the mouse TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE:

<221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 242 uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 243 <400> SEQUENCE: 243 000 <210> SEQ ID NO 244 <211> LENGTH: 4405 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, and 3 UTR of ALB <400> SEQUENCE: 244 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg 120 atgggcagtc atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa 180 cacagacaga cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga 240 aacagcagaa gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa 300 cagaatctgc tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt 360 cttccacaga ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc 420 gatcttcgga aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca 480 cctgagaaag aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc 540 actggcacac atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga 600 caacagcgac gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga 660 agaaaacccg atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag 720 caagagcaga agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact 780 gttcggaaac ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga 840 cctggcagaa gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa 900 cctgctggca cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag 960 cgacgcaatc ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct 1020 gagcgcaagc atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc 1080 actggtcaga cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa 1140 cggatacgca ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa 1200 gccgatcctg gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga 1260 cctgctgaga aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg 1320 agaactgcac gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag 1380 agaaaagatc gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag 1440 aggaaacagc agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa 1500 cttcgaagaa gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa 1560 cttcgacaag aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata 1620 cttcacagtc tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc 1680 ggcattcctg agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag 1740 aaaggtcaca gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag 1800 cgtcgaaatc agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct 1860 gctgaagatc atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga 1920 agacatcgtc ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa 1980 gacatacgca cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac 2040 aggatgggga agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa 2100 gacaatcctg gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat 2160 ccacgacgac agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg 2220 agacagcctg cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat 2280 cctgcagaca gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga 2340 aaacatcgtc atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag 2400 cagagaaaga atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa 2460 ggaacacccg gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca 2520 gaacggaaga gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga 2580 cgtcgaccac atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct 2640 gacaagaagc gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa 2700 gaagatgaag aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt 2760 cgacaacctg acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat 2820 caagagacag ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag 2880 cagaatgaac acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac 2940 actgaagagc aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga 3000 aatcaacaac taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact 3060 gatcaagaag tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga 3120 cgtcagaaag atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt 3180 cttctacagc aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat 3240 cagaaagaga ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg 3300 aagagacttc gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa 3360 gacagaagtc cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga 3420 caagctgatc gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc 3480 gacagtcgca tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct 3540 gaagagcgtc aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa 3600 cccgatcgac ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa 3660 gctgccgaag tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc 3720 aggagaactg cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta 3780 cctggcaagc cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct 3840 gttcgtcgaa cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag 3900 caagagagtc atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca 3960 cagagacaag ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa 4020 cctgggagca ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac 4080 aagcacaaag gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga 4140 aacaagaatc gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag 4200 aaaggtctag ctagccatca catttaaaag catctcagcc taccatgaga ataagagaaa 4260 gaaaatgaag atcaatagct tattcatctc tttttctttt tcgttggtgt aaagccaaca 4320 ccctgtctaa aaaacataaa tttctttaat cattttgcct cttttctctg tgcttcaatt 4380 aataaaaaat ggaaagaacc tcgag 4405 <210> SEQ ID NO 245 <211> LENGTH: 4188 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Alternative Cas9 ORF with 19.36% U content <400> SEQUENCE: 245 atggataaga agtactcgat cgggctggat atcggaacta attccgtggg ttgggcagtg 60 atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa caccgataga 120 cacagcatca agaagaatct catcggagcc ctgctgtttg actccggcga aaccgcagaa 180 gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag aaatcttttc gaacgaaatg gcaaaggtgg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtgg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcaactga caaggccgac ctcagattga tctacttggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga taactccgac 540 gtggataagc tgttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcca gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaga agaacggact tttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatt ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc cagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga tctgctccgc 1200 aaacagagaa cctttgacaa cggaagcatt ccacaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa tttcgaggaa 1440 gttgtggata agggagcttc ggcacaatcc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620

agcggagaac agaagaaagc gattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc agtggaaatc 1740 agcggagtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcagttgat ccacgacgac 2100 agcctgacct ttaaggagga catccagaaa gcacaagtga gcggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat cctgcaaact 2220 gtgaaggtgg tggacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaagaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tccatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcctggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gcaccgccct gatcaagaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga acttcttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa aaccgaagtg 3300 caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaagaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctggagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actgcccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 cagaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta tcttgcttcg 3720 cactacgaga aactcaaagg gtcaccggaa gataacgaac agaagcagct tttcgtggag 3780 cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc aaagcgcgtg 3840 atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca tagagataag 3900 ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa cctgggagct 3960 ccagccgcct tcaagtactt cgatactact atcgaccgca aaagatacac gtccaccaag 4020 gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga aactaggatc 4080 gatctgtcgc agctgggtgg cgatggtggc ggtggatcct acccatacga cgtgcctgac 4140 tacgcctccg gaggtggtgg ccccaagaag aaacggaagg tgtgatag 4188 <210> SEQ ID NO 246 <211> LENGTH: 4459 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 245, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 246 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatct gccaccatgg ataagaagta ctcgatcggg ctggatatcg gaactaattc 120 cgtgggttgg gcagtgatca cggatgaata caaagtgccg tccaagaagt tcaaggtcct 180 ggggaacacc gatagacaca gcatcaagaa gaatctcatc ggagccctgc tgtttgactc 240 cggcgaaacc gcagaagcga cccggctcaa acgtaccgcg aggcgacgct acacccggcg 300 gaagaatcgc atctgctatc tgcaagaaat cttttcgaac gaaatggcaa aggtggacga 360 cagcttcttc caccgcctgg aagaatcttt cctggtggag gaggacaaga agcatgaacg 420 gcatcctatc tttggaaaca tcgtggacga agtggcgtac cacgaaaagt acccgaccat 480 ctaccatctg cggaagaagt tggttgactc aactgacaag gccgacctca gattgatcta 540 cttggccctc gcccatatga tcaaattccg cggacacttc ctgatcgaag gcgatctgaa 600 ccctgataac tccgacgtgg ataagctgtt cattcaactg gtgcagacct acaaccaact 660 gttcgaagaa aacccaatca atgccagcgg cgtcgatgcc aaggccatcc tgtccgcccg 720 gctgtcgaag tcgcggcgcc tcgaaaacct gatcgcacag ctgccgggag agaagaagaa 780 cggacttttc ggcaacttga tcgctctctc actgggactc actcccaatt tcaagtccaa 840 ttttgacctg gccgaggacg cgaagctgca actctcaaag gacacctacg acgacgactt 900 ggacaatttg ctggcacaaa ttggcgatca gtacgcggat ctgttccttg ccgctaagaa 960 cctttcggac gcaatcttgc tgtccgatat cctgcgcgtg aacaccgaaa taaccaaagc 1020 gccgcttagc gcctcgatga ttaagcggta cgacgagcat caccaggatc tcacgctgct 1080 caaagcgctc gtgagacagc aactgcctga aaagtacaag gagattttct tcgaccagtc 1140 caagaatggg tacgcagggt acatcgatgg aggcgccagc caggaagagt tctataagtt 1200 catcaagcca atcctggaaa agatggacgg aaccgaagaa ctgctggtca agctgaacag 1260 ggaggatctg ctccgcaaac agagaacctt tgacaacgga agcattccac accagatcca 1320 tctgggtgag ctgcacgcca tcttgcggcg ccaggaggac ttttacccat tcctcaagga 1380 caaccgggaa aagatcgaga aaattctgac gttccgcatc ccgtattacg tgggcccact 1440 ggcgcgcggc aattcgcgct tcgcgtggat gactagaaaa tcagaggaaa ccatcactcc 1500 ttggaatttc gaggaagttg tggataaggg agcttcggca caatccttca tcgaacgaat 1560 gaccaacttc gacaagaatc tcccaaacga gaaggtgctt cctaagcaca gcctccttta 1620 cgaatacttc actgtctaca acgaactgac taaagtgaaa tacgttactg aaggaatgag 1680 gaagccggcc tttctgagcg gagaacagaa gaaagcgatt gtcgatctgc tgttcaagac 1740 caaccgcaag gtgaccgtca agcagcttaa agaggactac ttcaagaaga tcgagtgttt 1800 cgactcagtg gaaatcagcg gagtggagga cagattcaac gcttcgctgg gaacctatca 1860 tgatctcctg aagatcatca aggacaagga cttccttgac aacgaggaga acgaggacat 1920 cctggaagat atcgtcctga ccttgaccct tttcgaggat cgcgagatga tcgaggagag 1980 gcttaagacc tacgctcatc tcttcgacga taaggtcatg aaacaactca agcgccgccg 2040 gtacactggt tggggccgcc tctcccgcaa gctgatcaac ggtattcgcg ataaacagag 2100 cggtaaaact atcctggatt tcctcaaatc ggatggcttc gctaatcgta acttcatgca 2160 gttgatccac gacgacagcc tgacctttaa ggaggacatc cagaaagcac aagtgagcgg 2220 acagggagac tcactccatg aacacatcgc gaatctggcc ggttcgccgg cgattaagaa 2280 gggaatcctg caaactgtga aggtggtgga cgagctggtg aaggtcatgg gacggcacaa 2340 accggagaat atcgtgattg aaatggcccg agaaaaccag actacccaga agggccagaa 2400 gaactcccgc gaaaggatga agcggatcga agaaggaatc aaggagctgg gcagccagat 2460 cctgaaagag cacccggtgg aaaacacgca gctgcagaac gagaagctct acctgtacta 2520 tttgcaaaat ggacgggaca tgtacgtgga ccaagagctg gacatcaatc ggttgtctga 2580 ttacgacgtg gaccacatcg ttccacagtc ctttctgaag gatgactcca tcgataacaa 2640 ggtgttgact cgcagcgaca agaacagagg gaagtcagat aatgtgccat cggaggaggt 2700 cgtgaagaag atgaagaatt actggcggca gctcctgaat gcgaagctga ttacccagag 2760 aaagtttgac aatctcacta aagccgagcg cggcggactc tcagagctgg ataaggctgg 2820 attcatcaaa cggcagctgg tcgagactcg gcagattacc aagcacgtgg cgcagatcct 2880 ggactcccgc atgaacacta aatacgacga gaacgataag ctcatccggg aagtgaaggt 2940 gattaccctg aaaagcaaac ttgtgtcgga ctttcggaag gactttcagt tttacaaagt 3000 gagagaaatc aacaactacc atcacgcgca tgacgcatac ctcaacgctg tggtcggcac 3060 cgccctgatc aagaagtacc ctaaacttga atcggagttt gtgtacggag actacaaggt 3120 ctacgacgtg aggaagatga tagccaagtc cgaacaggaa atcgggaaag caactgcgaa 3180 atacttcttt tactcaaaca tcatgaactt cttcaagact gaaattacgc tggccaatgg 3240 agaaatcagg aagaggccac tgatcgaaac taacggagaa acgggcgaaa tcgtgtggga 3300 caagggcagg gacttcgcaa ctgttcgcaa agtgctctct atgccgcaag tcaatattgt 3360 gaagaaaacc gaagtgcaaa ccggcggatt ttcaaaggaa tcgatcctcc caaagagaaa 3420 tagcgacaag ctcattgcac gcaagaaaga ctgggacccg aagaagtacg gaggattcga 3480 ttcgccgact gtcgcatact ccgtcctcgt ggtggccaag gtggagaagg gaaagagcaa 3540 gaagctcaaa tccgtcaaag agctgctggg gattaccatc atggaacgat cctcgttcga 3600 gaagaacccg attgatttcc tggaggcgaa gggttacaag gaggtgaaga aggatctgat 3660 catcaaactg cccaagtact cactgttcga actggaaaat ggtcggaagc gcatgctggc 3720 ttcggccgga gaactccaga aaggaaatga gctggccttg cctagcaagt acgtcaactt 3780 cctctatctt gcttcgcact acgagaaact caaagggtca ccggaagata acgaacagaa 3840 gcagcttttc gtggagcagc acaagcatta tctggatgaa atcatcgaac aaatctccga 3900 gttttcaaag cgcgtgatcc tcgccgacgc caacctcgac aaagtcctgt cggcctacaa 3960 taagcataga gataagccga tcagagaaca ggccgagaac attatccact tgttcaccct 4020 gactaacctg ggagctccag ccgccttcaa gtacttcgat actactatcg accgcaaaag 4080 atacacgtcc accaaggaag ttctggacgc gaccctgatc caccaaagca tcactggact 4140 ctacgaaact aggatcgatc tgtcgcagct gggtggcgat ggtggcggtg gatcctaccc 4200 atacgacgtg cctgactacg cctccggagg tggtggcccc aagaagaaac ggaaggtgtg 4260 atagctagcc atcacattta aaagcatctc agcctaccat gagaataaga gaaagaaaat 4320 gaagatcaat agcttattca tctctttttc tttttcgttg gtgtaaagcc aacaccctgt 4380 ctaaaaaaca taaatttctt taatcatttt gcctcttttc tctgtgcttc aattaataaa 4440 aaatggaaag aacctcgag 4459 <210> SEQ ID NO 247

