THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

Description

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT Application No. PCT/US2014/033082, filed Apr. 4, 2014, which claims the benefit of U.S. Provisional Application Ser. No. 61/808,594, filed Apr. 4, 2013, the teachings of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N).sub.20NGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, CRISPR/Cas systems could be useful tools for therapeutic applications, but unfortunately prior published reports have demonstrated an efficiency of allele targeting of only 2%-4% in human stem cells (Mali et al., Science 339:823-826 (2013)).

SUMMARY OF THE INVENTION

[0004] Work described herein demonstrates methods of allele targeting using CRISPR/Cas systems resulting in mutant cells with efficiencies of up to 80%. In particular, work described herein surprisingly and unexpectedly demonstrates that a multiple guide strategy (e.g., using two or more ribonucleic acids which guide Cas protein to and hybridize to a target polynucleotide sequence) efficiently and effectively deletes target polynucleotide sequences (e.g., B2M, HPRT, CCR5 and/or CXCR4) in primary somatic cells (e.g., human blood cells, e.g., CD34+ and T cells), in contrast to a single guide strategy which has been demonstrated by the inventors to efficiently delete target polynucleotide sequences in cell lines (e.g., 293T) but not in primary somatic cells. These vastly improved methods permit CRISPR/Cas systems to be utilized effectively for the first time for therapeutic purposes. Methods of delivery of CRISPR/Cas systems to human stem cells are provided. In addition, methods of specifically identifying useful RNA guide sequences are provided, along with particular guide sequences useful in targeting specific genes (e.g., B2M, HPRT, CCR5 and/or CXCR4). Moreover, methods of treatment (e.g., methods of treating HIV infection) utilizing the compositions and methods disclosed herein are provided.

[0005] In some aspects, the present invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0006] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0007] In some aspects, the present invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0008] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0009] In some embodiments, the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex.

[0010] In some embodiments, the Cas protein is Cas9 protein from any bacterial species or functional portion thereof. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex.

[0011] In some embodiments, the Cas protein is complexed with the one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with the multiple ribonucleic acids.

[0012] In some embodiments, the target motif is a 20-nucleotide DNA sequence. In some embodiments, each target motif is a 20-nucleotide DNA sequence. In some embodiments, the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is G(N).sub.19NGG. In some embodiments, each target motif is G(N).sub.19NGG. In some embodiments, the target motif is (N).sub.20NGG. In some embodiments, each target motif is (N).sub.20NGG.

[0013] In some embodiments, the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, each target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, the target polynucleotide sequence is cleaved such that a single-strand) break results. In some embodiments, each target polynucleotide sequence is cleaved such that a single-strand break results.

[0014] In some embodiments, the alteration is an indel. In some embodiments, the alteration results in reduced expression of the target polynucleotide sequence. In some embodiments, the alteration results in reduced expression of the target polynucleotide sequences. In some embodiments, the alteration results in a knock out of the target polynucleotide sequence.

[0015] In some embodiments, the alteration results in a knock out of the target polynucleotide sequences. In some embodiments, the alteration results in correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. In some embodiments, the alteration results in correction of the target polynucleotide sequences from undesired sequences to desired sequences. In some embodiments, the alteration is a homozygous alteration. In some embodiments, each alteration is a homozygous alteration.

[0016] In some embodiments, subsequent to cleavage of the target polynucleotide sequence, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. In some embodiments, the exogenously introduced DNA repair template is single-stranded. In some embodiments, the exogenously introduced DNA repair template is double-stranded.

[0017] In some embodiments, subsequent to cleavage of the target polynucleotide sequences, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. In some embodiments, the exogenously introduced DNA repair template is single-stranded. In some embodiments, the exogenously introduced DNA repair template is double-stranded.

[0018] In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a stem cell or a pluripotent cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34.sup.+ cell. In some embodiments, the cell is a CD34.sup.+ mobilized peripheral blood cell. In some embodiments, the cell is a CD34.sup.+ cord blood cell. In some embodiments, the cell is a CD34.sup.+ bone marrow cell. In some embodiments, the cell is a CD34.sup.+CD38-Lineage-CD90.sup.+CD45RA.sup.- cell. In some embodiments, the cell is a hepatocyte.

[0019] In some embodiments, the target polynucleotide sequence is CCR5. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0020] In some embodiments, the target polynucleotide sequence is CXCR4. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0021] In some embodiments, the target polynucleotide sequences comprise multiple different portions of CCR5. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0022] In some embodiments, the target polynucleotide sequences comprise multiple different portions of CXCR4. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0023] In some embodiments, the target polynucleotide sequences comprise at least a portion of CCR5 and at least a portion of CXCR4. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1 and the ribonucleic acid sequences of FIG. 2. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1 and the ribonucleic acid sequences of FIG. 2.

[0024] In some embodiments, the disorder is a genetic disorder. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immunodeficiency syndrome (AIDS).

[0025] In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs. In some embodiments, the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein. In some embodiments, the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank mutant alleles located between the target motifs. In some embodiments, the one to two ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence.

[0026] In some embodiments, the target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.

[0027] In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell.

[0028] In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least one mismatch when compared with all other genomic nucleotide sequences in the cell.

[0029] In some embodiments, the efficiency of alteration at each loci is from about 50% to about 80%. In some embodiments, the efficiency of alteration is at least about 5%. In some embodiments, the efficiency of alteration is at least about 10%. In some embodiments, the efficiency of alteration is from about 50% to about 80%.

[0030] In some embodiments, the Cas protein is encoded by a modified nucleic acid. In some embodiments, the modified nucleic acid comprises a ribonucleic acid containing at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments, at least one of the ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0031] In some embodiments, any of the Cas protein or the ribonucleic acids are expressed from a plasmid.

[0032] In some embodiments, any of the Cas protein or the ribonucleic acids are expressed using a promoter optimized for increased expression in stem cells. In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter.

[0033] In some embodiments, the method further comprises selecting cells that express the Cas protein. In some embodiments, selecting cells comprises FACS. In some embodiments, FACS is used to select cells which co-express Cas and a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein.

[0034] In some aspects, the present invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0035] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0036] In some aspects, the present invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0037] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Gas protein is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0038] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0039] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0040] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0041] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0042] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2.

[0043] In some embodiments, the composition further comprises a nucleic acid sequence encoding a Cas protein. In some embodiments, the composition further comprises a nucleic acid sequence encoding a Cas9 protein or a functional portion thereof. In some embodiments, the nucleic acid comprises a modified ribonucleic acid comprising at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0044] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0045] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0046] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0047] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0048] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2.

[0049] In some embodiments, the composition further comprises a nucleic acid sequence encoding a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein.

[0050] In some embodiments, the composition further comprises a promoter operably linked to the chimeric nucleic acid. In some embodiments, the promoter is optimized for increased expression in human stem cells. In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter.

[0051] In some embodiments, the Cas protein comprises a Cas9 protein or a functional portion thereof.

[0052] In some aspects, the present invention provides a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2. In some embodiments, the kit further comprises one or more cell lines, cultures, or populations selected from the group consisting of human pluripotent cells, primary human cells, and non-transformed cells. In some embodiments, the kit further comprises a DNA repair template.