<211> LENGTH: 4453 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 245, and 3 UTR of ALB <400> SEQUENCE: 247 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatct atggataaga agtactcgat cgggctggat atcggaacta attccgtggg 120 ttgggcagtg atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa 180 caccgataga cacagcatca agaagaatct catcggagcc ctgctgtttg actccggcga 240 aaccgcagaa gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa 300 tcgcatctgc tatctgcaag aaatcttttc gaacgaaatg gcaaaggtgg acgacagctt 360 cttccaccgc ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc 420 tatctttgga aacatcgtgg acgaagtggc gtaccacgaa aagtacccga ccatctacca 480 tctgcggaag aagttggttg actcaactga caaggccgac ctcagattga tctacttggc 540 cctcgcccat atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga 600 taactccgac gtggataagc tgttcattca actggtgcag acctacaacc aactgttcga 660 agaaaaccca atcaatgcca gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc 720 gaagtcgcgg cgcctcgaaa acctgatcgc acagctgccg ggagagaaga agaacggact 780 tttcggcaac ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga 840 cctggccgag gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa 900 tttgctggca caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc 960 ggacgcaatc ttgctgtccg atatcctgcg cgtgaacacc gaaataacca aagcgccgct 1020 tagcgcctcg atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc 1080 gctcgtgaga cagcaactgc ctgaaaagta caaggagatt ttcttcgacc agtccaagaa 1140 tgggtacgca gggtacatcg atggaggcgc cagccaggaa gagttctata agttcatcaa 1200 gccaatcctg gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggagga 1260 tctgctccgc aaacagagaa cctttgacaa cggaagcatt ccacaccaga tccatctggg 1320 tgagctgcac gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg 1380 ggaaaagatc gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg 1440 cggcaattcg cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa 1500 tttcgaggaa gttgtggata agggagcttc ggcacaatcc ttcatcgaac gaatgaccaa 1560 cttcgacaag aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata 1620 cttcactgtc tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc 1680 ggcctttctg agcggagaac agaagaaagc gattgtcgat ctgctgttca agaccaaccg 1740 caaggtgacc gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gtttcgactc 1800 agtggaaatc agcggagtgg aggacagatt caacgcttcg ctgggaacct atcatgatct 1860 cctgaagatc atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga 1920 agatatcgtc ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa 1980 gacctacgct catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac 2040 tggttggggc cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa 2100 aactatcctg gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcagttgat 2160 ccacgacgac agcctgacct ttaaggagga catccagaaa gcacaagtga gcggacaggg 2220 agactcactc catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat 2280 cctgcaaact gtgaaggtgg tggacgagct ggtgaaggtc atgggacggc acaaaccgga 2340 gaatatcgtg attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaagaactc 2400 ccgcgaaagg atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa 2460 agagcacccg gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca 2520 aaatggacgg gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga 2580 cgtggaccac atcgttccac agtcctttct gaaggatgac tccatcgata acaaggtgtt 2640 gactcgcagc gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa 2700 gaagatgaag aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt 2760 tgacaatctc actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat 2820 caaacggcag ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcctggactc 2880 ccgcatgaac actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac 2940 cctgaaaagc aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga 3000 aatcaacaac taccatcacg cgcatgacgc atacctcaac gctgtggtcg gcaccgccct 3060 gatcaagaag taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga 3120 cgtgaggaag atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt 3180 cttttactca aacatcatga acttcttcaa gactgaaatt acgctggcca atggagaaat 3240 caggaagagg ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg 3300 cagggacttc gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa 3360 aaccgaagtg caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga 3420 caagctcatt gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc 3480 gactgtcgca tactccgtcc tcgtggtggc caaggtggag aagggaaaga gcaagaagct 3540 caaatccgtc aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa 3600 cccgattgat ttcctggagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa 3660 actgcccaag tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc 3720 cggagaactc cagaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta 3780 tcttgcttcg cactacgaga aactcaaagg gtcaccggaa gataacgaac agaagcagct 3840 tttcgtggag cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc 3900 aaagcgcgtg atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca 3960 tagagataag ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa 4020 cctgggagct ccagccgcct tcaagtactt cgatactact atcgaccgca aaagatacac 4080 gtccaccaag gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga 4140 aactaggatc gatctgtcgc agctgggtgg cgatggtggc ggtggatcct acccatacga 4200 cgtgcctgac tacgcctccg gaggtggtgg ccccaagaag aaacggaagg tgtgatagct 4260 agccatcaca tttaaaagca tctcagccta ccatgagaat aagagaaaga aaatgaagat 4320 caatagctta ttcatctctt tttctttttc gttggtgtaa agccaacacc ctgtctaaaa 4380 aacataaatt tctttaatca ttttgcctct tttctctgtg cttcaattaa taaaaaatgg 4440 aaagaacctc gag 4453 <210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210> SEQ ID NO 249 <211> LENGTH: 4409 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript comprising Kozak sequence with Cas9 ORF using codons with generally high expression in humans <400> SEQUENCE: 249 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgc ctaagaaaaa gcggaaggtc gacggggata agaagtactc 120 aatcgggctg gatatcggaa ctaattccgt gggttgggca gtgatcacgg atgaatacaa 180 agtgccgtcc aagaagttca aggtcctggg gaacaccgat agacacagca tcaagaaaaa 240 tctcatcgga gccctgctgt ttgactccgg cgaaaccgca gaagcgaccc ggctcaaacg 300 taccgcgagg cgacgctaca cccggcggaa gaatcgcatc tgctatctgc aagagatctt 360 ttcgaacgaa atggcaaagg tcgacgacag cttcttccac cgcctggaag aatctttcct 420 ggtggaggag gacaagaagc atgaacggca tcctatcttt ggaaacatcg tcgacgaagt 480 ggcgtaccac gaaaagtacc cgaccatcta ccatctgcgg aagaagttgg ttgactcaac 540 tgacaaggcc gacctcagat tgatctactt ggccctcgcc catatgatca aattccgcgg 600 acacttcctg atcgaaggcg atctgaaccc tgataactcc gacgtggata agcttttcat 660 tcaactggtg cagacctaca accaactgtt cgaagaaaac ccaatcaatg ctagcggcgt 720 cgatgccaag gccatcctgt ccgcccggct gtcgaagtcg cggcgcctcg aaaacctgat 780 cgcacagctg ccgggagaga aaaagaacgg acttttcggc aacttgatcg ctctctcact 840 gggactcact cccaatttca agtccaattt tgacctggcc gaggacgcga agctgcaact 900 ctcaaaggac acctacgacg acgacttgga caatttgctg gcacaaattg gcgatcagta 960 cgcggatctg ttccttgccg ctaagaacct ttcggacgca atcttgctgt ccgatatcct 1020 gcgcgtgaac accgaaataa ccaaagcgcc gcttagcgcc tcgatgatta agcggtacga 1080 cgagcatcac caggatctca cgctgctcaa agcgctcgtg agacagcaac tgcctgaaaa 1140 gtacaaggag atcttcttcg accagtccaa gaatgggtac gcagggtaca tcgatggagg 1200 cgctagccag gaagagttct ataagttcat caagccaatc ctggaaaaga tggacggaac 1260 cgaagaactg ctggtcaagc tgaacaggga ggatctgctc cggaaacaga gaacctttga 1320 caacggatcc attccccacc agatccatct gggtgagctg cacgccatct tgcggcgcca 1380 ggaggacttt tacccattcc tcaaggacaa ccgggaaaag atcgagaaaa ttctgacgtt 1440 ccgcatcccg tattacgtgg gcccactggc gcgcggcaat tcgcgcttcg cgtggatgac 1500 tagaaaatca gaggaaacca tcactccttg gaatttcgag gaagttgtgg ataagggagc 1560 ttcggcacaa agcttcatcg aacgaatgac caacttcgac aagaatctcc caaacgagaa 1620 ggtgcttcct aagcacagcc tcctttacga atacttcact gtctacaacg aactgactaa 1680 agtgaaatac gttactgaag gaatgaggaa gccggccttt ctgtccggag aacagaagaa 1740 agcaattgtc gatctgctgt tcaagaccaa ccgcaaggtg accgtcaagc agcttaaaga 1800 ggactacttc aagaagatcg agtgtttcga ctcagtggaa atcagcgggg tggaggacag 1860 attcaacgct tcgctgggaa cctatcatga tctcctgaag atcatcaagg acaaggactt 1920 ccttgacaac gaggagaacg aggacatcct ggaagatatc gtcctgacct tgaccctttt 1980 cgaggatcgc gagatgatcg aggagaggct taagacctac gctcatctct tcgacgataa 2040 ggtcatgaaa caactcaagc gccgccggta cactggttgg ggccgcctct cccgcaagct 2100 gatcaacggt attcgcgata aacagagcgg taaaactatc ctggatttcc tcaaatcgga 2160

tggcttcgct aatcgtaact tcatgcaatt gatccacgac gacagcctga cctttaagga 2220 ggacatccaa aaagcacaag tgtccggaca gggagactca ctccatgaac acatcgcgaa 2280 tctggccggt tcgccggcga ttaagaaggg aattctgcaa actgtgaagg tggtcgacga 2340 gctggtgaag gtcatgggac ggcacaaacc ggagaatatc gtgattgaaa tggcccgaga 2400 aaaccagact acccagaagg gccagaaaaa ctcccgcgaa aggatgaagc ggatcgaaga 2460 aggaatcaag gagctgggca gccagatcct gaaagagcac ccggtggaaa acacgcagct 2520 gcagaacgag aagctctacc tgtactattt gcaaaatgga cgggacatgt acgtggacca 2580 agagctggac atcaatcggt tgtctgatta cgacgtggac cacatcgttc cacagtcctt 2640 tctgaaggat gactcgatcg ataacaaggt gttgactcgc agcgacaaga acagagggaa 2700 gtcagataat gtgccatcgg aggaggtcgt gaagaagatg aagaattact ggcggcagct 2760 cctgaatgcg aagctgatta cccagagaaa gtttgacaat ctcactaaag ccgagcgcgg 2820 cggactctca gagctggata aggctggatt catcaaacgg cagctggtcg agactcggca 2880 gattaccaag cacgtggcgc agatcttgga ctcccgcatg aacactaaat acgacgagaa 2940 cgataagctc atccgggaag tgaaggtgat taccctgaaa agcaaacttg tgtcggactt 3000 tcggaaggac tttcagtttt acaaagtgag agaaatcaac aactaccatc acgcgcatga 3060 cgcatacctc aacgctgtgg tcggtaccgc cctgatcaaa aagtacccta aacttgaatc 3120 ggagtttgtg tacggagact acaaggtcta cgacgtgagg aagatgatag ccaagtccga 3180 acaggaaatc gggaaagcaa ctgcgaaata cttcttttac tcaaacatca tgaacttttt 3240 caagactgaa attacgctgg ccaatggaga aatcaggaag aggccactga tcgaaactaa 3300 cggagaaacg ggcgaaatcg tgtgggacaa gggcagggac ttcgcaactg ttcgcaaagt 3360 gctctctatg ccgcaagtca atattgtgaa gaaaaccgaa gtgcaaaccg gcggattttc 3420 aaaggaatcg atcctcccaa agagaaatag cgacaagctc attgcacgca agaaagactg 3480 ggacccgaag aagtacggag gattcgattc gccgactgtc gcatactccg tcctcgtggt 3540 ggccaaggtg gagaagggaa agagcaaaaa gctcaaatcc gtcaaagagc tgctggggat 3600 taccatcatg gaacgatcct cgttcgagaa gaacccgatt gatttcctcg aggcgaaggg 3660 ttacaaggag gtgaagaagg atctgatcat caaactcccc aagtactcac tgttcgaact 3720 ggaaaatggt cggaagcgca tgctggcttc ggccggagaa ctccaaaaag gaaatgagct 3780 ggccttgcct agcaagtacg tcaacttcct ctatcttgct tcgcactacg aaaaactcaa 3840 agggtcaccg gaagataacg aacagaagca gcttttcgtg gagcagcaca agcattatct 3900 ggatgaaatc atcgaacaaa tctccgagtt ttcaaagcgc gtgatcctcg ccgacgccaa 3960 cctcgacaaa gtcctgtcgg cctacaataa gcatagagat aagccgatca gagaacaggc 4020 cgagaacatt atccacttgt tcaccctgac taacctggga gccccagccg ccttcaagta 4080 cttcgatact actatcgatc gcaaaagata cacgtccacc aaggaagttc tggacgcgac 4140 cctgatccac caaagcatca ctggactcta cgaaactagg atcgatctgt cgcagctggg 4200 tggcgattga tagtctagcc atcacattta aaagcatctc agcctaccat gagaataaga 4260 gaaagaaaat gaagatcaat agcttattca tctctttttc tttttcgttg gtgtaaagcc 4320 aacaccctgt ctaaaaaaca taaatttctt taatcatttt gcctcttttc tctgtgcttc 4380 aattaataaa aaatggaaag aacctcgag 4409 <210> SEQ ID NO 250 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with splice junctions removed; 12.75% U content <400> SEQUENCE: 250 atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg atgggcagtc 60 atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa cacagacaga 120 cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga aacagcagaa 180 gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa cagaatctgc 240 tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt cttccaccgg 300 ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc actggcacac 480 atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga caacagcgac 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact gttcggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc ggcattcctg 1620 agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag aaaggtcaca 1680 gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag cgtcgaaatc 1740 agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac aggatgggga 1980 agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa gacaatcctg 2040 gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg agacagcctg 2160 cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga aaacatcgtc 2280 atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca aaacggaaga 2460 gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagatgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt cgacaacctg 2700 acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat caagagacag 2760 ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac actgaagagc 2880 aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga aatcaacaac 2940 taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact gatcaagaag 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa gacagaagtc 3300 cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga caagctgatc 3360 gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc gacagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa cccgatcgac 3540 ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc aggagaactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag caagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac aagcacaaag 4020 gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag aaaggtctag 4140 <210> SEQ ID NO 251 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 250, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 251 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360

cagcttcttc caccggctgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcaaaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 252 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with minimal uridine codons frequently used in humans in general; 12.75% U content <400> SEQUENCE: 252 atggacaaga agtacagcat cggcctggac atcggcacca acagcgtggg ctgggccgtg 60 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgacaga 120 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag 180 gccaccagac tgaagagaac cgccagaaga agatacacca gaagaaagaa cagaatctgc 240 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 300 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agagacaccc catcttcggc 360 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 420 aagctggtgg acagcaccga caaggccgac ctgagactga tctacctggc cctggcccac 480 atgatcaagt tcagaggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg ccagactgag caagagcaga 660 agactggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc 900 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgccagc 960 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgaga 1020 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc 1080 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg 1140 gagaagatgg acggcaccga ggagctgctg gtgaagctga acagagagga cctgctgaga 1200 aagcagagaa ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac 1260 gccatcctga gaagacagga ggacttctac cccttcctga aggacaacag agagaagatc 1320 gagaagatcc tgaccttcag aatcccctac tacgtgggcc ccctggccag aggcaacagc 1380 agattcgcct ggatgaccag aaagagcgag gagaccatca ccccctggaa cttcgaggag 1440 gtggtggaca agggcgccag cgcccagagc ttcatcgaga gaatgaccaa cttcgacaag 1500 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgagaaagcc cgccttcctg 1620 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaacag aaaggtgacc 1680 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc 1740 agcggcgtgg aggacagatt caacgccagc ctgggcacct accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860 ctgaccctga ccctgttcga ggacagagag atgatcgagg agagactgaa gacctacgcc 1920 cacctgttcg acgacaaggt gatgaagcag ctgaagagaa gaagatacac cggctggggc 1980 agactgagca gaaagctgat caacggcatc agagacaagc agagcggcaa gaccatcctg 2040 gacttcctga agagcgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg 2160 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc 2220 gtgaaggtgg tggacgagct ggtgaaggtg atgggcagac acaagcccga gaacatcgtg 2280 atcgagatgg ccagagagaa ccagaccacc cagaagggcc agaagaacag cagagagaga 2340 atgaagagaa tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc 2400 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggcaga 2460 gacatgtacg tggaccagga gctggacatc aacagactga gcgactacga cgtggaccac 2520 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gaccagaagc 2580 gacaagaaca gaggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag 2640 aactactgga gacagctgct gaacgccaag ctgatcaccc agagaaagtt cgacaacctg 2700 accaaggccg agagaggcgg cctgagcgag ctggacaagg ccggcttcat caagagacag 2760 ctggtggaga ccagacagat caccaagcac gtggcccaga tcctggacag cagaatgaac 2820 accaagtacg acgagaacga caagctgatc agagaggtga aggtgatcac cctgaagagc 2880 aagctggtga gcgacttcag aaaggacttc cagttctaca aggtgagaga gatcaacaac 2940 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgagaaag 3060 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc 3120

aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat cagaaagaga 3180 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg cagagacttc 3240 gccaccgtga gaaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300 cagaccggcg gcttcagcaa ggagagcatc ctgcccaaga gaaacagcga caagctgatc 3360 gccagaaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc 3420 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg 3480 aaggagctgc tgggcatcac catcatggag agaagcagct tcgagaagaa ccccatcgac 3540 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600 tacagcctgt tcgagctgga gaacggcaga aagagaatgc tggccagcgc cggcgagctg 3660 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc 3720 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag 3780 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagagagtg 3840 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca cagagacaag 3900 cccatcagag agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960 cccgccgcct tcaagtactt cgacaccacc atcgacagaa agagatacac cagcaccaag 4020 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gaccagaatc 4080 gacctgagcc agctgggcgg cgacggcggc ggcagcccca agaagaagag aaaggtgtga 4140 <210> SEQ ID NO 253 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 252, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 253 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcggc ctggacatcg gcaccaacag 120 cgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaagt tcaaggtgct 180 gggcaacacc gacagacaca gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag 240 cggcgagacc gccgaggcca ccagactgaa gagaaccgcc agaagaagat acaccagaag 300 aaagaacaga atctgctacc tgcaggagat cttcagcaac gagatggcca aggtggacga 360 cagcttcttc cacagactgg aggagagctt cctggtggag gaggacaaga agcacgagag 420 acaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat 480 ctaccacctg agaaagaagc tggtggacag caccgacaag gccgacctga gactgatcta 540 cctggccctg gcccacatga tcaagttcag aggccacttc ctgatcgagg gcgacctgaa 600 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct 660 gttcgaggag aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgagcgccag 720 actgagcaag agcagaagac tggagaacct gatcgcccag ctgcccggcg agaagaagaa 780 cggcctgttc ggcaacctga tcgccctgag cctgggcctg acccccaact tcaagagcaa 840 cttcgacctg gccgaggacg ccaagctgca gctgagcaag gacacctacg acgacgacct 900 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgttcctgg ccgccaagaa 960 cctgagcgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc 1020 ccccctgagc gccagcatga tcaagagata cgacgagcac caccaggacc tgaccctgct 1080 gaaggccctg gtgagacagc agctgcccga gaagtacaag gagatcttct tcgaccagag 1140 caagaacggc tacgccggct acatcgacgg cggcgccagc caggaggagt tctacaagtt 1200 catcaagccc atcctggaga agatggacgg caccgaggag ctgctggtga agctgaacag 1260 agaggacctg ctgagaaagc agagaacctt cgacaacggc agcatccccc accagatcca 1320 cctgggcgag ctgcacgcca tcctgagaag acaggaggac ttctacccct tcctgaagga 1380 caacagagag aagatcgaga agatcctgac cttcagaatc ccctactacg tgggccccct 1440 ggccagaggc aacagcagat tcgcctggat gaccagaaag agcgaggaga ccatcacccc 1500 ctggaacttc gaggaggtgg tggacaaggg cgccagcgcc cagagcttca tcgagagaat 1560 gaccaacttc gacaagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta 1620 cgagtacttc accgtgtaca acgagctgac caaggtgaag tacgtgaccg agggcatgag 1680 aaagcccgcc ttcctgagcg gcgagcagaa gaaggccatc gtggacctgc tgttcaagac 1740 caacagaaag gtgaccgtga agcagctgaa ggaggactac ttcaagaaga tcgagtgctt 1800 cgacagcgtg gagatcagcg gcgtggagga cagattcaac gccagcctgg gcacctacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaggaga acgaggacat 1920 cctggaggac atcgtgctga ccctgaccct gttcgaggac agagagatga tcgaggagag 1980 actgaagacc tacgcccacc tgttcgacga caaggtgatg aagcagctga agagaagaag 2040 atacaccggc tggggcagac tgagcagaaa gctgatcaac ggcatcagag acaagcagag 2100 cggcaagacc atcctggact tcctgaagag cgacggcttc gccaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgaccttcaa ggaggacatc cagaaggccc aggtgagcgg 2220 ccagggcgac agcctgcacg agcacatcgc caacctggcc ggcagccccg ccatcaagaa 2280 gggcatcctg cagaccgtga aggtggtgga cgagctggtg aaggtgatgg gcagacacaa 2340 gcccgagaac atcgtgatcg agatggccag agagaaccag accacccaga agggccagaa 2400 gaacagcaga gagagaatga agagaatcga ggagggcatc aaggagctgg gcagccagat 2460 cctgaaggag caccccgtgg agaacaccca gctgcagaac gagaagctgt acctgtacta 2520 cctgcagaac ggcagagaca tgtacgtgga ccaggagctg gacatcaaca gactgagcga 2580 ctacgacgtg gaccacatcg tgccccagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtgctgacc agaagcgaca agaacagagg caagagcgac aacgtgccca gcgaggaggt 2700 ggtgaagaag atgaagaact actggagaca gctgctgaac gccaagctga tcacccagag 2760 aaagttcgac aacctgacca aggccgagag aggcggcctg agcgagctgg acaaggccgg 2820 cttcatcaag agacagctgg tggagaccag acagatcacc aagcacgtgg cccagatcct 2880 ggacagcaga atgaacacca agtacgacga gaacgacaag ctgatcagag aggtgaaggt 2940 gatcaccctg aagagcaagc tggtgagcga cttcagaaag gacttccagt tctacaaggt 3000 gagagagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tggtgggcac 3060 cgccctgatc aagaagtacc ccaagctgga gagcgagttc gtgtacggcg actacaaggt 3120 gtacgacgtg agaaagatga tcgccaagag cgagcaggag atcggcaagg ccaccgccaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagacc gagatcaccc tggccaacgg 3240 cgagatcaga aagagacccc tgatcgagac caacggcgag accggcgaga tcgtgtggga 3300 caagggcaga gacttcgcca ccgtgagaaa ggtgctgagc atgccccagg tgaacatcgt 3360 gaagaagacc gaggtgcaga ccggcggctt cagcaaggag agcatcctgc ccaagagaaa 3420 cagcgacaag ctgatcgcca gaaagaagga ctgggacccc aagaagtacg gcggcttcga 3480 cagccccacc gtggcctaca gcgtgctggt ggtggccaag gtggagaagg gcaagagcaa 3540 gaagctgaag agcgtgaagg agctgctggg catcaccatc atggagagaa gcagcttcga 3600 gaagaacccc atcgacttcc tggaggccaa gggctacaag gaggtgaaga aggacctgat 3660 catcaagctg cccaagtaca gcctgttcga gctggagaac ggcagaaaga gaatgctggc 3720 cagcgccggc gagctgcaga agggcaacga gctggccctg cccagcaagt acgtgaactt 3780 cctgtacctg gccagccact acgagaagct gaagggcagc cccgaggaca acgagcagaa 3840 gcagctgttc gtggagcagc acaagcacta cctggacgag atcatcgagc agatcagcga 3900 gttcagcaag agagtgatcc tggccgacgc caacctggac aaggtgctga gcgcctacaa 3960 caagcacaga gacaagccca tcagagagca ggccgagaac atcatccacc tgttcaccct 4020 gaccaacctg ggcgcccccg ccgccttcaa gtacttcgac accaccatcg acagaaagag 4080 atacaccagc accaaggagg tgctggacgc caccctgatc caccagagca tcaccggcct 4140 gtacgagacc agaatcgacc tgagccagct gggcggcgac ggcggcggca gccccaagaa 4200 gaagagaaag gtgtgactag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 254 <211> LENGTH: 4140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 ORF with minimal uridine codons infrequently used in humans in general; 12.75% U content <400> SEQUENCE: 254 atggacaaaa aatacagcat agggctagac atagggacga acagcgtagg gtgggcggta 60 ataacggacg aatacaaagt accgagcaaa aaattcaaag tactagggaa cacggaccga 120 cacagcataa aaaaaaacct aataggggcg ctactattcg acagcgggga aacggcggaa 180 gcgacgcgac taaaacgaac ggcgcgacga cgatacacgc gacgaaaaaa ccgaatatgc 240 tacctacaag aaatattcag caacgaaatg gcgaaagtag acgacagctt cttccaccga 300 ctagaagaaa gcttcctagt agaagaagac aaaaaacacg aacgacaccc gatattcggg 360 aacatagtag acgaagtagc gtaccacgaa aaatacccga cgatatacca cctacgaaaa 420 aaactagtag acagcacgga caaagcggac ctacgactaa tatacctagc gctagcgcac 480 atgataaaat tccgagggca cttcctaata gaaggggacc taaacccgga caacagcgac 540 gtagacaaac tattcataca actagtacaa acgtacaacc aactattcga agaaaacccg 600 ataaacgcga gcggggtaga cgcgaaagcg atactaagcg cgcgactaag caaaagccga 660 cgactagaaa acctaatagc gcaactaccg ggggaaaaaa aaaacgggct attcgggaac 720 ctaatagcgc taagcctagg gctaacgccg aacttcaaaa gcaacttcga cctagcggaa 780 gacgcgaaac tacaactaag caaagacacg tacgacgacg acctagacaa cctactagcg 840 caaatagggg accaatacgc ggacctattc ctagcggcga aaaacctaag cgacgcgata 900 ctactaagcg acatactacg agtaaacacg gaaataacga aagcgccgct aagcgcgagc 960 atgataaaac gatacgacga acaccaccaa gacctaacgc tactaaaagc gctagtacga 1020 caacaactac cggaaaaata caaagaaata ttcttcgacc aaagcaaaaa cgggtacgcg 1080 gggtacatag acgggggggc gagccaagaa gaattctaca aattcataaa accgatacta 1140 gaaaaaatgg acgggacgga agaactacta gtaaaactaa accgagaaga cctactacga 1200 aaacaacgaa cgttcgacaa cgggagcata ccgcaccaaa tacacctagg ggaactacac 1260 gcgatactac gacgacaaga agacttctac ccgttcctaa aagacaaccg agaaaaaata 1320 gaaaaaatac taacgttccg aataccgtac tacgtagggc cgctagcgcg agggaacagc 1380

cgattcgcgt ggatgacgcg aaaaagcgaa gaaacgataa cgccgtggaa cttcgaagaa 1440 gtagtagaca aaggggcgag cgcgcaaagc ttcatagaac gaatgacgaa cttcgacaaa 1500 aacctaccga acgaaaaagt actaccgaaa cacagcctac tatacgaata cttcacggta 1560 tacaacgaac taacgaaagt aaaatacgta acggaaggga tgcgaaaacc ggcgttccta 1620 agcggggaac aaaaaaaagc gatagtagac ctactattca aaacgaaccg aaaagtaacg 1680 gtaaaacaac taaaagaaga ctacttcaaa aaaatagaat gcttcgacag cgtagaaata 1740 agcggggtag aagaccgatt caacgcgagc ctagggacgt accacgacct actaaaaata 1800 ataaaagaca aagacttcct agacaacgaa gaaaacgaag acatactaga agacatagta 1860 ctaacgctaa cgctattcga agaccgagaa atgatagaag aacgactaaa aacgtacgcg 1920 cacctattcg acgacaaagt aatgaaacaa ctaaaacgac gacgatacac ggggtggggg 1980 cgactaagcc gaaaactaat aaacgggata cgagacaaac aaagcgggaa aacgatacta 2040 gacttcctaa aaagcgacgg gttcgcgaac cgaaacttca tgcaactaat acacgacgac 2100 agcctaacgt tcaaagaaga catacaaaaa gcgcaagtaa gcgggcaagg ggacagccta 2160 cacgaacaca tagcgaacct agcggggagc ccggcgataa aaaaagggat actacaaacg 2220 gtaaaagtag tagacgaact agtaaaagta atggggcgac acaaaccgga aaacatagta 2280 atagaaatgg cgcgagaaaa ccaaacgacg caaaaagggc aaaaaaacag ccgagaacga 2340 atgaaacgaa tagaagaagg gataaaagaa ctagggagcc aaatactaaa agaacacccg 2400 gtagaaaaca cgcaactaca aaacgaaaaa ctatacctat actacctaca aaacgggcga 2460 gacatgtacg tagaccaaga actagacata aaccgactaa gcgactacga cgtagaccac 2520 atagtaccgc aaagcttcct aaaagacgac agcatagaca acaaagtact aacgcgaagc 2580 gacaaaaacc gagggaaaag cgacaacgta ccgagcgaag aagtagtaaa aaaaatgaaa 2640 aactactggc gacaactact aaacgcgaaa ctaataacgc aacgaaaatt cgacaaccta 2700 acgaaagcgg aacgaggggg gctaagcgaa ctagacaaag cggggttcat aaaacgacaa 2760 ctagtagaaa cgcgacaaat aacgaaacac gtagcgcaaa tactagacag ccgaatgaac 2820 acgaaatacg acgaaaacga caaactaata cgagaagtaa aagtaataac gctaaaaagc 2880 aaactagtaa gcgacttccg aaaagacttc caattctaca aagtacgaga aataaacaac 2940 taccaccacg cgcacgacgc gtacctaaac gcggtagtag ggacggcgct aataaaaaaa 3000 tacccgaaac tagaaagcga attcgtatac ggggactaca aagtatacga cgtacgaaaa 3060 atgatagcga aaagcgaaca agaaataggg aaagcgacgg cgaaatactt cttctacagc 3120 aacataatga acttcttcaa aacggaaata acgctagcga acggggaaat acgaaaacga 3180 ccgctaatag aaacgaacgg ggaaacgggg gaaatagtat gggacaaagg gcgagacttc 3240 gcgacggtac gaaaagtact aagcatgccg caagtaaaca tagtaaaaaa aacggaagta 3300 caaacggggg ggttcagcaa agaaagcata ctaccgaaac gaaacagcga caaactaata 3360 gcgcgaaaaa aagactggga cccgaaaaaa tacggggggt tcgacagccc gacggtagcg 3420 tacagcgtac tagtagtagc gaaagtagaa aaagggaaaa gcaaaaaact aaaaagcgta 3480 aaagaactac tagggataac gataatggaa cgaagcagct tcgaaaaaaa cccgatagac 3540 ttcctagaag cgaaagggta caaagaagta aaaaaagacc taataataaa actaccgaaa 3600 tacagcctat tcgaactaga aaacgggcga aaacgaatgc tagcgagcgc gggggaacta 3660 caaaaaggga acgaactagc gctaccgagc aaatacgtaa acttcctata cctagcgagc 3720 cactacgaaa aactaaaagg gagcccggaa gacaacgaac aaaaacaact attcgtagaa 3780 caacacaaac actacctaga cgaaataata gaacaaataa gcgaattcag caaacgagta 3840 atactagcgg acgcgaacct agacaaagta ctaagcgcgt acaacaaaca ccgagacaaa 3900 ccgatacgag aacaagcgga aaacataata cacctattca cgctaacgaa cctaggggcg 3960 ccggcggcgt tcaaatactt cgacacgacg atagaccgaa aacgatacac gagcacgaaa 4020 gaagtactag acgcgacgct aatacaccaa agcataacgg ggctatacga aacgcgaata 4080 gacctaagcc aactaggggg ggacgggggg gggagcccga aaaaaaaacg aaaagtatga 4140 <210> SEQ ID NO 255 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR of HSD, ORF corresponding to SEQ ID NO: 254, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 255 gggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaaaaaata cagcataggg ctagacatag ggacgaacag 120 cgtagggtgg gcggtaataa cggacgaata caaagtaccg agcaaaaaat tcaaagtact 180 agggaacacg gaccgacaca gcataaaaaa aaacctaata ggggcgctac tattcgacag 240 cggggaaacg gcggaagcga cgcgactaaa acgaacggcg cgacgacgat acacgcgacg 300 aaaaaaccga atatgctacc tacaagaaat attcagcaac gaaatggcga aagtagacga 360 cagcttcttc caccgactag aagaaagctt cctagtagaa gaagacaaaa aacacgaacg 420 acacccgata ttcgggaaca tagtagacga agtagcgtac cacgaaaaat acccgacgat 480 ataccaccta cgaaaaaaac tagtagacag cacggacaaa gcggacctac gactaatata 540 cctagcgcta gcgcacatga taaaattccg agggcacttc ctaatagaag gggacctaaa 600 cccggacaac agcgacgtag acaaactatt catacaacta gtacaaacgt acaaccaact 660 attcgaagaa aacccgataa acgcgagcgg ggtagacgcg aaagcgatac taagcgcgcg 720 actaagcaaa agccgacgac tagaaaacct aatagcgcaa ctaccggggg aaaaaaaaaa 780 cgggctattc gggaacctaa tagcgctaag cctagggcta acgccgaact tcaaaagcaa 840 cttcgaccta gcggaagacg cgaaactaca actaagcaaa gacacgtacg acgacgacct 900 agacaaccta ctagcgcaaa taggggacca atacgcggac ctattcctag cggcgaaaaa 960 cctaagcgac gcgatactac taagcgacat actacgagta aacacggaaa taacgaaagc 1020 gccgctaagc gcgagcatga taaaacgata cgacgaacac caccaagacc taacgctact 1080 aaaagcgcta gtacgacaac aactaccgga aaaatacaaa gaaatattct tcgaccaaag 1140 caaaaacggg tacgcggggt acatagacgg gggggcgagc caagaagaat tctacaaatt 1200 cataaaaccg atactagaaa aaatggacgg gacggaagaa ctactagtaa aactaaaccg 1260 agaagaccta ctacgaaaac aacgaacgtt cgacaacggg agcataccgc accaaataca 1320 cctaggggaa ctacacgcga tactacgacg acaagaagac ttctacccgt tcctaaaaga 1380 caaccgagaa aaaatagaaa aaatactaac gttccgaata ccgtactacg tagggccgct 1440 agcgcgaggg aacagccgat tcgcgtggat gacgcgaaaa agcgaagaaa cgataacgcc 1500 gtggaacttc gaagaagtag tagacaaagg ggcgagcgcg caaagcttca tagaacgaat 1560 gacgaacttc gacaaaaacc taccgaacga aaaagtacta ccgaaacaca gcctactata 1620 cgaatacttc acggtataca acgaactaac gaaagtaaaa tacgtaacgg aagggatgcg 1680 aaaaccggcg ttcctaagcg gggaacaaaa aaaagcgata gtagacctac tattcaaaac 1740 gaaccgaaaa gtaacggtaa aacaactaaa agaagactac ttcaaaaaaa tagaatgctt 1800 cgacagcgta gaaataagcg gggtagaaga ccgattcaac gcgagcctag ggacgtacca 1860 cgacctacta aaaataataa aagacaaaga cttcctagac aacgaagaaa acgaagacat 1920 actagaagac atagtactaa cgctaacgct attcgaagac cgagaaatga tagaagaacg 1980 actaaaaacg tacgcgcacc tattcgacga caaagtaatg aaacaactaa aacgacgacg 2040 atacacgggg tgggggcgac taagccgaaa actaataaac gggatacgag acaaacaaag 2100 cgggaaaacg atactagact tcctaaaaag cgacgggttc gcgaaccgaa acttcatgca 2160 actaatacac gacgacagcc taacgttcaa agaagacata caaaaagcgc aagtaagcgg 2220 gcaaggggac agcctacacg aacacatagc gaacctagcg gggagcccgg cgataaaaaa 2280 agggatacta caaacggtaa aagtagtaga cgaactagta aaagtaatgg ggcgacacaa 2340 accggaaaac atagtaatag aaatggcgcg agaaaaccaa acgacgcaaa aagggcaaaa 2400 aaacagccga gaacgaatga aacgaataga agaagggata aaagaactag ggagccaaat 2460 actaaaagaa cacccggtag aaaacacgca actacaaaac gaaaaactat acctatacta 2520 cctacaaaac gggcgagaca tgtacgtaga ccaagaacta gacataaacc gactaagcga 2580 ctacgacgta gaccacatag taccgcaaag cttcctaaaa gacgacagca tagacaacaa 2640 agtactaacg cgaagcgaca aaaaccgagg gaaaagcgac aacgtaccga gcgaagaagt 2700 agtaaaaaaa atgaaaaact actggcgaca actactaaac gcgaaactaa taacgcaacg 2760 aaaattcgac aacctaacga aagcggaacg aggggggcta agcgaactag acaaagcggg 2820 gttcataaaa cgacaactag tagaaacgcg acaaataacg aaacacgtag cgcaaatact 2880 agacagccga atgaacacga aatacgacga aaacgacaaa ctaatacgag aagtaaaagt 2940 aataacgcta aaaagcaaac tagtaagcga cttccgaaaa gacttccaat tctacaaagt 3000 acgagaaata aacaactacc accacgcgca cgacgcgtac ctaaacgcgg tagtagggac 3060 ggcgctaata aaaaaatacc cgaaactaga aagcgaattc gtatacgggg actacaaagt 3120 atacgacgta cgaaaaatga tagcgaaaag cgaacaagaa atagggaaag cgacggcgaa 3180 atacttcttc tacagcaaca taatgaactt cttcaaaacg gaaataacgc tagcgaacgg 3240 ggaaatacga aaacgaccgc taatagaaac gaacggggaa acgggggaaa tagtatggga 3300 caaagggcga gacttcgcga cggtacgaaa agtactaagc atgccgcaag taaacatagt 3360 aaaaaaaacg gaagtacaaa cgggggggtt cagcaaagaa agcatactac cgaaacgaaa 3420 cagcgacaaa ctaatagcgc gaaaaaaaga ctgggacccg aaaaaatacg gggggttcga 3480 cagcccgacg gtagcgtaca gcgtactagt agtagcgaaa gtagaaaaag ggaaaagcaa 3540 aaaactaaaa agcgtaaaag aactactagg gataacgata atggaacgaa gcagcttcga 3600 aaaaaacccg atagacttcc tagaagcgaa agggtacaaa gaagtaaaaa aagacctaat 3660 aataaaacta ccgaaataca gcctattcga actagaaaac gggcgaaaac gaatgctagc 3720 gagcgcgggg gaactacaaa aagggaacga actagcgcta ccgagcaaat acgtaaactt 3780 cctataccta gcgagccact acgaaaaact aaaagggagc ccggaagaca acgaacaaaa 3840 acaactattc gtagaacaac acaaacacta cctagacgaa ataatagaac aaataagcga 3900 attcagcaaa cgagtaatac tagcggacgc gaacctagac aaagtactaa gcgcgtacaa 3960 caaacaccga gacaaaccga tacgagaaca agcggaaaac ataatacacc tattcacgct 4020 aacgaaccta ggggcgccgg cggcgttcaa atacttcgac acgacgatag accgaaaacg 4080 atacacgagc acgaaagaag tactagacgc gacgctaata caccaaagca taacggggct 4140 atacgaaacg cgaatagacc taagccaact agggggggac ggggggggga gcccgaaaaa 4200 aaaacgaaaa gtatgactag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380

tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 256 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR of HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 256 aggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 257 <211> LENGTH: 4481 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from CMV, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 257 gggcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac 60 cgatccagcc tccgcggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt 120 gactcaccgt ccttgacacg gccaccatgg acaagaagta cagcatcgga ctggacatcg 180 gaacaaacag cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt 240 tcaaggtcct gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc 300 tgttcgacag cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat 360 acacaagaag aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa 420 aggtcgacga cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga 480 agcacgaaag acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt 540 acccgacaat ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga 600 gactgatcta cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag 660 gagacctgaa cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat 720 acaaccagct gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc 780 tgagcgcaag actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag 840 aaaagaagaa cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact 900 tcaagagcaa cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg 960 acgacgacct ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg 1020 cagcaaagaa cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa 1080 tcacaaaggc accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc 1140 tgacactgct gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct 1200 tcgaccagag caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat 1260 tctacaagtt catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca 1320 agctgaacag agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc 1380 accagatcca cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt 1440 tcctgaagga caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg 1500 tcggaccgct ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa 1560 caatcacacc gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca 1620 tcgaaagaat gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca 1680 gcctgctgta cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag 1740 aaggaatgag aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc 1800 tgttcaagac aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga 1860 tcgaatgctt cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg 1920 gaacatacca cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa 1980 acgaagacat cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga 2040 tcgaagaaag actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga 2100 agagaagaag atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag 2160 acaagcagag cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa 2220 acttcatgca gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac 2280

aggtcagcgg acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg 2340 caatcaagaa gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg 2400 gaagacacaa gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga 2460 agggacagaa gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg 2520 gaagccagat cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt 2580 acctgtacta cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca 2640 gactgagcga ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca 2700 tcgacaacaa ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga 2760 gcgaagaagt cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga 2820 tcacacagag aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg 2880 acaaggcagg attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg 2940 cacagatcct ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag 3000 aagtcaaggt catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt 3060 tctacaaggt cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag 3120 tcgtcggaac agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag 3180 actacaaggt ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg 3240 caacagcaaa gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac 3300 tggcaaacgg agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa 3360 tcgtctggga caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg 3420 tcaacatcgt caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc 3480 cgaagagaaa cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg 3540 gaggattcga cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg 3600 gaaagagcaa gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa 3660 gcagcttcga aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga 3720 aggacctgat catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga 3780 gaatgctggc aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt 3840 acgtcaactt cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca 3900 acgaacagaa gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac 3960 agatcagcga attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga 4020 gcgcatacaa caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc 4080 tgttcacact gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg 4140 acagaaagag atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca 4200 tcacaggact gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa 4260 gcccgaagaa gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc 4320 atgagaataa gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt 4380 tggtgtaaag ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt 4440 tctctgtgct tcaattaata aaaaatggaa agaacctcga g 4481 <210> SEQ ID NO 258 <211> LENGTH: 4348 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from HBB, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of HBB <400> SEQUENCE: 258 gggacatttg cttctgacac aactgtgttc actagcaacc tcaaacagac accggatctg 60 ccaccatgga caagaagtac agcatcggac tggacatcgg aacaaacagc gtcggatggg 120 cagtcatcac agacgaatac aaggtcccga gcaagaagtt caaggtcctg ggaaacacag 180 acagacacag catcaagaag aacctgatcg gagcactgct gttcgacagc ggagaaacag 240 cagaagcaac aagactgaag agaacagcaa gaagaagata cacaagaaga aagaacagaa 300 tctgctacct gcaggaaatc ttcagcaacg aaatggcaaa ggtcgacgac agcttcttcc 360 acagactgga agaaagcttc ctggtcgaag aagacaagaa gcacgaaaga cacccgatct 420 tcggaaacat cgtcgacgaa gtcgcatacc acgaaaagta cccgacaatc taccacctga 480 gaaagaagct ggtcgacagc acagacaagg cagacctgag actgatctac ctggcactgg 540 cacacatgat caagttcaga ggacacttcc tgatcgaagg agacctgaac ccggacaaca 600 gcgacgtcga caagctgttc atccagctgg tccagacata caaccagctg ttcgaagaaa 660 acccgatcaa cgcaagcgga gtcgacgcaa aggcaatcct gagcgcaaga ctgagcaaga 720 gcagaagact ggaaaacctg atcgcacagc tgccgggaga aaagaagaac ggactgttcg 780 gaaacctgat cgcactgagc ctgggactga caccgaactt caagagcaac ttcgacctgg 840 cagaagacgc aaagctgcag ctgagcaagg acacatacga cgacgacctg gacaacctgc 900 tggcacagat cggagaccag tacgcagacc tgttcctggc agcaaagaac ctgagcgacg 960 caatcctgct gagcgacatc ctgagagtca acacagaaat cacaaaggca ccgctgagcg 1020 caagcatgat caagagatac gacgaacacc accaggacct gacactgctg aaggcactgg 1080 tcagacagca gctgccggaa aagtacaagg aaatcttctt cgaccagagc aagaacggat 1140 acgcaggata catcgacgga ggagcaagcc aggaagaatt ctacaagttc atcaagccga 1200 tcctggaaaa gatggacgga acagaagaac tgctggtcaa gctgaacaga gaagacctgc 1260 tgagaaagca gagaacattc gacaacggaa gcatcccgca ccagatccac ctgggagaac 1320 tgcacgcaat cctgagaaga caggaagact tctacccgtt cctgaaggac aacagagaaa 1380 agatcgaaaa gatcctgaca ttcagaatcc cgtactacgt cggaccgctg gcaagaggaa 1440 acagcagatt cgcatggatg acaagaaaga gcgaagaaac aatcacaccg tggaacttcg 1500 aagaagtcgt cgacaaggga gcaagcgcac agagcttcat cgaaagaatg acaaacttcg 1560 acaagaacct gccgaacgaa aaggtcctgc cgaagcacag cctgctgtac gaatacttca 1620 cagtctacaa cgaactgaca aaggtcaagt acgtcacaga aggaatgaga aagccggcat 1680 tcctgagcgg agaacagaag aaggcaatcg tcgacctgct gttcaagaca aacagaaagg 1740 tcacagtcaa gcagctgaag gaagactact tcaagaagat cgaatgcttc gacagcgtcg 1800 aaatcagcgg agtcgaagac agattcaacg caagcctggg aacataccac gacctgctga 1860 agatcatcaa ggacaaggac ttcctggaca acgaagaaaa cgaagacatc ctggaagaca 1920 tcgtcctgac actgacactg ttcgaagaca gagaaatgat cgaagaaaga ctgaagacat 1980 acgcacacct gttcgacgac aaggtcatga agcagctgaa gagaagaaga tacacaggat 2040 ggggaagact gagcagaaag ctgatcaacg gaatcagaga caagcagagc ggaaagacaa 2100 tcctggactt cctgaagagc gacggattcg caaacagaaa cttcatgcag ctgatccacg 2160 acgacagcct gacattcaag gaagacatcc agaaggcaca ggtcagcgga cagggagaca 2220 gcctgcacga acacatcgca aacctggcag gaagcccggc aatcaagaag ggaatcctgc 2280 agacagtcaa ggtcgtcgac gaactggtca aggtcatggg aagacacaag ccggaaaaca 2340 tcgtcatcga aatggcaaga gaaaaccaga caacacagaa gggacagaag aacagcagag 2400 aaagaatgaa gagaatcgaa gaaggaatca aggaactggg aagccagatc ctgaaggaac 2460 acccggtcga aaacacacag ctgcagaacg aaaagctgta cctgtactac ctgcagaacg 2520 gaagagacat gtacgtcgac caggaactgg acatcaacag actgagcgac tacgacgtcg 2580 accacatcgt cccgcagagc ttcctgaagg acgacagcat cgacaacaag gtcctgacaa 2640 gaagcgacaa gaacagagga aagagcgaca acgtcccgag cgaagaagtc gtcaagaaga 2700 tgaagaacta ctggagacag ctgctgaacg caaagctgat cacacagaga aagttcgaca 2760 acctgacaaa ggcagagaga ggaggactga gcgaactgga caaggcagga ttcatcaaga 2820 gacagctggt cgaaacaaga cagatcacaa agcacgtcgc acagatcctg gacagcagaa 2880 tgaacacaaa gtacgacgaa aacgacaagc tgatcagaga agtcaaggtc atcacactga 2940 agagcaagct ggtcagcgac ttcagaaagg acttccagtt ctacaaggtc agagaaatca 3000 acaactacca ccacgcacac gacgcatacc tgaacgcagt cgtcggaaca gcactgatca 3060 agaagtaccc gaagctggaa agcgaattcg tctacggaga ctacaaggtc tacgacgtca 3120 gaaagatgat cgcaaagagc gaacaggaaa tcggaaaggc aacagcaaag tacttcttct 3180 acagcaacat catgaacttc ttcaagacag aaatcacact ggcaaacgga gaaatcagaa 3240 agagaccgct gatcgaaaca aacggagaaa caggagaaat cgtctgggac aagggaagag 3300 acttcgcaac agtcagaaag gtcctgagca tgccgcaggt caacatcgtc aagaagacag 3360 aagtccagac aggaggattc agcaaggaaa gcatcctgcc gaagagaaac agcgacaagc 3420 tgatcgcaag aaagaaggac tgggacccga agaagtacgg aggattcgac agcccgacag 3480 tcgcatacag cgtcctggtc gtcgcaaagg tcgaaaaggg aaagagcaag aagctgaaga 3540 gcgtcaagga actgctggga atcacaatca tggaaagaag cagcttcgaa aagaacccga 3600 tcgacttcct ggaagcaaag ggatacaagg aagtcaagaa ggacctgatc atcaagctgc 3660 cgaagtacag cctgttcgaa ctggaaaacg gaagaaagag aatgctggca agcgcaggag 3720 aactgcagaa gggaaacgaa ctggcactgc cgagcaagta cgtcaacttc ctgtacctgg 3780 caagccacta cgaaaagctg aagggaagcc cggaagacaa cgaacagaag cagctgttcg 3840 tcgaacagca caagcactac ctggacgaaa tcatcgaaca gatcagcgaa ttcagcaaga 3900 gagtcatcct ggcagacgca aacctggaca aggtcctgag cgcatacaac aagcacagag 3960 acaagccgat cagagaacag gcagaaaaca tcatccacct gttcacactg acaaacctgg 4020 gagcaccggc agcattcaag tacttcgaca caacaatcga cagaaagaga tacacaagca 4080 caaaggaagt cctggacgca acactgatcc accagagcat cacaggactg tacgaaacaa 4140 gaatcgacct gagccagctg ggaggagacg gaggaggaag cccgaagaag aagagaaagg 4200 tctagctagc gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa 4260 gtccaactac taaactgggg gatattatga agggccttga gcatctggat tctgcctaat 4320 aaaaaacatt tattttcatt gcctcgag 4348 <210> SEQ ID NO 259 <211> LENGTH: 4325 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with 5 UTR from XBG, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of XBG <400> SEQUENCE: 259 gggaagctca gaataaacgc tcaactttgg ccggatctgc caccatggac aagaagtaca 60 gcatcggact ggacatcgga acaaacagcg tcggatgggc agtcatcaca gacgaataca 120 aggtcccgag caagaagttc aaggtcctgg gaaacacaga cagacacagc atcaagaaga 180