[0053] In some embodiments, the cell comprises a primary cell. In some embodiments, the cell comprises a primary somatic cell. In some embodiments, the cell comprises an autologous primary somatic cell. In some embodiments, the cell comprises an allogeneic primary somatic cell. In some embodiments, the target polynucleotide sequence is B2M. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence optimized to target the B2M gene. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence optimized to target the B2M gene. In some embodiments, the target polynucleotide sequences comprises multiple different portions of B2M. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence optimized to target the B2M gene. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence optimized to target the B2M gene. In some embodiments, the one to two ribonucleic acids comprise two guide ribonucleic acid sequences.

[0054] In some embodiments, the one to two ribonucleic acids comprise two guide ribonucleic acid sequences. In some embodiments, the target polynucleotide sequence comprises CCR5. In some embodiments, the cell comprises a primary CD34+ hematopoietic progenitor cell. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to a different sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to offset sequences selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which hybridize to and target Cas protein to offset target sites in CCR5 selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences from SEQ ID NOs: 298-303. In some embodiments, the two guide ribonucleic acid sequences comprise a pair of guide ribonucleic acids selected from the group consisting of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298 and 300, SEQ ID NOs: 299 and 300, SEQ ID NOs: 298 and 303, SEQ ID NOs: 299 and 301, SEQ ID NOs: 298 and 299, SEQ ID NOs: 301 and 303, SEQ ID NOs: 298 and 302, and SEQ ID NOs: 298 and 301. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to a different sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to offset sequences selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which hybridize to and target Cas protein to offset target sites in CCR5 selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the target polynucleotide sequence comprises CXCR4. In some embodiments, the cell comprises a primary CD34+ hematopoietic progenitor cell. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to a different sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to offset sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which hybridize to and target Cas protein to offset target sites in CXCR4 selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the target polynucleotide sequence comprises B2M. In some embodiments, the cell comprises a primary cell. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to different sequences in the B2M gene. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which are complementary to offset sequences in the B2M gene. In some embodiments, the two guide ribonucleic acid sequences comprise any combination of two guide ribonucleic acid sequences which hybridize to and target Cas protein to offset target sites in B2M.

[0055] In some aspects, the invention provides a method for altering a target polynucleotide sequence in a primary cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least two ribonucleic acids, wherein the at least two ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0056] In some aspects, the invention provides a method for altering a target polynucleotide sequence in a primary cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least two ribonucleic acids, wherein the at least two ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0057] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a primary cell ex vivo by contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least two ribonucleic acids, wherein the at least two ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0058] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a primary cell ex vivo by contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least two ribonucleic acids, wherein the at least two ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0059] In some aspects, the invention provides, a method for simultaneously altering multiple target polynucleotide sequences in a primary cell comprising contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0060] In some aspects, the invention provides, a method for simultaneously altering multiple target polynucleotide sequences in a primary cell comprising contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0061] In some aspects, the disclosure provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a primary cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0062] In some aspects, the disclosure provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a primary cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids comprise guide ribonucleic acids which direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0063] In some embodiments, the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the Cas protein is Cas9 protein from any bacterial species or functional portion thereof. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the Cas protein is complexed with the one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with the multiple ribonucleic acids.

[0064] In some embodiments, the target motif is a 20-nucleotide DNA sequence. In some embodiments, each target motif is a 20-nucleotide DNA sequence. In some embodiments, the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is G(N).sub.19NGG. In some embodiments, each target motif is G(N).sub.19NGG. In some embodiments, the target motif is (N).sub.20NGG. In some embodiments, each target motif is (N).sub.20NGG. In some embodiments, the target motif comprises a sequence selected from the group consisting of SEQ ID NOs: 1-297 or 304-333. In some embodiments, the target motif comprises a sequence selected from the group consisting of SEQ ID NOs: 1-297 or 304-333. In some embodiments, the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, each target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, the target polynucleotide sequence is cleaved such that a single-strand break results. In some embodiments, each target polynucleotide sequence is cleaved such that a single-strand break results. In some embodiments, the alteration is an indel. In some embodiments, the alteration results in reduced expression of the target polynucleotide sequence. In some embodiments, the alteration results in reduced expression of the target polynucleotide sequences. In some embodiments, the alteration results in a knock out of the target polynucleotide sequence. In some embodiments, the alteration results in a knock out of the target polynucleotide sequences. In some embodiments, the alteration results in correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. In some embodiments, the alteration results in correction of the target polynucleotide sequences from undesired sequences to desired sequences. In some embodiments, the alteration is a homozygous alteration.

[0065] In some embodiments, each alteration is a homozygous alteration. In some embodiments, subsequent to cleavage of the target polynucleotide sequence, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. In some embodiments, the exogenously introduced DNA repair template is single-stranded. In some embodiments, the exogenously introduced DNA repair template is double-stranded. In some embodiments, subsequent to cleavage of the target polynucleotide sequences, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. In some embodiments, the exogenously introduced DNA repair template is single-stranded. In some embodiments, the exogenously introduced DNA repair template is double-stranded. In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a stem cell or a pluripotent cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34.sup.+ cell. In some embodiments, the cell is a CD34.sup.+ mobilized peripheral blood cell. In some embodiments, the cell is a CD34.sup.+ cord blood cell. In some embodiments, the cell is a CD34.sup.+ bone marrow cell. In some embodiments, the cell is a CD34.sup.+CD38-Lineage-CD90.sup.+CD45RA.sup.- cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a primary cell. In some embodiments, the target polynucleotide sequence is CCR5.

[0066] In some embodiments, the two ribonucleic acids comprise a different sequence selected from the group consisting of SEQ ID NOs: 298-303. In some embodiments, the two guide ribonucleic acid sequences comprise a pair of guide ribonucleic acids selected from the group consisting of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298 and 300, SEQ ID NOs: 299 and 300, SEQ ID NOs: 298 and 303, SEQ ID NOs: 299 and 301, SEQ ID NOs: 298 and 299, SEQ ID NOs: 301 and 303, SEQ ID NOs: 298 and 302, and SEQ ID NOs: 298 and 301. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to different sequences selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to different sequences with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridizes to offset sequences selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to offsets sequences with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the target polynucleotide sequence is CXCR4. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to different sequences selected from the group consisting of SEQ ID NO: 140-297. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to different sequences with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 140-297. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to offset sequences selected from the group consisting of SEQ ID NO: 140-297. In some embodiments, the two ribonucleic acids comprise sequences which are complementary to and/or hybridize to offset sequences with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 140-297. In some embodiments, the target polynucleotide sequences comprise multiple different portions of CCR5. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to an offset sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0067] In some embodiments, the target polynucleotide sequences comprise multiple different portions of CXCR4. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 140-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 140-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of SEQ ID NOs: 140-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to an offset sequence selected from the group consisting of SEQ ID NOs: 140-297 and 304-333.