acctgatcgg agcactgctg ttcgacagcg gagaaacagc agaagcaaca agactgaaga 240 gaacagcaag aagaagatac acaagaagaa agaacagaat ctgctacctg caggaaatct 300 tcagcaacga aatggcaaag gtcgacgaca gcttcttcca cagactggaa gaaagcttcc 360 tggtcgaaga agacaagaag cacgaaagac acccgatctt cggaaacatc gtcgacgaag 420 tcgcatacca cgaaaagtac ccgacaatct accacctgag aaagaagctg gtcgacagca 480 cagacaaggc agacctgaga ctgatctacc tggcactggc acacatgatc aagttcagag 540 gacacttcct gatcgaagga gacctgaacc cggacaacag cgacgtcgac aagctgttca 600 tccagctggt ccagacatac aaccagctgt tcgaagaaaa cccgatcaac gcaagcggag 660 tcgacgcaaa ggcaatcctg agcgcaagac tgagcaagag cagaagactg gaaaacctga 720 tcgcacagct gccgggagaa aagaagaacg gactgttcgg aaacctgatc gcactgagcc 780 tgggactgac accgaacttc aagagcaact tcgacctggc agaagacgca aagctgcagc 840 tgagcaagga cacatacgac gacgacctgg acaacctgct ggcacagatc ggagaccagt 900 acgcagacct gttcctggca gcaaagaacc tgagcgacgc aatcctgctg agcgacatcc 960 tgagagtcaa cacagaaatc acaaaggcac cgctgagcgc aagcatgatc aagagatacg 1020 acgaacacca ccaggacctg acactgctga aggcactggt cagacagcag ctgccggaaa 1080 agtacaagga aatcttcttc gaccagagca agaacggata cgcaggatac atcgacggag 1140 gagcaagcca ggaagaattc tacaagttca tcaagccgat cctggaaaag atggacggaa 1200 cagaagaact gctggtcaag ctgaacagag aagacctgct gagaaagcag agaacattcg 1260 acaacggaag catcccgcac cagatccacc tgggagaact gcacgcaatc ctgagaagac 1320 aggaagactt ctacccgttc ctgaaggaca acagagaaaa gatcgaaaag atcctgacat 1380 tcagaatccc gtactacgtc ggaccgctgg caagaggaaa cagcagattc gcatggatga 1440 caagaaagag cgaagaaaca atcacaccgt ggaacttcga agaagtcgtc gacaagggag 1500 caagcgcaca gagcttcatc gaaagaatga caaacttcga caagaacctg ccgaacgaaa 1560 aggtcctgcc gaagcacagc ctgctgtacg aatacttcac agtctacaac gaactgacaa 1620 aggtcaagta cgtcacagaa ggaatgagaa agccggcatt cctgagcgga gaacagaaga 1680 aggcaatcgt cgacctgctg ttcaagacaa acagaaaggt cacagtcaag cagctgaagg 1740 aagactactt caagaagatc gaatgcttcg acagcgtcga aatcagcgga gtcgaagaca 1800 gattcaacgc aagcctggga acataccacg acctgctgaa gatcatcaag gacaaggact 1860 tcctggacaa cgaagaaaac gaagacatcc tggaagacat cgtcctgaca ctgacactgt 1920 tcgaagacag agaaatgatc gaagaaagac tgaagacata cgcacacctg ttcgacgaca 1980 aggtcatgaa gcagctgaag agaagaagat acacaggatg gggaagactg agcagaaagc 2040 tgatcaacgg aatcagagac aagcagagcg gaaagacaat cctggacttc ctgaagagcg 2100 acggattcgc aaacagaaac ttcatgcagc tgatccacga cgacagcctg acattcaagg 2160 aagacatcca gaaggcacag gtcagcggac agggagacag cctgcacgaa cacatcgcaa 2220 acctggcagg aagcccggca atcaagaagg gaatcctgca gacagtcaag gtcgtcgacg 2280 aactggtcaa ggtcatggga agacacaagc cggaaaacat cgtcatcgaa atggcaagag 2340 aaaaccagac aacacagaag ggacagaaga acagcagaga aagaatgaag agaatcgaag 2400 aaggaatcaa ggaactggga agccagatcc tgaaggaaca cccggtcgaa aacacacagc 2460 tgcagaacga aaagctgtac ctgtactacc tgcagaacgg aagagacatg tacgtcgacc 2520 aggaactgga catcaacaga ctgagcgact acgacgtcga ccacatcgtc ccgcagagct 2580 tcctgaagga cgacagcatc gacaacaagg tcctgacaag aagcgacaag aacagaggaa 2640 agagcgacaa cgtcccgagc gaagaagtcg tcaagaagat gaagaactac tggagacagc 2700 tgctgaacgc aaagctgatc acacagagaa agttcgacaa cctgacaaag gcagagagag 2760 gaggactgag cgaactggac aaggcaggat tcatcaagag acagctggtc gaaacaagac 2820 agatcacaaa gcacgtcgca cagatcctgg acagcagaat gaacacaaag tacgacgaaa 2880 acgacaagct gatcagagaa gtcaaggtca tcacactgaa gagcaagctg gtcagcgact 2940 tcagaaagga cttccagttc tacaaggtca gagaaatcaa caactaccac cacgcacacg 3000 acgcatacct gaacgcagtc gtcggaacag cactgatcaa gaagtacccg aagctggaaa 3060 gcgaattcgt ctacggagac tacaaggtct acgacgtcag aaagatgatc gcaaagagcg 3120 aacaggaaat cggaaaggca acagcaaagt acttcttcta cagcaacatc atgaacttct 3180 tcaagacaga aatcacactg gcaaacggag aaatcagaaa gagaccgctg atcgaaacaa 3240 acggagaaac aggagaaatc gtctgggaca agggaagaga cttcgcaaca gtcagaaagg 3300 tcctgagcat gccgcaggtc aacatcgtca agaagacaga agtccagaca ggaggattca 3360 gcaaggaaag catcctgccg aagagaaaca gcgacaagct gatcgcaaga aagaaggact 3420 gggacccgaa gaagtacgga ggattcgaca gcccgacagt cgcatacagc gtcctggtcg 3480 tcgcaaaggt cgaaaaggga aagagcaaga agctgaagag cgtcaaggaa ctgctgggaa 3540 tcacaatcat ggaaagaagc agcttcgaaa agaacccgat cgacttcctg gaagcaaagg 3600 gatacaagga agtcaagaag gacctgatca tcaagctgcc gaagtacagc ctgttcgaac 3660 tggaaaacgg aagaaagaga atgctggcaa gcgcaggaga actgcagaag ggaaacgaac 3720 tggcactgcc gagcaagtac gtcaacttcc tgtacctggc aagccactac gaaaagctga 3780 agggaagccc ggaagacaac gaacagaagc agctgttcgt cgaacagcac aagcactacc 3840 tggacgaaat catcgaacag atcagcgaat tcagcaagag agtcatcctg gcagacgcaa 3900 acctggacaa ggtcctgagc gcatacaaca agcacagaga caagccgatc agagaacagg 3960 cagaaaacat catccacctg ttcacactga caaacctggg agcaccggca gcattcaagt 4020 acttcgacac aacaatcgac agaaagagat acacaagcac aaaggaagtc ctggacgcaa 4080 cactgatcca ccagagcatc acaggactgt acgaaacaag aatcgacctg agccagctgg 4140 gaggagacgg aggaggaagc ccgaagaaga agagaaaggt ctagctagca ccagcctcaa 4200 gaacacccga atggagtctc taagctacat aataccaact tacactttac aaaatgttgt 4260 cccccaaaat gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacattctc 4320 tcgag 4325 <210> SEQ ID NO 260 <211> LENGTH: 4325 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR from XBG, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of XBG <400> SEQUENCE: 260 aggaagctca gaataaacgc tcaactttgg ccggatctgc caccatggac aagaagtaca 60 gcatcggact ggacatcgga acaaacagcg tcggatgggc agtcatcaca gacgaataca 120 aggtcccgag caagaagttc aaggtcctgg gaaacacaga cagacacagc atcaagaaga 180 acctgatcgg agcactgctg ttcgacagcg gagaaacagc agaagcaaca agactgaaga 240 gaacagcaag aagaagatac acaagaagaa agaacagaat ctgctacctg caggaaatct 300 tcagcaacga aatggcaaag gtcgacgaca gcttcttcca cagactggaa gaaagcttcc 360 tggtcgaaga agacaagaag cacgaaagac acccgatctt cggaaacatc gtcgacgaag 420 tcgcatacca cgaaaagtac ccgacaatct accacctgag aaagaagctg gtcgacagca 480 cagacaaggc agacctgaga ctgatctacc tggcactggc acacatgatc aagttcagag 540 gacacttcct gatcgaagga gacctgaacc cggacaacag cgacgtcgac aagctgttca 600 tccagctggt ccagacatac aaccagctgt tcgaagaaaa cccgatcaac gcaagcggag 660 tcgacgcaaa ggcaatcctg agcgcaagac tgagcaagag cagaagactg gaaaacctga 720 tcgcacagct gccgggagaa aagaagaacg gactgttcgg aaacctgatc gcactgagcc 780 tgggactgac accgaacttc aagagcaact tcgacctggc agaagacgca aagctgcagc 840 tgagcaagga cacatacgac gacgacctgg acaacctgct ggcacagatc ggagaccagt 900 acgcagacct gttcctggca gcaaagaacc tgagcgacgc aatcctgctg agcgacatcc 960 tgagagtcaa cacagaaatc acaaaggcac cgctgagcgc aagcatgatc aagagatacg 1020 acgaacacca ccaggacctg acactgctga aggcactggt cagacagcag ctgccggaaa 1080 agtacaagga aatcttcttc gaccagagca agaacggata cgcaggatac atcgacggag 1140 gagcaagcca ggaagaattc tacaagttca tcaagccgat cctggaaaag atggacggaa 1200 cagaagaact gctggtcaag ctgaacagag aagacctgct gagaaagcag agaacattcg 1260 acaacggaag catcccgcac cagatccacc tgggagaact gcacgcaatc ctgagaagac 1320 aggaagactt ctacccgttc ctgaaggaca acagagaaaa gatcgaaaag atcctgacat 1380 tcagaatccc gtactacgtc ggaccgctgg caagaggaaa cagcagattc gcatggatga 1440 caagaaagag cgaagaaaca atcacaccgt ggaacttcga agaagtcgtc gacaagggag 1500 caagcgcaca gagcttcatc gaaagaatga caaacttcga caagaacctg ccgaacgaaa 1560 aggtcctgcc gaagcacagc ctgctgtacg aatacttcac agtctacaac gaactgacaa 1620 aggtcaagta cgtcacagaa ggaatgagaa agccggcatt cctgagcgga gaacagaaga 1680 aggcaatcgt cgacctgctg ttcaagacaa acagaaaggt cacagtcaag cagctgaagg 1740 aagactactt caagaagatc gaatgcttcg acagcgtcga aatcagcgga gtcgaagaca 1800 gattcaacgc aagcctggga acataccacg acctgctgaa gatcatcaag gacaaggact 1860 tcctggacaa cgaagaaaac gaagacatcc tggaagacat cgtcctgaca ctgacactgt 1920 tcgaagacag agaaatgatc gaagaaagac tgaagacata cgcacacctg ttcgacgaca 1980 aggtcatgaa gcagctgaag agaagaagat acacaggatg gggaagactg agcagaaagc 2040 tgatcaacgg aatcagagac aagcagagcg gaaagacaat cctggacttc ctgaagagcg 2100 acggattcgc aaacagaaac ttcatgcagc tgatccacga cgacagcctg acattcaagg 2160 aagacatcca gaaggcacag gtcagcggac agggagacag cctgcacgaa cacatcgcaa 2220 acctggcagg aagcccggca atcaagaagg gaatcctgca gacagtcaag gtcgtcgacg 2280 aactggtcaa ggtcatggga agacacaagc cggaaaacat cgtcatcgaa atggcaagag 2340 aaaaccagac aacacagaag ggacagaaga acagcagaga aagaatgaag agaatcgaag 2400 aaggaatcaa ggaactggga agccagatcc tgaaggaaca cccggtcgaa aacacacagc 2460 tgcagaacga aaagctgtac ctgtactacc tgcagaacgg aagagacatg tacgtcgacc 2520 aggaactgga catcaacaga ctgagcgact acgacgtcga ccacatcgtc ccgcagagct 2580 tcctgaagga cgacagcatc gacaacaagg tcctgacaag aagcgacaag aacagaggaa 2640 agagcgacaa cgtcccgagc gaagaagtcg tcaagaagat gaagaactac tggagacagc 2700 tgctgaacgc aaagctgatc acacagagaa agttcgacaa cctgacaaag gcagagagag 2760 gaggactgag cgaactggac aaggcaggat tcatcaagag acagctggtc gaaacaagac 2820 agatcacaaa gcacgtcgca cagatcctgg acagcagaat gaacacaaag tacgacgaaa 2880 acgacaagct gatcagagaa gtcaaggtca tcacactgaa gagcaagctg gtcagcgact 2940