[0068] In some embodiments, the target polynucleotide sequences comprise at least a portion of CCR5 and at least a portion of CXCR4. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to an offset sequence selected from the group consisting of SEQ ID NOs: 1-297 and 304-333. In some embodiments, the multiple ribonucleic acids comprise at least two ribonucleic acid sequences which are complementary to and/or hybridize to offset sequences selected from the group consisting of SEQ ID NOs: 1-139 and 304-333, and at least two ribonucleic acid sequences which are complementary to and/or hybridize to offset sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the multiple ribonucleic acids comprises at least two ribonucleic acid sequences which are complementary to and/or hybridize to different sequences with a single nucleotide mismatch to an offset sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333, and at least two ribonucleic acid sequences which are complementary to and/or hybridize to different sequences with a single nucleotide mismatch to an offset sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0069] In some embodiments, the disorder is a genetic disorder. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immunodeficiency syndrome (AIDS). In some embodiments, the two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs. In some embodiments, the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein. In some embodiments, the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank mutant alleles located between the target motifs. In some embodiments, the two ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least two mismatches when compared with all other genomic nucleotide sequences in the cell.

[0070] In some embodiments, the two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the efficiency of alteration at each loci is from about 50% to about 80%.

[0071] In some embodiments, the Cas protein is encoded by a modified nucleic acid. In some embodiments, the modified nucleic acid comprises a ribonucleic acid containing at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments, at least one of the two ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments, the two ribonucleic acids comprise modified ribonucleic acids comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0072] In some embodiments, any of the Cas protein or the ribonucleic acids are expressed from a plasmid. In some embodiments, any of the Cas protein or the ribonucleic acids are expressed using a promoter optimized for increased expression in stem cells. In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter. In some embodiments, the method comprises selecting cells that express the Cas protein. In some embodiments, selecting cells comprises FACS. In some embodiments, FACs is used to select cells which co-express Cas and a fluorescent protein.

[0073] In some aspects, the invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0074] In some aspects, the invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0075] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence. In some embodiments, the efficiency of alteration is from about 8% to about 80%.

[0076] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0077] In some aspects, the invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0078] In some aspects, the invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0079] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences. In some embodiments, the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0080] In some aspects, the invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0081] In some aspects, the disclosure provides a composition comprising at least two ribonucleic acids each comprising a different sequence selected from the group consisting of SEQ ID NOs: 298-303.

[0082] In some aspects, the disclosure provides a composition comprising at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0083] In some aspects, the disclosure provides a composition comprising at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0084] In some aspects, the disclosure provides a composition comprising at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0085] In some aspects, the disclosure provides a composition comprising at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0086] In some embodiments, at least one of the two ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0087] In some embodiments, the two ribonucleic acids comprise modified ribonucleic acids comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments, the composition includes a nucleic acid sequence encoding a Cas protein. In some embodiments, the composition includes a nucleic acid sequence encoding a Cas9 protein or a functional portion thereof. In some embodiments, the nucleic acid comprises a modified ribonucleic acid comprising at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0088] In some aspects, the invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acids each having a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303.

[0089] In some aspects, the invention provides a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acids each having a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0090] In some aspects, the invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acids each having a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0091] In some aspects, the invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acid sequences each comprising a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0092] In some aspects, the invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acid sequences each comprising a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0093] In some embodiments, the composition includes a nucleic acid sequence encoding a detectable marker. In some embodiments, the composition includes a nucleic acid sequence encoding a fluorescent protein. In some embodiments, the composition includes a promoter operably linked to the chimeric nucleic acid. In some embodiments, the promoter is optimized for increased expression in human stem cells. In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter. In some embodiments, the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments, the Cas protein comprises a Cas9 protein or a functional portion thereof.

[0094] In some aspects, the invention provides a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acids each comprising a different sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303.

[0095] In some aspects, the invention provides a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333.

[0096] In some aspects, the invention provides a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acids each comprising a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0097] In some embodiments, the kit includes one or more cell lines, cultures, or populations selected from the group consisting of human pluripotent cells, primary human cells, and non-transformed cells. In some aspects, the kit includes a DNA repair template.

[0098] In some aspects, the invention provides a method of administering cells to a subject in need of such cells, the method comprising: (a) contacting a cell or population of cells ex vivo with a Cas protein and two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence is cleaved; and (b) administering the resulting cells from (a) to a subject in need of such cells.

[0099] In some aspects, the invention provides a method of administering cells to a subject in need of such cells, the method comprising: (a) contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, and (iii) at least two additional ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence in the cell or population of cells, wherein the target polynucleotide sequences are cleaved; and (b) administering the resulting cell or cells from (a) to a subject in need of such cells.

[0100] In some embodiments, cleavage of the target polynucleotide sequence encoding B2M in the cell or population of cells reduces the likelihood that the resulting cell or cells will trigger a host immune response when the cells are administered to the subject. In some aspects, the target polynucleotide sequence comprises CCR5. In some embodiments, the at least two ribonucleic acids comprise two different sequences selected from the group consisting of SEQ ID NOs: 298-303. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to sequences comprising at least one nucleotide mismatch to different sequences selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the target polynucleotide sequence comprises CXCR4. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to a different sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to sequences comprising at least one nucleotide mismatch to different sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the cell or population of cells comprises primary cells. In some embodiments, the subject in need of administration of cells is suffering from a disorder. In some embodiments, the disorder comprises a genetic disorder. In some embodiments, the disorder comprises an infection. In some embodiments, the disorder comprises HIV or AIDs. In some embodiments, the disorder comprises cancer.

[0101] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with a Cas protein and two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M is cleaved, thereby reducing the likelihood that cells administered to the subject will trigger a host immune response in the subject; and (b) administering the resulting cells from (a) to a subject in need of such cells.

[0102] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M in the cell or population of cells is cleaved, thereby reducing the likelihood that the cell or population of cells will trigger a host immune response in the subject, and (iii) at least two additional ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence in the cell or population of cells, wherein the target polynucleotide sequence is cleaved; and (b) administering the resulting cell or cells from (a) to a subject in need of such cells.

[0103] In some embodiments, the target polynucleotide sequence comprises CCR5. In some embodiments, the at least two ribonucleic acids comprise two different sequences selected from the group consisting of SEQ ID NOs: 298-303. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to sequences comprising at least one nucleotide mismatch to different sequences selected from the group consisting of SEQ ID NOs: 1-139 and 304-333. In some embodiments, the target polynucleotide sequence comprises CXCR4. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to a different sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the at least two ribonucleic acids each comprise sequences which are complementary to and/or hybridize to sequences comprising at least one nucleotide mismatch to different sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the cell or population of cells comprises primary cells. In some embodiments, the subject in need of administration of cells is suffering from a disorder. In some embodiments, the disorder comprises a genetic disorder. In some embodiments, the disorder comprises an infection. In some embodiments, the disorder comprises HIV or AIDs. In some embodiments, the disorder comprises cancer.