tcagaaagga cttccagttc tacaaggtca gagaaatcaa caactaccac cacgcacacg 3000 acgcatacct gaacgcagtc gtcggaacag cactgatcaa gaagtacccg aagctggaaa 3060 gcgaattcgt ctacggagac tacaaggtct acgacgtcag aaagatgatc gcaaagagcg 3120 aacaggaaat cggaaaggca acagcaaagt acttcttcta cagcaacatc atgaacttct 3180 tcaagacaga aatcacactg gcaaacggag aaatcagaaa gagaccgctg atcgaaacaa 3240 acggagaaac aggagaaatc gtctgggaca agggaagaga cttcgcaaca gtcagaaagg 3300 tcctgagcat gccgcaggtc aacatcgtca agaagacaga agtccagaca ggaggattca 3360 gcaaggaaag catcctgccg aagagaaaca gcgacaagct gatcgcaaga aagaaggact 3420 gggacccgaa gaagtacgga ggattcgaca gcccgacagt cgcatacagc gtcctggtcg 3480 tcgcaaaggt cgaaaaggga aagagcaaga agctgaagag cgtcaaggaa ctgctgggaa 3540 tcacaatcat ggaaagaagc agcttcgaaa agaacccgat cgacttcctg gaagcaaagg 3600 gatacaagga agtcaagaag gacctgatca tcaagctgcc gaagtacagc ctgttcgaac 3660 tggaaaacgg aagaaagaga atgctggcaa gcgcaggaga actgcagaag ggaaacgaac 3720 tggcactgcc gagcaagtac gtcaacttcc tgtacctggc aagccactac gaaaagctga 3780 agggaagccc ggaagacaac gaacagaagc agctgttcgt cgaacagcac aagcactacc 3840 tggacgaaat catcgaacag atcagcgaat tcagcaagag agtcatcctg gcagacgcaa 3900 acctggacaa ggtcctgagc gcatacaaca agcacagaga caagccgatc agagaacagg 3960 cagaaaacat catccacctg ttcacactga caaacctggg agcaccggca gcattcaagt 4020 acttcgacac aacaatcgac agaaagagat acacaagcac aaaggaagtc ctggacgcaa 4080 cactgatcca ccagagcatc acaggactgt acgaaacaag aatcgacctg agccagctgg 4140 gaggagacgg aggaggaagc ccgaagaaga agagaaaggt ctagctagca ccagcctcaa 4200 gaacacccga atggagtctc taagctacat aataccaact tacactttac aaaatgttgt 4260 cccccaaaat gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacattctc 4320 tcgag 4325 <210> SEQ ID NO 261 <211> LENGTH: 4411 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Cas9 transcript with AGG as first three nucleotides for use with CleanCapTM, 5 UTR from HSD, ORF corresponding to SEQ ID NO: 204, Kozak sequence, and 3 UTR of ALB <400> SEQUENCE: 261 aggtcccgca gtcggcgtcc agcggctctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcggatcc gccaccatgg acaagaagta cagcatcgga ctggacatcg gaacaaacag 120 cgtcggatgg gcagtcatca cagacgaata caaggtcccg agcaagaagt tcaaggtcct 180 gggaaacaca gacagacaca gcatcaagaa gaacctgatc ggagcactgc tgttcgacag 240 cggagaaaca gcagaagcaa caagactgaa gagaacagca agaagaagat acacaagaag 300 aaagaacaga atctgctacc tgcaggaaat cttcagcaac gaaatggcaa aggtcgacga 360 cagcttcttc cacagactgg aagaaagctt cctggtcgaa gaagacaaga agcacgaaag 420 acacccgatc ttcggaaaca tcgtcgacga agtcgcatac cacgaaaagt acccgacaat 480 ctaccacctg agaaagaagc tggtcgacag cacagacaag gcagacctga gactgatcta 540 cctggcactg gcacacatga tcaagttcag aggacacttc ctgatcgaag gagacctgaa 600 cccggacaac agcgacgtcg acaagctgtt catccagctg gtccagacat acaaccagct 660 gttcgaagaa aacccgatca acgcaagcgg agtcgacgca aaggcaatcc tgagcgcaag 720 actgagcaag agcagaagac tggaaaacct gatcgcacag ctgccgggag aaaagaagaa 780 cggactgttc ggaaacctga tcgcactgag cctgggactg acaccgaact tcaagagcaa 840 cttcgacctg gcagaagacg caaagctgca gctgagcaag gacacatacg acgacgacct 900 ggacaacctg ctggcacaga tcggagacca gtacgcagac ctgttcctgg cagcaaagaa 960 cctgagcgac gcaatcctgc tgagcgacat cctgagagtc aacacagaaa tcacaaaggc 1020 accgctgagc gcaagcatga tcaagagata cgacgaacac caccaggacc tgacactgct 1080 gaaggcactg gtcagacagc agctgccgga aaagtacaag gaaatcttct tcgaccagag 1140 caagaacgga tacgcaggat acatcgacgg aggagcaagc caggaagaat tctacaagtt 1200 catcaagccg atcctggaaa agatggacgg aacagaagaa ctgctggtca agctgaacag 1260 agaagacctg ctgagaaagc agagaacatt cgacaacgga agcatcccgc accagatcca 1320 cctgggagaa ctgcacgcaa tcctgagaag acaggaagac ttctacccgt tcctgaagga 1380 caacagagaa aagatcgaaa agatcctgac attcagaatc ccgtactacg tcggaccgct 1440 ggcaagagga aacagcagat tcgcatggat gacaagaaag agcgaagaaa caatcacacc 1500 gtggaacttc gaagaagtcg tcgacaaggg agcaagcgca cagagcttca tcgaaagaat 1560 gacaaacttc gacaagaacc tgccgaacga aaaggtcctg ccgaagcaca gcctgctgta 1620 cgaatacttc acagtctaca acgaactgac aaaggtcaag tacgtcacag aaggaatgag 1680 aaagccggca ttcctgagcg gagaacagaa gaaggcaatc gtcgacctgc tgttcaagac 1740 aaacagaaag gtcacagtca agcagctgaa ggaagactac ttcaagaaga tcgaatgctt 1800 cgacagcgtc gaaatcagcg gagtcgaaga cagattcaac gcaagcctgg gaacatacca 1860 cgacctgctg aagatcatca aggacaagga cttcctggac aacgaagaaa acgaagacat 1920 cctggaagac atcgtcctga cactgacact gttcgaagac agagaaatga tcgaagaaag 1980 actgaagaca tacgcacacc tgttcgacga caaggtcatg aagcagctga agagaagaag 2040 atacacagga tggggaagac tgagcagaaa gctgatcaac ggaatcagag acaagcagag 2100 cggaaagaca atcctggact tcctgaagag cgacggattc gcaaacagaa acttcatgca 2160 gctgatccac gacgacagcc tgacattcaa ggaagacatc cagaaggcac aggtcagcgg 2220 acagggagac agcctgcacg aacacatcgc aaacctggca ggaagcccgg caatcaagaa 2280 gggaatcctg cagacagtca aggtcgtcga cgaactggtc aaggtcatgg gaagacacaa 2340 gccggaaaac atcgtcatcg aaatggcaag agaaaaccag acaacacaga agggacagaa 2400 gaacagcaga gaaagaatga agagaatcga agaaggaatc aaggaactgg gaagccagat 2460 cctgaaggaa cacccggtcg aaaacacaca gctgcagaac gaaaagctgt acctgtacta 2520 cctgcagaac ggaagagaca tgtacgtcga ccaggaactg gacatcaaca gactgagcga 2580 ctacgacgtc gaccacatcg tcccgcagag cttcctgaag gacgacagca tcgacaacaa 2640 ggtcctgaca agaagcgaca agaacagagg aaagagcgac aacgtcccga gcgaagaagt 2700 cgtcaagaag atgaagaact actggagaca gctgctgaac gcaaagctga tcacacagag 2760 aaagttcgac aacctgacaa aggcagagag aggaggactg agcgaactgg acaaggcagg 2820 attcatcaag agacagctgg tcgaaacaag acagatcaca aagcacgtcg cacagatcct 2880 ggacagcaga atgaacacaa agtacgacga aaacgacaag ctgatcagag aagtcaaggt 2940 catcacactg aagagcaagc tggtcagcga cttcagaaag gacttccagt tctacaaggt 3000 cagagaaatc aacaactacc accacgcaca cgacgcatac ctgaacgcag tcgtcggaac 3060 agcactgatc aagaagtacc cgaagctgga aagcgaattc gtctacggag actacaaggt 3120 ctacgacgtc agaaagatga tcgcaaagag cgaacaggaa atcggaaagg caacagcaaa 3180 gtacttcttc tacagcaaca tcatgaactt cttcaagaca gaaatcacac tggcaaacgg 3240 agaaatcaga aagagaccgc tgatcgaaac aaacggagaa acaggagaaa tcgtctggga 3300 caagggaaga gacttcgcaa cagtcagaaa ggtcctgagc atgccgcagg tcaacatcgt 3360 caagaagaca gaagtccaga caggaggatt cagcaaggaa agcatcctgc cgaagagaaa 3420 cagcgacaag ctgatcgcaa gaaagaagga ctgggacccg aagaagtacg gaggattcga 3480 cagcccgaca gtcgcataca gcgtcctggt cgtcgcaaag gtcgaaaagg gaaagagcaa 3540 gaagctgaag agcgtcaagg aactgctggg aatcacaatc atggaaagaa gcagcttcga 3600 aaagaacccg atcgacttcc tggaagcaaa gggatacaag gaagtcaaga aggacctgat 3660 catcaagctg ccgaagtaca gcctgttcga actggaaaac ggaagaaaga gaatgctggc 3720 aagcgcagga gaactgcaga agggaaacga actggcactg ccgagcaagt acgtcaactt 3780 cctgtacctg gcaagccact acgaaaagct gaagggaagc ccggaagaca acgaacagaa 3840 gcagctgttc gtcgaacagc acaagcacta cctggacgaa atcatcgaac agatcagcga 3900 attcagcaag agagtcatcc tggcagacgc aaacctggac aaggtcctga gcgcatacaa 3960 caagcacaga gacaagccga tcagagaaca ggcagaaaac atcatccacc tgttcacact 4020 gacaaacctg ggagcaccgg cagcattcaa gtacttcgac acaacaatcg acagaaagag 4080 atacacaagc acaaaggaag tcctggacgc aacactgatc caccagagca tcacaggact 4140 gtacgaaaca agaatcgacc tgagccagct gggaggagac ggaggaggaa gcccgaagaa 4200 gaagagaaag gtctagctag ccatcacatt taaaagcatc tcagcctacc atgagaataa 4260 gagaaagaaa atgaagatca atagcttatt catctctttt tctttttcgt tggtgtaaag 4320 ccaacaccct gtctaaaaaa cataaatttc tttaatcatt ttgcctcttt tctctgtgct 4380 tcaattaata aaaaatggaa agaacctcga g 4411 <210> SEQ ID NO 262 <400> SEQUENCE: 262 000 <210> SEQ ID NO 263 <211> LENGTH: 93 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: poly-A 100 sequence <400> SEQUENCE: 263 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 93 <210> SEQ ID NO 264 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G209 single guide RNA targeting the mouse TTR gene <400> SEQUENCE: 264 aaataagaga gaaaagaaga gtaagaagaa atataagagc cacc 44 <210> SEQ ID NO 265 <211> LENGTH: 3312 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: ORF encoding Neisseria

meningitidis Cas9 using minimal uridine codons, with start and stop codons <400> SEQUENCE: 265 atggcagcat tcaagccgaa ctcgatcaac tacatcctgg gactggacat cggaatcgca 60 tcggtcggat gggcaatggt cgaaatcgac gaagaagaaa acccgatcag actgatcgac 120 ctgggagtca gagtcttcga aagagcagaa gtcccgaaga caggagactc gctggcaatg 180 gcaagaagac tggcaagatc ggtcagaaga ctgacaagaa gaagagcaca cagactgctg 240 agaacaagaa gactgctgaa gagagaagga gtcctgcagg cagcaaactt cgacgaaaac 300 ggactgatca agtcgctgcc gaacacaccg tggcagctga gagcagcagc actggacaga 360 aagctgacac cgctggaatg gtcggcagtc ctgctgcacc tgatcaagca cagaggatac 420 ctgtcgcaga gaaagaacga aggagaaaca gcagacaagg aactgggagc actgctgaag 480 ggagtcgcag gaaacgcaca cgcactgcag acaggagact tcagaacacc ggcagaactg 540 gcactgaaca agttcgaaaa ggaatcggga cacatcagaa accagagatc ggactactcg 600 cacacattct cgagaaagga cctgcaggca gaactgatcc tgctgttcga aaagcagaag 660 gaattcggaa acccgcacgt ctcgggagga ctgaaggaag gaatcgaaac actgctgatg 720 acacagagac cggcactgtc gggagacgca gtccagaaga tgctgggaca ctgcacattc 780 gaaccggcag aaccgaaggc agcaaagaac acatacacag cagaaagatt catctggctg 840 acaaagctga acaacctgag aatcctggaa cagggatcgg aaagaccgct gacagacaca 900 gaaagagcaa cactgatgga cgaaccgtac agaaagtcga agctgacata cgcacaggca 960 agaaagctgc tgggactgga agacacagca ttcttcaagg gactgagata cggaaaggac 1020 aacgcagaag catcgacact gatggaaatg aaggcatacc acgcaatctc gagagcactg 1080 gaaaaggaag gactgaagga caagaagtcg ccgctgaacc tgtcgccgga actgcaggac 1140 gaaatcggaa cagcattctc gctgttcaag acagacgaag acatcacagg aagactgaag 1200 gacagaatcc agccggaaat cctggaagca ctgctgaagc acatctcgtt cgacaagttc 1260 gtccagatct cgctgaaggc actgagaaga atcgtcccgc tgatggaaca gggaaagaga 1320 tacgacgaag catgcgcaga aatctacgga gaccactacg gaaagaagaa cacagaagaa 1380 aagatctacc tgccgccgat cccggcagac gaaatcagaa acccggtcgt cctgagagca 1440 ctgtcgcagg caagaaaggt catcaacgga gtcgtcagaa gatacggatc gccggcaaga 1500 atccacatcg aaacagcaag agaagtcgga aagtcgttca aggacagaaa ggaaatcgaa 1560 aagagacagg aagaaaacag aaaggacaga gaaaaggcag cagcaaagtt cagagaatac 1620 ttcccgaact tcgtcggaga accgaagtcg aaggacatcc tgaagctgag actgtacgaa 1680 cagcagcacg gaaagtgcct gtactcggga aaggaaatca acctgggaag actgaacgaa 1740 aagggatacg tcgaaatcga ccacgcactg ccgttctcga gaacatggga cgactcgttc 1800 aacaacaagg tcctggtcct gggatcggaa aaccagaaca agggaaacca gacaccgtac 1860 gaatacttca acggaaagga caactcgaga gaatggcagg aattcaaggc aagagtcgaa 1920 acatcgagat tcccgagatc gaagaagcag agaatcctgc tgcagaagtt cgacgaagac 1980 ggattcaagg aaagaaacct gaacgacaca agatacgtca acagattcct gtgccagttc 2040 gtcgcagaca gaatgagact gacaggaaag ggaaagaaga gagtcttcgc atcgaacgga 2100 cagatcacaa acctgctgag aggattctgg ggactgagaa aggtcagagc agaaaacgac 2160 agacaccacg cactggacgc agtcgtcgtc gcatgctcga cagtcgcaat gcagcagaag 2220 atcacaagat tcgtcagata caaggaaatg aacgcattcg acggaaagac aatcgacaag 2280 gaaacaggag aagtcctgca ccagaagaca cacttcccgc agccgtggga attcttcgca 2340 caggaagtca tgatcagagt cttcggaaag ccggacggaa agccggaatt cgaagaagca 2400 gacacactgg aaaagctgag aacactgctg gcagaaaagc tgtcgtcgag accggaagca 2460 gtccacgaat acgtcacacc gctgttcgtc tcgagagcac cgaacagaaa gatgtcggga 2520 cagggacaca tggaaacagt caagtcggca aagagactgg acgaaggagt ctcggtcctg 2580 agagtcccgc tgacacagct gaagctgaag gacctggaaa agatggtcaa cagagaaaga 2640 gaaccgaagc tgtacgaagc actgaaggca agactggaag cacacaagga cgacccggca 2700 aaggcattcg cagaaccgtt ctacaagtac gacaaggcag gaaacagaac acagcaggtc 2760 aaggcagtca gagtcgaaca ggtccagaag acaggagtct gggtcagaaa ccacaacgga 2820 atcgcagaca acgcaacaat ggtcagagta gacgtcttcg aaaagggaga caagtactac 2880 ctggtcccga tctactcgtg gcaggtcgca aagggaatcc tgccggacag agcagtcgtc 2940 cagggaaagg acgaagaaga ctggcagctg atcgacgact cgttcaactt caagttctcg 3000 ctgcacccga acgacctggt cgaagtcatc acaaagaagg caagaatgtt cggatacttc 3060 gcatcgtgcc acagaggaac aggaaacatc aacatcagaa tccacgacct ggaccacaag 3120 atcggaaaga acggaatcct ggaaggaatc ggagtcaaga cagcactgtc gttccagaag 3180 taccagatcg acgaactggg aaaggaaatc agaccgtgca gactgaagaa gagaccgccg 3240 gtcagatccg gaaagagaac agcagacgga tcggaattcg aatcgccgaa gaagaagaga 3300 aaggtcgaat ga 3312 <210> SEQ ID NO 266 <211> LENGTH: 3306 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: ORF encoding Neisseria meningitidis Cas9 using minimal uridine codons (no start or stop codons; suitable for inclusion in fusion protein coding sequence) <400> SEQUENCE: 266 gcagcattca agccgaactc gatcaactac atcctgggac tggacatcgg aatcgcatcg 60 gtcggatggg caatggtcga aatcgacgaa gaagaaaacc cgatcagact gatcgacctg 120 ggagtcagag tcttcgaaag agcagaagtc ccgaagacag gagactcgct ggcaatggca 180 agaagactgg caagatcggt cagaagactg acaagaagaa gagcacacag actgctgaga 240 acaagaagac tgctgaagag agaaggagtc ctgcaggcag caaacttcga cgaaaacgga 300 ctgatcaagt cgctgccgaa cacaccgtgg cagctgagag cagcagcact ggacagaaag 360 ctgacaccgc tggaatggtc ggcagtcctg ctgcacctga tcaagcacag aggatacctg 420 tcgcagagaa agaacgaagg agaaacagca gacaaggaac tgggagcact gctgaaggga 480 gtcgcaggaa acgcacacgc actgcagaca ggagacttca gaacaccggc agaactggca 540 ctgaacaagt tcgaaaagga atcgggacac atcagaaacc agagatcgga ctactcgcac 600 acattctcga gaaaggacct gcaggcagaa ctgatcctgc tgttcgaaaa gcagaaggaa 660 ttcggaaacc cgcacgtctc gggaggactg aaggaaggaa tcgaaacact gctgatgaca 720 cagagaccgg cactgtcggg agacgcagtc cagaagatgc tgggacactg cacattcgaa 780 ccggcagaac cgaaggcagc aaagaacaca tacacagcag aaagattcat ctggctgaca 840 aagctgaaca acctgagaat cctggaacag ggatcggaaa gaccgctgac agacacagaa 900 agagcaacac tgatggacga accgtacaga aagtcgaagc tgacatacgc acaggcaaga 960 aagctgctgg gactggaaga cacagcattc ttcaagggac tgagatacgg aaaggacaac 1020 gcagaagcat cgacactgat ggaaatgaag gcataccacg caatctcgag agcactggaa 1080 aaggaaggac tgaaggacaa gaagtcgccg ctgaacctgt cgccggaact gcaggacgaa 1140 atcggaacag cattctcgct gttcaagaca gacgaagaca tcacaggaag actgaaggac 1200 agaatccagc cggaaatcct ggaagcactg ctgaagcaca tctcgttcga caagttcgtc 1260 cagatctcgc tgaaggcact gagaagaatc gtcccgctga tggaacaggg aaagagatac 1320 gacgaagcat gcgcagaaat ctacggagac cactacggaa agaagaacac agaagaaaag 1380 atctacctgc cgccgatccc ggcagacgaa atcagaaacc cggtcgtcct gagagcactg 1440 tcgcaggcaa gaaaggtcat caacggagtc gtcagaagat acggatcgcc ggcaagaatc 1500 cacatcgaaa cagcaagaga agtcggaaag tcgttcaagg acagaaagga aatcgaaaag 1560 agacaggaag aaaacagaaa ggacagagaa aaggcagcag caaagttcag agaatacttc 1620 ccgaacttcg tcggagaacc gaagtcgaag gacatcctga agctgagact gtacgaacag 1680 cagcacggaa agtgcctgta ctcgggaaag gaaatcaacc tgggaagact gaacgaaaag 1740 ggatacgtcg aaatcgacca cgcactgccg ttctcgagaa catgggacga ctcgttcaac 1800 aacaaggtcc tggtcctggg atcggaaaac cagaacaagg gaaaccagac accgtacgaa 1860 tacttcaacg gaaaggacaa ctcgagagaa tggcaggaat tcaaggcaag agtcgaaaca 1920 tcgagattcc cgagatcgaa gaagcagaga atcctgctgc agaagttcga cgaagacgga 1980 ttcaaggaaa gaaacctgaa cgacacaaga tacgtcaaca gattcctgtg ccagttcgtc 2040 gcagacagaa tgagactgac aggaaaggga aagaagagag tcttcgcatc gaacggacag 2100 atcacaaacc tgctgagagg attctgggga ctgagaaagg tcagagcaga aaacgacaga 2160 caccacgcac tggacgcagt cgtcgtcgca tgctcgacag tcgcaatgca gcagaagatc 2220 acaagattcg tcagatacaa ggaaatgaac gcattcgacg gaaagacaat cgacaaggaa 2280 acaggagaag tcctgcacca gaagacacac ttcccgcagc cgtgggaatt cttcgcacag 2340 gaagtcatga tcagagtctt cggaaagccg gacggaaagc cggaattcga agaagcagac 2400 acactggaaa agctgagaac actgctggca gaaaagctgt cgtcgagacc ggaagcagtc 2460 cacgaatacg tcacaccgct gttcgtctcg agagcaccga acagaaagat gtcgggacag 2520 ggacacatgg aaacagtcaa gtcggcaaag agactggacg aaggagtctc ggtcctgaga 2580 gtcccgctga cacagctgaa gctgaaggac ctggaaaaga tggtcaacag agaaagagaa 2640 ccgaagctgt acgaagcact gaaggcaaga ctggaagcac acaaggacga cccggcaaag 2700 gcattcgcag aaccgttcta caagtacgac aaggcaggaa acagaacaca gcaggtcaag 2760 gcagtcagag tcgaacaggt ccagaagaca ggagtctggg tcagaaacca caacggaatc 2820 gcagacaacg caacaatggt cagagtagac gtcttcgaaa agggagacaa gtactacctg 2880 gtcccgatct actcgtggca ggtcgcaaag ggaatcctgc cggacagagc agtcgtccag 2940 ggaaaggacg aagaagactg gcagctgatc gacgactcgt tcaacttcaa gttctcgctg 3000 cacccgaacg acctggtcga agtcatcaca aagaaggcaa gaatgttcgg atacttcgca 3060 tcgtgccaca gaggaacagg aaacatcaac atcagaatcc acgacctgga ccacaagatc 3120 ggaaagaacg gaatcctgga aggaatcgga gtcaagacag cactgtcgtt ccagaagtac 3180 cagatcgacg aactgggaaa ggaaatcaga ccgtgcagac tgaagaagag accgccggtc 3240 agatccggaa agagaacagc agacggatcg gaattcgaat cgccgaagaa gaagagaaag 3300 gtcgaa 3306 <210> SEQ ID NO 267 <211> LENGTH: 3636 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Transcript comprising SEQ ID NO: 265 (encoding Neisseria meningitidis Cas9)