[0104] In some embodiments of the method disclosed herein, at least at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0105] In some embodiments of the methods disclosed herein, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0106] In some embodiments of the methods disclosed herein, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0107] In some embodiments of the methods disclosed herein, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0108] In some embodiments of the methods disclosed herein, the at least one ribonucleic acid has a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0109] In some embodiments of the compositions disclosed herein, the at least one additional ribonucleic acid has a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0110] In some embodiments of the kits disclosed herein, the at least one ribonucleic acid sequence is selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0111] In some embodiments of the methods disclosed herein, the two guide ribonucleic acid sequences comprise any combination of two ribonucleic acid sequences selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0112] In some embodiments of the methods disclosed herein, the two guide ribonucleic acid sequences comprise any combination of two ribonucleic acid sequences with a single nucleotide mismatch to a ribonucleic acid sequence selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0113] In some embodiments of the methods disclosed herein, the two guide ribonucleic acid sequences comprise any combination of two ribonucleic acid sequences with at least two nucleotide mismatches to a ribonucleic acid sequence selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0114] In some embodiments of the methods disclosed herein, the two guide ribonucleic acid sequences comprise a pair of guide ribonucleic acids selected from the group consisting of SEQ ID NOs: 309 and 317, SEQ ID NO: 309 and SEQ ID NO: 331, SEQ ID NO: 309 and SEQ ID NO: 332, SEQ ID NO: 309 and SEQ ID NO: 316, SEQ ID NO: 317 and SEQ ID NO: 331, SEQ ID NO: 317 and SEQ ID NO: 332, SEQ ID NO: 317 and SEQ ID NO: 316, SEQ ID NO: 331 and SEQ ID NO: 332, SEQ ID NO: 331 and SEQ ID NO: 316, and SEQ ID NO: 332 and SEQ ID NO: 316.

[0115] In some embodiments of the methods disclosed herein, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0116] In some aspects, the disclosure provides a composition comprising any combination of two ribonucleic acids selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0117] In some aspects, the disclosure provides a composition comprising any combination of two ribonucleic acids having a single nucleotide mismatch to the ribonucleic acid sequences selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0118] In some aspects, the disclosure provides a composition comprising any combination of two ribonucleic acids having two nucleotide mismatches to the ribonucleic acid sequences selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0119] In some aspects, the disclosure provides a composition comprising any combination of two ribonucleic acids having a single nucleotide mismatch to the ribonucleic acid sequences selected from the group consisting of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0120] In some embodiments of the compositions disclosed herein, at least one of the two ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments of the compositions disclosed herein, the two ribonucleic acids comprise modified ribonucleic acids comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments of the compositions disclosed herein, the composition includes a nucleic acid sequence encoding a Cas9 protein or a functional portion thereof. In some embodiments of the compositions disclosed herein, the nucleic acid comprises a modified ribonucleic acid comprising at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0121] In some aspects, the disclosure provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acids each having a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0122] In some aspects, the disclosure provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acid sequences each comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0123] In some aspects, the disclosure provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acid sequences each comprising a sequence with at least two nucleotide mismatches to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0124] In some embodiments of the compositions disclosed herein, the composition includes a nucleic acid sequence encoding a detectable marker. In some embodiments of the compositions disclosed herein, the composition includes a nucleic acid sequence encoding a fluorescent protein. In some embodiments of the compositions disclosed herein, the composition includes a promoter operably linked to the chimeric nucleic acid. In some embodiments of the compositions disclosed herein, the promoter is optimized for increased expression in human stem cells. In some embodiments of the compositions disclosed herein, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter. In some embodiments of the compositions disclosed herein, the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In some embodiments of the compositions disclosed herein, the Cas protein comprises a Cas9 protein or a functional portion thereof.

[0125] In some aspects, the disclosure provides a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acids each selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 309, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 331 and SEQ ID NO: 332.

[0126] In some embodiments of the kits disclosed herein, the nucleic acid encoding the Cas9 protein comprises a modified ribonucleic acid. In some embodiments of the kits disclosed herein, the modified ribonucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0127] In some embodiments of the kits disclosed herein, the kit includes one or more cell lines, cultures, or populations selected from the group consisting of human pluripotent cells, primary human cells, and non-transformed cells. In some embodiments, the primary human cell comprises a primary CD34+ HSPC. In some embodiments of the kits disclosed herein, the kit includes a DNA repair template.

BRIEF DESCRIPTION OF THE DRAWINGS

[0128] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0129] FIG. 1 shows exemplary guide RNA sequences useful when the target polynucleotide sequence is human CCR5.

[0130] FIG. 2 shows exemplary guide RNA sequences useful when the target polynucleotide sequence is human CXCR4.

[0131] FIG. 3 shows an exemplary amino acid sequence of a Cas protein. Yellow highlights indicate Ruv-C-like domain. Underlining indicates HNH nuclease domain.

[0132] FIGS. 4A, 4B, 4C, 4D and 4E demonstrate that a single guide strategy achieves high efficiency genome editing in cell lines, but not in clinically relevant primary somatic cells. FIG. 4A is a table showing CRISPR-targeting sites in the CCR5 locus (single guides), which were found by scanning the human chemokine receptor CCR5 gene for optimized guide RNA sequences using a CRISPR design program (available on the world wide web at http://CRISPR.mit.edu) (left panel). A total of 11 guide RNAs having a score greater than 50 was tested for editing efficiency in a K562 cell line. FIG. 4A (right panel) shows the editing efficiency of 7 of selected guides (% indels) was measured by a CEL surveyor assay. FIG. 4B shows a comparative analysis of genome-editing efficiency in cell lines 293T, K562 (left two panels) and primary human CD34+ HSPCs (right two panels) illustrating inefficient genome editing efficiency in primary CD34+ cells. Cells were transfected with Cas9 (lane 1) together with guide RNA (lane 2) or expression plasmids (lane 3). FIG. 4C is a schematic illustrating CRISPR-targeting sites in the CCR5 locus (single guides). FIG. 4D shows the results of targeting the B2M locus with single guide RNAs in 293T cells. FIG. 4E shows the results of flow cytometry analysis using a single guide strategy targeting B2M in 293T cell, which demonstrate that B2M CRISPRs ablate B2M surface expression with high efficiency.

[0133] FIGS. 5A, 5B and 5C demonstrate that a double guide strategy achieves genome editing with high efficacy in clinically relevant cells. FIG. 5A shows that as compared to single guide (A or B), 2-guide combination (A+B) showed robust editing efficiency in targeting CCR5 in K562 cell line. FIG. 5B shows various guide combinations and spacing between each guide pair with orientation (upper panel). The PCR results (bottom left panel) and CEL assay (bottom right) show robust genome editing for tested guide pairs. FIG. 5C shows the results of PCR analysis indicating that with 2-guide combination wild-type Cas9 effectively deleted the DNA sequence between the two guides, in contrast to Nickase (D10A) which did not effectively delete the DNA sequence between the two guides. FIG. 5D is a schematic showing double B2M CRISPR combinations.

[0134] FIGS. 6A and 6B demonstrate effective genome-editing in human CD34+ HSPC using a two-guide approach. FIG. 6A is a representative gel picture showing efficient clonal deletion frequency using two guides. Clonal deletion efficiency was determined by PCR carried on individual colony grown on methyl cellulose. FIG. 6B is a Table showing data obtained from two independent clonal deletion experiments, which suggests efficacious genome-editing in primary human CD34+ cells using a two-guide approach.