<400> SEQUENCE: 267 gggagaccca agctggctag cgtttaaact taagcttgga tccgccacca tggcagcatt 60 caagccgaac tcgatcaact acatcctggg actggacatc ggaatcgcat cggtcggatg 120 ggcaatggtc gaaatcgacg aagaagaaaa cccgatcaga ctgatcgacc tgggagtcag 180 agtcttcgaa agagcagaag tcccgaagac aggagactcg ctggcaatgg caagaagact 240 ggcaagatcg gtcagaagac tgacaagaag aagagcacac agactgctga gaacaagaag 300 actgctgaag agagaaggag tcctgcaggc agcaaacttc gacgaaaacg gactgatcaa 360 gtcgctgccg aacacaccgt ggcagctgag agcagcagca ctggacagaa agctgacacc 420 gctggaatgg tcggcagtcc tgctgcacct gatcaagcac agaggatacc tgtcgcagag 480 aaagaacgaa ggagaaacag cagacaagga actgggagca ctgctgaagg gagtcgcagg 540 aaacgcacac gcactgcaga caggagactt cagaacaccg gcagaactgg cactgaacaa 600 gttcgaaaag gaatcgggac acatcagaaa ccagagatcg gactactcgc acacattctc 660 gagaaaggac ctgcaggcag aactgatcct gctgttcgaa aagcagaagg aattcggaaa 720 cccgcacgtc tcgggaggac tgaaggaagg aatcgaaaca ctgctgatga cacagagacc 780 ggcactgtcg ggagacgcag tccagaagat gctgggacac tgcacattcg aaccggcaga 840 accgaaggca gcaaagaaca catacacagc agaaagattc atctggctga caaagctgaa 900 caacctgaga atcctggaac agggatcgga aagaccgctg acagacacag aaagagcaac 960 actgatggac gaaccgtaca gaaagtcgaa gctgacatac gcacaggcaa gaaagctgct 1020 gggactggaa gacacagcat tcttcaaggg actgagatac ggaaaggaca acgcagaagc 1080 atcgacactg atggaaatga aggcatacca cgcaatctcg agagcactgg aaaaggaagg 1140 actgaaggac aagaagtcgc cgctgaacct gtcgccggaa ctgcaggacg aaatcggaac 1200 agcattctcg ctgttcaaga cagacgaaga catcacagga agactgaagg acagaatcca 1260 gccggaaatc ctggaagcac tgctgaagca catctcgttc gacaagttcg tccagatctc 1320 gctgaaggca ctgagaagaa tcgtcccgct gatggaacag ggaaagagat acgacgaagc 1380 atgcgcagaa atctacggag accactacgg aaagaagaac acagaagaaa agatctacct 1440 gccgccgatc ccggcagacg aaatcagaaa cccggtcgtc ctgagagcac tgtcgcaggc 1500 aagaaaggtc atcaacggag tcgtcagaag atacggatcg ccggcaagaa tccacatcga 1560 aacagcaaga gaagtcggaa agtcgttcaa ggacagaaag gaaatcgaaa agagacagga 1620 agaaaacaga aaggacagag aaaaggcagc agcaaagttc agagaatact tcccgaactt 1680 cgtcggagaa ccgaagtcga aggacatcct gaagctgaga ctgtacgaac agcagcacgg 1740 aaagtgcctg tactcgggaa aggaaatcaa cctgggaaga ctgaacgaaa agggatacgt 1800 cgaaatcgac cacgcactgc cgttctcgag aacatgggac gactcgttca acaacaaggt 1860 cctggtcctg ggatcggaaa accagaacaa gggaaaccag acaccgtacg aatacttcaa 1920 cggaaaggac aactcgagag aatggcagga attcaaggca agagtcgaaa catcgagatt 1980 cccgagatcg aagaagcaga gaatcctgct gcagaagttc gacgaagacg gattcaagga 2040 aagaaacctg aacgacacaa gatacgtcaa cagattcctg tgccagttcg tcgcagacag 2100 aatgagactg acaggaaagg gaaagaagag agtcttcgca tcgaacggac agatcacaaa 2160 cctgctgaga ggattctggg gactgagaaa ggtcagagca gaaaacgaca gacaccacgc 2220 actggacgca gtcgtcgtcg catgctcgac agtcgcaatg cagcagaaga tcacaagatt 2280 cgtcagatac aaggaaatga acgcattcga cggaaagaca atcgacaagg aaacaggaga 2340 agtcctgcac cagaagacac acttcccgca gccgtgggaa ttcttcgcac aggaagtcat 2400 gatcagagtc ttcggaaagc cggacggaaa gccggaattc gaagaagcag acacactgga 2460 aaagctgaga acactgctgg cagaaaagct gtcgtcgaga ccggaagcag tccacgaata 2520 cgtcacaccg ctgttcgtct cgagagcacc gaacagaaag atgtcgggac agggacacat 2580 ggaaacagtc aagtcggcaa agagactgga cgaaggagtc tcggtcctga gagtcccgct 2640 gacacagctg aagctgaagg acctggaaaa gatggtcaac agagaaagag aaccgaagct 2700 gtacgaagca ctgaaggcaa gactggaagc acacaaggac gacccggcaa aggcattcgc 2760 agaaccgttc tacaagtacg acaaggcagg aaacagaaca cagcaggtca aggcagtcag 2820 agtcgaacag gtccagaaga caggagtctg ggtcagaaac cacaacggaa tcgcagacaa 2880 cgcaacaatg gtcagagtag acgtcttcga aaagggagac aagtactacc tggtcccgat 2940 ctactcgtgg caggtcgcaa agggaatcct gccggacaga gcagtcgtcc agggaaagga 3000 cgaagaagac tggcagctga tcgacgactc gttcaacttc aagttctcgc tgcacccgaa 3060 cgacctggtc gaagtcatca caaagaaggc aagaatgttc ggatacttcg catcgtgcca 3120 cagaggaaca ggaaacatca acatcagaat ccacgacctg gaccacaaga tcggaaagaa 3180 cggaatcctg gaaggaatcg gagtcaagac agcactgtcg ttccagaagt accagatcga 3240 cgaactggga aaggaaatca gaccgtgcag actgaagaag agaccgccgg tcagatccgg 3300 aaagagaaca gcagacggat cggaattcga atcgccgaag aagaagagaa aggtcgaatg 3360 atagctagct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 3420 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 3480 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 3540 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 3600 atagcaggca tgctggggat gcggtgggct ctatgg 3636 <210> SEQ ID NO 268 <211> LENGTH: 1103 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Amino acid sequence of Neisseria meningitidis Cas9 <400> SEQUENCE: 268 Met Ala Ala Phe Lys Pro Asn Ser Ile Asn Tyr Ile Leu Gly Leu Asp 1 5 10 15 Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Glu 20 25 30 Glu Asn Pro Ile Arg Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg 35 40 45 Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu 50 55 60 Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu 65 70 75 80 Arg Thr Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asn 85 90 95 Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln 100 105 110 Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser 115 120 125 Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg 130 135 140 Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys 145 150 155 160 Gly Val Ala Gly Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr 165 170 175 Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile 180 185 190 Arg Asn Gln Arg Ser Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu 195 200 205 Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn 210 215 220 Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met 225 230 235 240 Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly 245 250 255 His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr 260 265 270 Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile 275 280 285 Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr 290 295 300 Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala 305 310 315 320 Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg 325 330 335 Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala 340 345 350 Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys 355 360 365 Lys Ser Pro Leu Asn Leu Ser Pro Glu Leu Gln Asp Glu Ile Gly Thr 370 375 380 Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys 385 390 395 400 Asp Arg Ile Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser 405 410 415 Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val 420 425 430 Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile 435 440 445 Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu 450 455 460 Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala 465 470 475 480 Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly 485 490 495 Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser 500 505 510 Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys 515 520 525 Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe 530 535 540 Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu 545 550 555 560 Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Gly 565 570 575 Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe 580 585 590 Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly 595 600 605

Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn 610 615 620 Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu 625 630 635 640 Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys 645 650 655 Phe Asp Glu Asp Gly Phe Lys Glu Arg Asn Leu Asn Asp Thr Arg Tyr 660 665 670 Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp Arg Met Arg Leu Thr 675 680 685 Gly Lys Gly Lys Lys Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn 690 695 700 Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp 705 710 715 720 Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala 725 730 735 Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala 740 745 750 Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Glu Val Leu His Gln 755 760 765 Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met 770 775 780 Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala 785 790 795 800 Asp Thr Leu Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser 805 810 815 Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg 820 825 830 Ala Pro Asn Arg Lys Met Ser Gly Gln Gly His Met Glu Thr Val Lys 835 840 845 Ser Ala Lys Arg Leu Asp Glu Gly Val Ser Val Leu Arg Val Pro Leu 850 855 860 Thr Gln Leu Lys Leu Lys Asp Leu Glu Lys Met Val Asn Arg Glu Arg 865 870 875 880 Glu Pro Lys Leu Tyr Glu Ala Leu Lys Ala Arg Leu Glu Ala His Lys 885 890 895 Asp Asp Pro Ala Lys Ala Phe Ala Glu Pro Phe Tyr Lys Tyr Asp Lys 900 905 910 Ala Gly Asn Arg Thr Gln Gln Val Lys Ala Val Arg Val Glu Gln Val 915 920 925 Gln Lys Thr Gly Val Trp Val Arg Asn His Asn Gly Ile Ala Asp Asn 930 935 940 Ala Thr Met Val Arg Val Asp Val Phe Glu Lys Gly Asp Lys Tyr Tyr 945 950 955 960 Leu Val Pro Ile Tyr Ser Trp Gln Val Ala Lys Gly Ile Leu Pro Asp 965 970 975 Arg Ala Val Val Gln Gly Lys Asp Glu Glu Asp Trp Gln Leu Ile Asp 980 985 990 Asp Ser Phe Asn Phe Lys Phe Ser Leu His Pro Asn Asp Leu Val Glu 995 1000 1005 Val Ile Thr Lys Lys Ala Arg Met Phe Gly Tyr Phe Ala Ser Cys 1010 1015 1020 His Arg Gly Thr Gly Asn Ile Asn Ile Arg Ile His Asp Leu Asp 1025 1030 1035 His Lys Ile Gly Lys Asn Gly Ile Leu Glu Gly Ile Gly Val Lys 1040 1045 1050 Thr Ala Leu Ser Phe Gln Lys Tyr Gln Ile Asp Glu Leu Gly Lys 1055 1060 1065 Glu Ile Arg Pro Cys Arg Leu Lys Lys Arg Pro Pro Val Arg Ser 1070 1075 1080 Gly Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys 1085 1090 1095 Lys Arg Lys Val Glu 1100 <210> SEQ ID NO 269 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G390 single guide RNA targeting the rat TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 269 gccgagucug gagagcugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 270 <211> LENGTH: 74 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: trRNA <400> SEQUENCE: 270 aacagcauag caaguuaaaa uaaggcuagu ccguuaucaa cuugaaaaag uggcaccgag 60 ucggugcuuu uuuu 74 <210> SEQ ID NO 271 <400> SEQUENCE: 271 000 <210> SEQ ID NO 272 <211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G534 single guide RNA targeting the rat TTR gene <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (29)..(40) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (69)..(96) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (97)..(100) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 272 acgcaaauau caguccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 273 <211> LENGTH: 95 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: G000395 5 truncated inactive sgRNA modified sequence <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (1)..(3) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(35) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (64)..(91) <223> OTHER INFORMATION: 2'-O-Me nucleotide <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (92)..(95) <223> OTHER INFORMATION: PS linkage, 2'-O-Me nucleotide <400> SEQUENCE: 273 gcaauggugu agcggguuuu agagcuagaa auagcaaguu aaaauaaggc uaguccguua 60 ucaacuugaa aaaguggcac cgagucggug cuuuu 95 <210> SEQ ID NO 274 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: SV40 NLS <400> SEQUENCE: 274 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 275 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Alternate SV40 NLS <400> SEQUENCE: 275 Pro Lys Lys Lys Arg Arg Val 1 5 <210> SEQ ID NO 276 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Nucleoplasmin NLS <400> SEQUENCE: 276 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15

<210> SEQ ID NO 277 <211> LENGTH: 10 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Exemplary Kozak sequence <400> SEQUENCE: 277 gccrccaugg 10 <210> SEQ ID NO 278 <211> LENGTH: 13 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Synthetic: Exemplary Kozak sequence <400> SEQUENCE: 278 gccgccrcca ugg 13

Claims

1. A method of inducing a double-stranded break (DSB) within the TTR gene, comprising delivering a composition to a cell, wherein the composition comprises a. a guide RNA comprising a guide sequence selected from SEQ ID NOs: 5-82; b. a guide RNA comprising at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

2. A method of modifying the TTR gene comprising delivering a composition to a cell, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

3. A method of treating amyloidosis associated with TTR (ATTR), comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby treating ATTR.

4. A method of reducing TTR serum concentration, comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby reducing TTR serum concentration.

5. A method for reducing or preventing the accumulation of amyloids or amyloid fibrils comprising TTR in a subject, comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82, thereby reducing accumulation of amyloids or amyloid fibrils.

6. A composition comprising a guide RNA comprising: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

7. A composition comprising a vector encoding a guide RNA, wherein the guide RNA comprises: a. a guide sequence selected from SEQ ID NOs: 5-82; b. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 5-82; or c. a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-82.

8. The composition of claim 6 or 7, for use in inducing a double-stranded break (DSB) within the TTR gene in a cell or subject.

9. The composition of claim 6 or 7, for use in modifying the TTR gene in a cell or subject.

10. The composition of claim 6 or 7, for use in treating amyloidosis associated with TTR (ATTR) in a subject.

11. The composition of claim 6 or 7, for use in reducing TTR serum concentration in a subject.

12. The composition of claim 6 or 7, for use in reducing or preventing the accumulation of amyloids or amyloid fibrils in a subject.

13. The method of any one of claims 1-5 or the composition for use of any one of claims 8-12, wherein the composition reduces serum TTR levels.

14. The method or composition for use of claim 13, wherein the serum TTR levels are reduced by at least 50% as compared to serum TTR levels before administration of the composition.