[0135] FIGS. 7A, 7B and 7C demonstrate that in contrast to primary cells, the double guide strategy does not improve B2M editing efficiency in 293T cells. FIG. 7A shows the gating strategy for flow cytometry analysis of 293T cells electroporated with 1 .mu.g Cas9 plus either 0.5 .mu.g gRNA or 0.25 .mu.g+0.25 .mu.g gRNA targeting B2M 72 hours post-transfection in a 6-well format. FIG. 7B shows the results of a SURVEYOR assay with B2M CRISPR gRNAs in 293T cells (72 h). FIG. 7C shows that the double guide strategy does not improve B2M cutting efficiency in 293T cells, in contrast to the double guide strategy which significantly improves B2M cutting efficiency in primary cells (FIG. 5).

[0136] FIGS. 8A, 8B, 8C and 8D demonstrate ablation of B2M surface expression in somatic cells (e.g., primary CD4+ T cells) using a double guide strategy. FIG. 8A shows the results of a flow cytometry analysis demonstrating B2M knock-out efficiency in CD4+ T cells (total live cells). FIG. 8B shows the results of a flow cytometry analysis demonstrating B2M knock-out efficiency in CD4+ T cells (gated on GFP+ cells). FIG. 8C shows a Table quantifying the results of a flow cytometry analysis demonstrating B2M knock-out efficiency in CD4+ T cells. FIG. 8D shows the results of a flow cytometry analysis of cells gated on live/7AAD neg/GFP+ cells, demonstrating that the double guide strategy results in ablation of B2M surface expression.

[0137] FIGS. 9A, 9B, 9C and 9D demonstrate targeted capture and extremely deep sequencing of on-target and predicted off-target sites in CD34+ HPSCs. FIG. 9A is a schematic overview of targeted capture and deep sequencing of on-target and predicted off-target sites (red bar). A 500 bp flanking cutting site (in yellow) were included in sequence analysis for detection of structural rearrangements, including translocations. Probe sets are indicated in blue. FIG. 9B features plots showing consistent sequencing depth coverage at both on-target (left panel) and off-target (right panel) sites, achieving a coverage exceeding 3,000.times. for all on-target sites. Decrease in sequencing depth at the on-target sites in dual-gRNA libraries is marked by arrow, supporting predicted deletions (bottom left; i=35 bp, ii=205 bp, iii=205 bp). FIG. 9C is a Table depicting the precise estimation of on-target mutation allele frequencies by capture sequencing. Notably, the observed rate of effective null mutation exceeds previous estimates by PCR validation of predictable deletions, as smaller InDels and inversions also occur at appreciable frequencies. FIG. 9D is a Table depicting the estimation of mutation frequencies at predicted off-target sites (*One off-target site was statistically different from controls following correction for multiple comparisons; p.ltoreq.7.6.times.10.sup.-11), N-fold enrichment is determined based on the ratio of non-reference reads in treated libraries compared to untreated library. Each value represents the average of all off-target sites for a given single gRNA or dual-gRNA experiment. Enrichment of 1 is equivalent to baseline (untreated control). **For reference to on-target enrichments, on-target combined represents the proportion of non-reference reads (including single and dual-gRNA treatments using a given gRNA) to total reads at on-target sites in treatment compared to control.

[0138] FIGS. 10A and 10B demonstrate potential off-target sites identified in CCR5 homologue CCR2 and analysis of events detected at the single off-target site in which mutagenesis was significantly detected above background. FIG. 10A depicts a sequence alignment of CCR5 gRNAs utilized in this study in relation to the closest homologous sequence in CCR2 showing mismatched nucleotides in bold. Noteworthy is the fact that guide crCCR5_B, which yielded the sole significantly detected off-target mutagenesis in CCR2 (detailed in panel B), has 3 nucleotide mismatches, which are distal to the PAM (underlined) and seed (grey box) sequences. FIG. 10B is a Table depicting in-depth analyses of all sequence reads at the single off-target site in which mutagenesis was significantly detected above background in both capture libraries treated with the associated gRNA (B; libraries treated with single gRNA crCCR5_B & dual-gRNA crCCR5_A+B), as well as the library treated with gRNA crCCR5_A as a comparison. Total off-target mutation frequency at this site was 0.6% in the single gRNA treatment (crCCR5_B) and notably decreased to 0.24% in the dual gRNA treatment (crCCR5_A+B) in which gRNA plasmid concentration of each gRNA was half of that utilized in single gRNA treatments.

[0139] FIGS. 11A and 11B demonstrate the generation of Firm knockout mice by a CRISPR/Cas system employing a modified Cas9 mRNA. FIG. 11A is a schematic illustrating the steps employed to generate Fgm knockout mice using the CRISPR/Cas system employing the Cas9 modified RNA. FIG. 11B shows part of a gel picture depicting results from PCR screening of surviving pups for genetic mutations resulting from genomic editing using the CRISPR/Cas system and the modified Cas9 mRNA.

[0140] FIG. 12 shows predicted gRNA mapping in Ensembl GRCh37v71.

[0141] FIG. 13 shows guide pair crCCR5_A+B on-target alleles.

[0142] FIG. 14 shows guide pair crCCR5_C+D on-target alleles.

[0143] FIG. 15 shows guide pair crCCR5_D+Q on-target alleles.

[0144] FIG. 16 shows off-target sites with statistically significant mutational burden.

[0145] FIG. 17 shows a comparison of on- and off-target mutational burdens.

DETAILED DESCRIPTION OF THE INVENTION

[0146] Work described herein demonstrates methods of allele targeting using CRISPR/Cas systems resulting in mutant cells with efficiencies of up to 80%. In particular, work described herein surprisingly and unexpectedly demonstrates that a multiple guide strategy (e.g., using two or more ribonucleic acids which guide Cas protein to and hybridize to a target polynucleotide sequence) efficiently and effectively deletes target polynucleotide sequences (e.g., B2M, HPRT, CCR5 and/or CXCR4) in primary somatic cells (e.g., human blood cells, e.g., CD34+ and T cells), in contrast to a single guide strategy which has been demonstrated by the inventors to efficiently delete target polynucleotide sequences in cell lines (e.g., 293T) but not in primary somatic cells. These vastly improved methods permit CRISPR/Cas systems to be utilized effectively for the first time for therapeutic purposes. Methods of delivery of CRISPR/Cas systems to human stem cells are provided. In addition, methods of specifically identifying useful RNA guide sequences are provided, along with particular guide sequences useful in targeting specific genes (e.g., B2M, HPRT, CCR5 and/or CXCR4). Moreover, methods of treatment (e.g., methods of treating HIV infection) utilizing the compositions and methods disclosed herein are provided. Moreover, methods of administering cells (e.g., methods of administering a cell that has a reduced likelihood of triggering a host immune response) utilizing the compositions and methods disclosed herein are provided.

[0147] In one aspect, the present invention provides a method for altering a target polynucleotide sequence in a cell.

[0148] An exemplary method for altering a target polynucleotide sequence in a cell comprises contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0149] As used herein, the term "contacting" (i.e., contacting a polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and/or ribonucleic acids) is intended to include incubating the Cas protein and/or the ribonucleic acids in the cell together in vitro (e.g., adding the Cas protein or nucleic acid encoding the Cas protein to cells in culture) or contacting a cell ex vivo. The step of contacting a target polynucleotide sequence with a Cas protein and/or ribonucleic acids as disclosed herein can be conducted in any suitable manner. For example, the cells may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with a Cas protein and/or ribonucleic acids as disclosed herein can also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further.

[0150] In another aspect, the present invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject.

[0151] The terms "treat", "treating", "treatment", etc., as applied to an isolated cell, include subjecting the cell to any kind of process or condition or performing any kind of manipulation or procedure on the cell. As applied to a subject, the terms refer to providing a cell in which a target polynucleotide sequence has been altered ex vivo according to the methods described herein to an individual. The individual is usually ill or injured, or at increased risk of becoming ill relative to an average member of the population and in need of such attention, care, or management.

[0152] As used herein, the term "treating" and "treatment" refers to administering to a subject an effective amount of cells with target polynucleotide sequences altered ex vivo according to the methods described herein so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with a disorder associated with expression of a polynucleotide sequence, as well as those likely to develop such a disorder due to genetic susceptibility or other factors.

[0153] By "treatment," "prevention" or "amelioration" of a disease or disorder is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of a condition associated with such a disease or disorder. In one embodiment, the symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0154] An exemplary method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject comprises (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0155] The present invention contemplates altering target polynucleotide sequences in any manner which is available to the skilled artisan utilizing a CRISPR/Cas system of the present invention. Any CRISPR/Cas system that is capable of altering a target polynucleotide sequence in a cell can be used. Such CRISPR-Cas systems can employ a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1(6)e60). The molecular machinery of such Cas proteins that allows the CRISPR/Cas system to alter target polynucleotide sequences in cells include RNA binding proteins, endo- and exo-nucleases, helicases, and polymerases. In some embodiments, the CRISPR/Cas system is a CRISPR type I system. In some embodiments, the CRISPR/Cas system is a CRISPR type II system.

[0156] The CRISPR/Cas systems of the present invention can be used to alter a target polynucleotide sequence in a cell. The present invention contemplates altering target polynucleotide sequences in a cell for any purpose. In some embodiments, the target polynucleotide sequence in a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell with a resulting genotype that differs from its original genotype. In some instances, a "mutant cell" exhibits a mutant phenotype, for example when a normally functioning gene is altered using the CRISPR/Cas systems of the present invention. In other instances, a "mutant cell" exhibits a wild-type phenotype, for example when a CRISPR/Cas system of the present invention is used to correct a mutant genotype. In some embodiments, the target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, the target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0157] In some embodiments, the alteration is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. A CRISPR/Cas system of the present invention can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence.

[0158] In some embodiments, the alteration results in a knock out of the target polynucleotide sequence or a portion thereof. Knocking out a target polynucleotide sequence or a portion thereof using a CRISPR/Cas system of the present invention can be useful for a variety of applications. For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes. For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing those cells comprising the knocked out mutant allele into a subject).

[0159] As used herein, "knock out" includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock out can be achieved by altering a target polynucleotide sequence by inducing an indel in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence (e.g., a DNA binding domain). Those skilled in the art will readily appreciate how to use the CRISPR/Cas systems of the present invention to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

[0160] In some embodiments, the alteration results in reduced expression of the target polynucleotide sequence. The terms "decrease," "reduced," "reduction," and "decrease" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, decrease," "reduced," "reduction," "decrease" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0161] The terms "increased", "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0162] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0163] In some embodiments, the alteration is a homozygous alteration. In some embodiments, the alteration is a heterozygous alteration.

[0164] In some embodiments, the alteration results in correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. The CRISPR/Cas systems of the present invention can be used to correct any type of mutation or error in a target polynucleotide sequence. For example, the CRISPR/Cas systems of the present invention can be used to insert a nucleotide sequence that is missing from a target polynucleotide sequence due to a deletion. The CRISPR/Cas systems of the present invention can also be used to delete or excise a nucleotide sequence from a target polynucleotide sequence due to an insertion mutation. In some instances, the CRISPR/Cas systems of the present invention can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (e.g., to restore function to a target polynucleotide sequence that is impaired due to a loss of function mutation, i.e., a SNP).

[0165] The CRISPR/Cas systems of the present invention can alter target polynucleotides with surprisingly high efficiency as compared to conventional CRISPR/Cas systems. In certain embodiments, the efficiency of alteration is at least about 5%. In certain embodiments, the efficiency of alteration is at least about 10%. In certain embodiments, the efficiency of alteration is from about 10% to about 80%. In certain embodiments, the efficiency of alteration is from about 30% to about 80%. In certain embodiments, the efficiency of alteration is from about 50% to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 80%.

[0166] The CRISPR/Cas systems of the present invention can be used to alter any target polynucleotide sequence in a cell. Those skilled in the art will readily appreciate that desirable target polynucleotide sequences to be altered in any particular cell may correspond to any genomic sequence for which expression of the genomic sequence is associated with a disorder or otherwise facilitates entry of a pathogen into the cell. For example, a desirable target polynucleotide sequence to alter in a cell may be a polynucleotide sequence corresponding to a genomic sequence which contains a disease associated single polynucleotide polymorphism. In such example, the CRISPR/Cas systems of the present invention can be used to correct the disease associated SNP in a cell by replacing it with a wild-type allele. As another example, a polynucleotide sequence of a target gene which is responsible for entry or proliferation of a pathogen into a cell may be a suitable target for deletion or insertion to disrupt the function of the target gene to prevent the pathogen from entering the cell or proliferating inside the cell.

[0167] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0168] In some embodiments, a target polynucleotide sequence is a pathogenic genomic sequence. Exemplary pathogenic genomic sequences include, but are not limited to a viral genomic sequence, a bacterial genomic sequence, a fungal genomic sequence, a toxin genomic sequence, or a parasitic genomic sequence. In such embodiments, the CRISPR/Cas systems of the present invention can be used to disrupt the function of a pathogen (e.g., to treat or prevent an infection by the pathogen) by cleaving a genomic sequence of the pathogen (e.g., a genomic sequence that is critical for entry into a cell, or responsible for multiplication, growth or survival once the pathogen is inside a cell).

[0169] In some embodiments, the target polynucleotide sequence is beta-2-microglobulin (B2M; Gene ID: 567). The B2M polynucleotide sequence encodes a serum protein associated with the heavy chain of the major histocompatibility complex (MHC) class I molecules which are expressed on the surface of virtually all nucleated cells. B2M protein comprises a beta-pleated sheet structure that has been found to form amyloid fibrils in certain pathological conditions. The B2M gene has 4 exons which span approximately 8 kb. B2M has been observed in the serum of normal individuals and in elevated amounts in urine from patients having Wilson disease, cadmium poisoning, and various conditions leading to renal tubular dysfunction. Other pathological conditions known to be associated with the B2M include, without limitation, a homozygous mutation (e.g., ala11pro) in the B2M gene has been reported in individuals having familial hypercatabolic hypoproteinemia, a heterozygous mutation (e.g., asp76asn) in the B2M gene has been reported in individuals having familial visceral amyloidosis

[0170] In some embodiments, the target polynucleotide sequence is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0171] In some embodiments, the target polynucleotide sequence is hypoxanthine phosphoribosyltransferase 1 (HPRT1; Gene ID: 3251).