15. The method or composition for use of claim 13, wherein the serum TTR levels are reduced by 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-98%, 98-99%, or 99-100% as compared to serum TTR levels before administration of the composition.

16. The method or composition for use of any one of claim 1-5 or 8-15, wherein the composition results in editing of the TTR gene.

17. The method or composition for use of claim 16, wherein the editing is calculated as a percentage of the population that is edited (percent editing).

18. The method or composition for use of claim 17, wherein the percent editing is between 30 and 99% of the population.

19. The method or composition for use of claim 17, wherein the percent editing is between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the population.

20. The method of any one of claims 1-5 or the composition for use of any one of claims 8-19, wherein the composition reduces amyloid deposition in at least one tissue.

21. The method or composition for use of claim 20, wherein the at least one tissue comprises one or more of stomach, colon, sciatic nerve, or dorsal root ganglion.

22. The method or composition for use of claim 20 or 21, wherein amyloid deposition is measured 8 weeks after administration of the composition.

23. The method or composition for use of any one of claims 20-22, wherein amyloid deposition is compared to a negative control or a level measured before administration of the composition.

24. The method or composition for use of any one of claims 20-23, wherein amyloid deposition is measured in a biopsy sample and/or by immunostaining.

25. The method or composition for use of any one of claims 20-24, wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the amyloid deposition seen in a negative control.

26. The method or composition for use of any one of claims 20-25, wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the amyloid deposition seen before administration of the composition.

27. The method or composition for use of any one of claim 1-5 or 8-26, wherein the composition is administered or delivered at least two times.

28. The method or composition for use of claim 27, wherein the composition is administered or delivered at least three times.

29. The method or composition for use of claim 27, wherein the composition is administered or delivered at least four times.

30. The method or composition for use of claim 27, wherein the composition is administered or delivered up to five, six, seven, eight, nine, or ten times.

31. The method or composition for use of any one of claims 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.

32. The method or composition for use of any one of claims 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks.

33. The method or composition for use of any one of claims 27-30, wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.

34. The method or composition of any one of the preceding claims, wherein the guide sequence is selected from SEQ ID NOs: 5-82.

35. The method or composition of any one of the preceding claims, wherein the guide RNA is at least partially complementary to a target sequence present in the human TTR gene.

36. The method or composition of claim 35, wherein the target sequence is in exon 1, 2, 3, or 4 of the human TTR gene.

37. The method or composition of claim 35, wherein the target sequence is in exon 1 of the human TTR gene.

38. The method or composition of claim 35, wherein the target sequence is in exon 2 of the human TTR gene.

39. The method or composition of claim 35, wherein the target sequence is in exon 3 of the human TTR gene.

40. The method or composition of claim 35, wherein the target sequence is in exon 4 of the human TTR gene.

41. The method or composition of any one of claims 1-40, wherein the guide sequence is complementary to a target sequence in the positive strand of TTR.

42. The method or composition of any one of claims 1-40, wherein the guide sequence is complementary to a target sequence in the negative strand of TTR.

43. The method or composition of any one of claims 1-40, wherein the first guide sequence is complementary to a first target sequence in the positive strand of the TTR gene, and wherein the composition further comprises a second guide sequence that is complementary to a second target sequence in the negative strand of the TTR gene.

44. The method or composition of any one of the preceding claims, wherein the guide RNA comprises a crRNA that comprises the guide sequence and further comprises a nucleotide sequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequence at its 3' end.

45. The method or composition of any one of the preceding claims, wherein the guide RNA is a dual guide (dgRNA).

46. The method or composition of claim 45, wherein the dual guide RNA comprises a crRNA comprising a nucleotide sequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequence at its 3' end, and a trRNA.

47. The method or composition of any one of claims 1-43, wherein the guide RNA is a single guide (sgRNA).

48. The method or composition of claim 47, wherein the sgRNA comprises a guide sequence that has the pattern of SEQ ID NO: 3.

49. The method or composition of claim 47, wherein the sgRNA comprises the sequence of SEQ ID NO: 3.

50. The method or composition of claim 48 or 49, wherein each N in SEQ ID NO: 3 is any natural or non-natural nucleotide, wherein the N's form the guide sequence, and the guide sequence targets Cas9 to the TTR gene.

51. The method or composition of any one of claims 47-50, wherein the sgRNA comprises any one of the guide sequences of SEQ ID NOs: 5-82 and the nucleotides of SEQ ID NO: 126.

52. The method or composition of any one of claims 47-51, wherein the sgRNA comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID Nos: 87-124.

53. The method or composition of claim 47, wherein the sgRNA comprises a sequence selected from SEQ ID Nos: 87-124.

54. The method or composition of any one of the preceding claims, wherein the guide RNA comprises at least one modification.

55. The method or composition of claim 54, wherein the at least one modification includes a 2'-O-methyl (2'-O-Me) modified nucleotide.

56. The method or composition of claim 54 or 55, wherein the at least one modification includes a phosphorothioate (PS) bond between nucleotides.

57. The method or composition of any one of claims 54-56, wherein the at least one modification includes a 2'-fluoro (2'-F) modified nucleotide.

58. The method or composition of any one of claims 54-57, wherein the at least one modification includes a modification at one or more of the first five nucleotides at the 5' end.

59. The method or composition of any one of claims 54-58, wherein the at least one modification includes a modification at one or more of the last five nucleotides at the 3' end.

60. The method or composition of any one of claims 54-59, wherein the at least one modification includes PS bonds between the first four nucleotides.

61. The method or composition of any one of claims 54-60, wherein the at least one modification includes PS bonds between the last four nucleotides.

62. The method or composition of any one of claims 54-61, wherein the at least one modification includes 2'-O-Me modified nucleotides at the first three nucleotides at the 5' end.

63. The method or composition of any one of claims 54-62, wherein the at least one modification includes 2'-O-Me modified nucleotides at the last three nucleotides at the 3' end.

64. The method or composition of any one of claims 54-63, wherein the guide RNA comprises the modified nucleotides of SEQ ID NO: 3.

65. The method or composition of any one of claims 1-64, wherein the composition further comprises a pharmaceutically acceptable excipient.

66. The method or composition of any one of claims 1-65, wherein the guide RNA is associated with a lipid nanoparticle (LNP).

67. The method or composition of claim 66, wherein the LNP comprises a CCD lipid.

68. The method or composition of claim 67, wherein the CCD lipid is Lipid A or Lipid B.

69. The method or composition of claim 66-68, wherein the LNP comprises a neutral lipid.

70. The method or composition of claim 69, wherein the neutral lipid is DSPC

71. The method or composition of any one of claims 66-70, wherein the LNP comprises a helper lipid.

72. The method or composition of claim 71, wherein the helper lipid is cholesterol.

73. The method or composition of any one of claims 66-72, wherein the LNP comprises a stealth lipid.

74. The method or composition of claim 73, wherein the stealth lipid is PEG2k-DMG.

75. The method or composition of any one of the preceding claims, wherein the composition further comprises an RNA-guided DNA binding agent.

76. The method or composition of any one of the preceding claims, wherein the composition further comprises an mRNA that encodes an RNA-guided DNA binding agent.

77. The method or composition of claim 75 or 76, wherein the RNA-guided DNA binding agent is a Cas cleavase.

78. The method or composition of claim 77, wherein the RNA-guided DNA binding agent is Cas9.

79. The method or composition of any one of claims 75-78, wherein the RNA-guided DNA binding agent is modified.

80. The method or composition of any one of claims 75-79, wherein the RNA-guided DNA binding agent is a nickase.

81. The method or composition of claim 79 or 80, wherein the modified RNA-guided DNA binding agent comprises a nuclear localization signal (NLS).

82. The method or composition of any one of claims 75-81, wherein the RNA-guided DNA binding agent is a Cas from a Type-II CRISPR/Cas system.

83. The method or composition of any one of the preceding claims, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.

84. The method or composition for use of any one of claim 1-5 or 8-83, wherein the composition reduces or prevents amyloids or amyloid fibrils comprising TTR.

85. The method or composition for use of claim 84, wherein the amyloids or amyloid fibrils are in the nerves, heart, or gastrointestinal track.

86. The method or composition for use of any one of claim 1-5 or 8-83, wherein non-homologous ending joining (NHEJ) leads to a mutation during repair of a DSB in the TTR gene.

87. The method or composition for use of claim 86, wherein NHEJ leads to a deletion or insertion of a nucleotide(s) during repair of a DSB in the TTR gene.

88. The method or composition for use of claim 87, wherein the deletion or insertion of a nucleotide(s) induces a frame shift or nonsense mutation in the TTR gene.

89. The method or composition for use of claim 87, wherein a frame shift or nonsense mutation is induced in the TTR gene of at least 50% of liver cells.

90. The method or composition for use of claim 89, wherein a frame shift or nonsense mutation is induced in the TTR gene of 50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or 99%-100% of liver cells.

91. The method or composition for use of any one of claims 87-90, wherein a deletion or insertion of a nucleotide(s) occurs in the TTR gene at least 50-fold or more than in off-target sites.

92. The method or composition for use of claim 91, wherein the deletion or insertion of a nucleotide(s) occurs in the TTR gene 50-fold to 150-fold, 150-fold to 500-fold, 500-fold to 1500-fold, 1500-fold to 5000-fold, 5000-fold to 15000-fold, 15000-fold to 30000-fold, or 30000-fold to 60000-fold more than in off-target sites.

93. The method or composition for use of any one of claims 87-92, wherein the deletion or insertion of a nucleotide(s) occurs at less than or equal to 3, 2, 1, or 0 off-target site(s) in primary human hepatocytes, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

94. The method or composition for use of claim 93, wherein the deletion or insertion of a nucleotide(s) occurs at a number of off-target sites in primary human hepatocytes that is less than the number of off-target sites at which a deletion or insertion of a nucleotide(s) occurs in Cas9-overexpressing cells, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

95. The method or composition for use of claim 94, wherein the Cas9-overexpressing cells are HEK293 cells stably expressing Cas9.

96. The method or composition for use of any one of claims 93-95, wherein the number of off-target sites in primary human hepatocytes is determined by analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA and the guide RNA, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

97. The method or composition for use of any one of claims 93-95, wherein the number of off-target sites in primary human hepatocytes is determined by an oligonucleotide insertion assay comprising analyzing genomic DNA from primary human hepatocytes transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide, optionally wherein the off-target site(s) does (do) not occur in a protein coding region in the genome of the primary human hepatocytes.

98. The method or composition of any one of claim 1-43 or 47-97, wherein the sequence of the guide RNA is: a) SEQ ID NO: 92 or 104; b) SEQ ID NO: 87, 89, 96, or 113; c) SEQ ID NO: 100, 102, 106, 111, or 112; or d) SEQ ID NO: 88, 90, 91, 93, 94, 95, 97, 101, 103, 108, or 109, optionally wherein the guide RNA does not produce indels at off-target site(s) that occur in a protein coding region in the genome of primary human hepatocytes.

99. The method or composition for use of any one of claim 1-5 or 8-98, wherein administering the composition reduces levels of TTR in the subject.

100. The method or composition for use of claim 99, wherein the levels of TTR are reduced by at least 50%.

101. The method or composition for use of claim 100, wherein the levels of TTR are reduced by 50%-60%, 60%-70%, 70% or 80%, 80%-90%, 90-95%, 95%-99%, or 99%-100%.

102. The method or composition for use of claim 100 or 101, wherein the levels of TTR are measured in serum, plasma, blood, cerebral spinal fluid, or sputum.

103. The method or composition for use of claim 100 or 101, wherein the levels of TTR are measured in liver, choroid plexus, and/or retina.

104. The method or composition for use of any one of claims 99-103, wherein the levels of TTR are measured via enzyme-linked immunosorbent assay (ELISA).

105. The method or composition for use of any one of claim 1-5 or 8-104, wherein the subject has ATTR.

106. The method or composition for use of any one of claim 1-5 or 8-105, wherein the subject is human.

107. The method or composition for use of claim 105 or 106, wherein the subject has ATTRwt.

108. The method or composition for use of claim 105 or 106, wherein the subject has hereditary ATTR.

109. The method or composition for use of any one of claim 1-5, 8-106, or 108, wherein the subject has a family history of ATTR.

110. The method or composition for use of any one of claim 1-5, 8-106, or 108-109, wherein the subject has familial amyloid polyneuropathy.

111. The method or composition for use of any one of claim 1-5 or 8-110, wherein the subject has only or predominantly nerve symptoms of ATTR.

112. The method or composition for use of any one of claim 1-5 or 8-110, wherein the subject has familial amyloid cardiomyopathy.

113. The method or composition for use of any one of claim 1-5, 8-109, or 112, wherein the subject has only or predominantly cardiac symptoms of ATTR.

114. The method or composition for use of any one of claim 1-5 or 8-113, wherein the subject expresses TTR having a V30 mutation.

115. The method or composition for use of claim 114, wherein the V30 mutation is V30A, V30G, V30L, or V30M.

116. The method or composition for use of claim any one of claim 1-5 or 8-113, wherein the subject expresses TTR having a T60 mutation.

117. The method or composition for use of claim 116, wherein the T60 mutation is T60A.

118. The method or composition for use of claim any one of claim 1-5 or 8-113, wherein the subject expresses TTR having a V122 mutation.

119. The method or composition for use of claim 118, wherein the V122 mutation is V122A, V122I, or V122(-).

120. The method or composition for use of any one of claim 1-5 or 8-119, wherein the subject expresses wild-type TTR.

121. The method or composition for use of any one of claim 1-5, 8-107, or 120, wherein the subject does not express TTR having a V30, T60, or V122 mutation.

122. The method or composition for use of any one of claim 1-5, 8-107, or 120-121, wherein the subject does not express TTR having a pathological mutation.

123. The method or composition for use of claim 121, wherein the subject is homozygous for wild-type TTR.

124. The method or composition for use of any one of claim 1-5 or 8-123, wherein after administration the subject has an improvement, stabilization, or slowing of change in symptoms of sensorimotor neuropathy.

125. The method or composition for use of claim 124, wherein the improvement, stabilization, or slowing of change in sensory neuropathy is measured using electromyogram, nerve conduction tests, or patient-reported outcomes.

126. The method or composition for use of any one of claim 1-5 or 8-125, wherein the subject has an improvement, stabilization, or slowing of change in symptoms of congestive heart failure.

127. The method or composition for use of claim 126, wherein the improvement, stabilization, or slowing of change in congestive heart failure is measured using cardiac biomarker tests, lung function tests, chest x-rays, or electrocardiography.

128. The method or composition for use of any one of claim 1-5 or 8-127, wherein the composition or pharmaceutical formulation is administered via a viral vector.

129. The method or composition for use of any one of claim 1-5 or 8-127, wherein the composition or pharmaceutical formulation is administered via lipid nanoparticles.

130. The method or composition for use of any one of claim 1-5 or 8-129, wherein the subject is tested for specific mutations in the TTR gene before administering the composition or formulation.

131. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 5.

132. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 6.

133. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 7.

134. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 8.

135. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 9.

136. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 10.

137. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 11.

138. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 12.

139. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 13.

140. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 14.

141. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 15.

142. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 16.

143. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 17.

144. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 18.

145. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 19.

146. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 20.

147. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 21.

148. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 22.

149. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 23.

150. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 24.

151. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 25.

152. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 26.

153. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 27.

154. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 28.

155. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 29.

156. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 30.

157. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 31.

158. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 32.

159. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 33.

160. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 34.

161. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 35.

162. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 36.

163. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 37.

164. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 38.

165. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 39.

166. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 40.

167. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 41.

168. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 42.

169. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 43.

170. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 44.

171. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 45.

172. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 46.

173. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 47.

174. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 48.

175. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 49.

176. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 50.

177. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 51.

178. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 52.

179. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 53.

180. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 54.

181. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 55.

182. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 56.

183. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 57.

184. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 58.

185. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 59.

186. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 60.

187. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 61.

188. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 62.

189. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 63.

190. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 64.

191. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 65.

192. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 66.

193. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 67.

194. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 68.

195. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 69.

196. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 70.

197. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 71.

198. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 72.

199. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 73.

200. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 74.

201. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 75.

202. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 76.

203. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 77.

204. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 78.

205. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 79.

206. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 80.

207. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 81.

208. The method or composition of any one of claims 1-130, wherein the sequence selected from SEQ ID NOs: 5-82 is SEQ ID NO: 82.

209. Use of a composition or formulation of any of claims 6-208 for the preparation of a medicament for treating a human subject having ATTR.

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