[0172] In some embodiments, the target polynucleotide sequence is CCR5 (Gene ID: 1234, also known as CC-CKR-5, CCCKR5, CCR-5, CD195, CKR-5, CKR5, CMKBR5, and IDDM22). In some embodiments, the target polynucleotide sequence is a variant of CCR5. In some embodiments, the target polynucleotide sequence is a homolog of CCR5. In some embodiments, the target polynucleotide sequence is an ortholog of CCR5.

[0173] In some embodiments, the target polynucleotide sequence is CXCR4 (Gene ID: 7852, also known as FB22; HM89; LAP3; LCR1; NPYR; WHIM; CD184; LESTR; NPY3R; NPYRL; HSY3RR; NPYY3R; and D2S201E). In some embodiments, the target polynucleotide sequence is a variant of CXCR4. In some embodiments, the target polynucleotide sequence is a homolog of CXCR4. In some embodiments, the target polynucleotide sequence is an ortholog of CXCR4. It should be appreciated that the CRISPR/Cas systems of the present invention can cleave target polynucleotide sequences in a variety of ways. In some embodiments, the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, the target polynucleotide sequence is cleaved such that a single-strand break results.

[0174] The methods of the present invention can be used to alter any target polynucleotide sequence in a cell, as long as the target polynucleotide sequence in the cell contains a suitable target motif that allows at least one ribonucleic acid of the CRISPR/Cas system to direct the Cas protein to and hybridize to the target motif. Those skilled in the art will appreciate that the target motif for targeting a particular polynucleotide depends on the CRISPR/Cas system being used, and the sequence of the polynucleotide to be targeted.

[0175] In some embodiments, the target motif is at least 20 bp in length. In some embodiments, the target motif is a 20-nucleotide DNA sequence. In some embodiments, the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is G(N).sub.19NGG. In some embodiments, the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, the target motif is (N).sub.20NGG.

[0176] The target motifs of the present invention can be selected to minimize off-target effects of the CRISPR/Cas systems of the present invention. In some embodiments, the target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the target motif is selected such that it contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. Those skilled in the art will appreciate that a variety of techniques can be used to select suitable target motifs for minimizing off-target effects (e.g., bioinformatics analyses).

[0177] In some embodiments, the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the target motif comprises a DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the target motif comprises a DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the target motif comprises a DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the target motif comprises a DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the target motif comprises a DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the target motif comprises a DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the CRISPR/Cas systems of the present invention utilize homology-directed repair to correct target polynucleotide sequences. In some embodiments, subsequent to cleavage of the target polynucleotide sequence, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. The exogenously introduced DNA repair template can be single-stranded or double-stranded. The DNA repair template can be of any length. Those skilled in the art will appreciate that the length of any particular DNA repair template will depend on the target polynucleotide sequence that is to be corrected. The DNA repair template can be designed to repair or replace any target polynucleotide sequence, particularly target polynucleotide sequences comprising disease associated polymorphisms (e.g., SNPs). For example, homology-directed repair of a mutant allele comprising such SNPs can be achieved with a CRISPR/Cas system by selecting two target motifs which flank the mutant allele, and an designing a DNA repair template to match the wild-type allele.

[0178] In some embodiments, a CRISPR/Cas system of the present invention includes a Cas protein and at least one to two one ribonucleic acids that are capable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence.

[0179] As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids) and include modified amino acids (e.g., phosphorylated, glycated, glycosolated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants, and analogs of the above.

[0180] In some embodiments, a Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprises a conservative amino acid substitution. In some instances, substitutions and/or modifications can prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in a cell. In some embodiments, the Cas protein can comprise a peptide bond replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.). In some embodiments, the Cas protein can comprise a naturally occurring amino acid. In some embodiments, the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0181] In some embodiments, a Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to Cas1, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8 and Cas9. In some embodiments, a Cas protein comprises a Cas protein of an E. coli subtype (also known as CASS2). Exemplary Cas proteins of the E. Coli subtype include, but are not limited to Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to Csy1, Csy2, Csy3, and Csy4. In some embodiments, a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to Csn1 and Csn2. In some embodiments, a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments, a Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to Csh1, Csh2, and Cas5h. In some embodiments, a Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, a Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0182] In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is Cas9 protein from any bacterial species or functional portion thereof. Cas9 protein is a member of the type II CRISPR systems which typically include a trans-coded small RNA (tracrRNA), endogenous ribonuclease 3 (rue) and a Cas protein. Cas 9 protein (also known as CRISPR-associated endonuclease Cas9/Csn1) is a polypeptide comprising 1368 amino acids. An exemplary amino acid sequence of a Cas9 protein (SEQ ID NO: 298) is shown in FIG. 3. Cas 9 contains 2 enconuclease domains, including an RuvC-like domain (residues 7-22, 759-766 and 982-989) which cleaves target DNA that is noncomplementary to crRNA, and an HNH nuclease domain (residues 810-872) which cleave target DNA complementary to crRNA. In FIG. 3, the RuvC-like domain is highlighted in yellow and the HNH nuclease domain is underlined.

[0183] As used herein, "functional portion" refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex.

[0184] In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain.

[0185] It should be appreciated that the present invention contemplates various of ways of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas9). In some embodiments, exogenous Cas protein can be introduced into the cell in polypeptide form. In certain embodiments, Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refers to a polypeptide or peptide, respectively, which facilitates the uptake of molecule into a cell. The cell-penetrating polypeptides can contain a detectable label.

[0186] In certain embodiments, Cas proteins can be conjugated to or fused to a charged protein (e.g., that carries a positive, negative or overall neutral electric charge). Such linkage may be covalent. In some embodiments, the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8):747-52).

[0187] In certain embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat, oligoarginine, and penetratin.

[0188] In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositively charged GFP.

[0189] In some embodiments, the Cas protein can be introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (e.g., Cas9). The process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA, as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA, as described herein (e.g., a synthetic, modified mRNA).

[0190] In some embodiments, the Cas protein is complexed with the one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).

[0191] The methods of the present invention contemplate the use of any ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises CRISPR RNA (crRNA). In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. The ribonucleic acids of the present invention can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. The one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.

[0192] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1.

[0193] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2.

[0194] In some embodiments, each of the one to two ribonucleic acids comprises guide RNAs that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences selected from the group consisting of SEQ ID NOs: 1-1.39. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 1-139.

[0195] In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 140-297.

[0196] In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences selected from the group consisting of SEQ ID NOs: 298-303. In some embodiments, the two guide ribonucleic acid sequences comprise a pair of guide ribonucleic acids selected from the group consisting of SEQ ID NOs: 299 and 303, SEQ ID NOs: 298 and 300, SEQ ID NOs: 299 and 300, SEQ ID NOs: 298 and 303, SEQ ID NOs: 299 and 301, SEQ ID NOs: 298 and 299, SEQ ID NOs: 301 and 303, SEQ ID NOs: 298 and 302, and SEQ ID NOs: 298 and 301. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which are complementary to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, the two guide RNA sequences comprise any combination of two guide ribonucleic acid sequences comprising RNA sequences which hybridize to two different offset sequences comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333.

[0197] In some embodiments, the two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, the two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on the opposite strands of a target polynucleotide sequence. In some embodiments, the two ribonucleic acids (e.g., guide RNAs) are not complementary to and/or do not hybrize to sequences on the opposite strands of a target polynucleotide sequence. In some embodiments, the two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, the two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to offset target motifs of a target polynucleotide sequence.

[0198] The present invention also contemplates multiplex genomic editing. Those skilled in the art will appreciate that the description above with respect to genomic editing of a single gene is equally applicable to the multiplex genomic editing embodiments described below.

[0199] In another aspect, the present invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell.

[0200] An exemplary method for simultaneously altering multiple target polynucleotide sequences in a cell comprises contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

[0201] In yet another aspect, the present invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject.

[0202] An exemplary method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject comprises (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0203] As used herein, the terms "administering," "introducing" and "transplanting" are used interchangeably in the context of the placement of cells, e.g. cells described herein comprising a target polynucleotide sequence altered according to the methods of the invention into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site. The cells can be implanted directly to the desired site, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g. twenty-four hours, to a few days, to as long as several years. In some instances, the cells can also be administered a location other than the desired site, such as in the liver or subcutaneously, for example, in a capsule to maintain the implanted cells at the implant location and avoid migration of the implanted cells.

[0204] For ex vivo methods, cells can include autologous cells, i.e., a cell or cells taken from a subject who is in need of altering a target polynucleotide sequence in the cell or cells (i.e., the donor and recipient are the same individual). Autologous cells have the advantage of avoiding any immunologically-based rejection of the cells. Alternatively, the cells can be heterologous, e.g., taken from a donor. The second subject can be of the same or different species. Typically, when the cells come from a donor, they will be from a donor who is sufficiently immunologically compatible with the recipient, i.e., will not be subject to transplant rejection, to lessen or remove the need for immunosuppression. In some embodiments, the cells are taken from a xenogeneic source, i.e., a non-human mammal that has been genetically engineered to be sufficiently immunologically compatible with the recipient, or the recipient's species. Methods for determining immunological compatibility are known in the art, and include tissue typing to assess donor-recipient compatibility for HLA and ABO determinants. See, e.g., Transplantation Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

[0205] Any suitable cell culture media can be used for ex vivo methods of the invention.

[0206] The terms "subject" and "individual" are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human subject, the term subject refers to that specific animal. The "non-human animals" and "non-human mammals" as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g. dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like.

[0207] In some embodiments, the alteration results in reduced expression of the target polynucleotide sequences. In some embodiments, the alteration results in a knock out of the target polynucleotide sequences. In some embodiments, the alteration results in correction of the target polynucleotide sequences from undesired sequences to desired sequences. In some embodiments, each alteration is a homozygous alteration. In some embodiments, the efficiency of alteration at each loci is from about 5% to about 80%. In some embodiments, the efficiency of alteration at each loci is from about 10% to about 80%. In some embodiments, the efficiency of alteration at each loci is from about 30% to about 80%. In some embodiments, the efficiency of alteration at each loci is from about 50% to about 80%. In some embodiments, the efficiency of alteration at each loci is from greater than or equal to about 80%.

[0208] In some embodiments, each target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, each target polynucleotide sequence is cleaved such that a single-strand break results.

[0209] In some embodiments, the target polynucleotide sequences comprise multiple different portions of B2M. In some embodiments, the target polynucleotide sequences comprise multiple different portions of CCR5. In some embodiments, the target polynucleotide sequences comprise multiple different portions of CXCR4. In some embodiments, the target polynucleotide sequences comprise at least a portion of CCR5 and at least a portion of CXCR4.

[0210] In some embodiments, each target motif is a 20-nucleotide DNA sequence. In some embodiments, each target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. In some embodiments, each target motif is G(N)19NGG. In some embodiments, each target motif is (N)20NGG. In some embodiments, each target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each target motif is selected such that it contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell.

[0211] In some embodiments, each target motif comprises a different DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, each target motif comprises a different DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, each target motif comprises a DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 1-139. In some embodiments, each target motif comprises a different DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, each target motif comprises a different DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, each target motif comprises a different DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 140-297. In some embodiments, each target motif comprises a different DNA sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, each target motif comprises a different DNA sequence comprising at least one nucleotide mismatch compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, each target motif comprises a different DNA sequence comprising at least two nucleotide mismatches compared to a DNA sequence selected from the group consisting of SEQ ID NOs: 304-333.

[0212] In some embodiments, subsequent to cleavage of the target polynucleotide sequences, homology-directed repair occurs. In some embodiments, homology-directed repair is performed using an exogenously introduced DNA repair template. In some embodiments, exogenously introduced DNA repair template is single-stranded. In some embodiments, exogenously introduced DNA repair template is double-stranded.

[0213] In some embodiments, the Cas protein (e.g., Cas9) is complexed with the multiple ribonucleic acids. In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence (e.g., multiple alterations of a single target polynucleotide sequence). In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequences (e.g., one or more alterations of multiple target polynucleotide sequences). In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids hybridize to target motifs that contain at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein. In some embodiments, each of the multiple ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank mutant alleles located between the target motifs.

[0214] In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1 and the ribonucleic acid sequences of FIG. 2. In some embodiments, each of the multiple ribonucleic acids comprises a sequence with a single nucleotide mismatch to a different sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1 and the ribonucleic acid sequences of FIG. 2.

[0215] In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2). In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2).

[0216] In some embodiments, each of the multiple ribonucleic acids comprises a different sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303. In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some embodiments, each of the multiple ribonucleic acids comprises a different ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 304-333.

[0217] It should be appreciated that any of the Cas protein or the ribonucleic acids can be expressed from a plasmid. In some embodiments, any of the Cas protein or the ribonucleic acids are expressed using a promoter optimized for increased expression in stem cells (e.g., human stem cells). In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter.

[0218] In some embodiments, the methods of the present invention further comprise selecting cells that express the Cas protein. The present invention contemplates any suitable method for selecting cells. In some embodiments, selecting cells comprises FACS. In some embodiments, FACs is used to select cells which co-express Cas and a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein.

[0219] The present invention contemplates treating and/or preventing a variety of disorders which are associated with expression of a target polynucleotide sequences. It should be appreciated that the methods and compositions described herein can be used to treat or prevent disorders associated with increased expression of a target polynucleotide sequence, as well as decreased expression of a target polynucleotide sequence in a cell. Increased and decreased expression of a target polynucleotide sequence includes circumstances where the expression levels of the target polynucleotide sequence are increased or decreased, respectively, as well as circumstances in which the function and/or level of activity of an expression product of the target polynucleotide sequence increases or decreases, respectively, compared to normal expression and/or activity levels. Those skilled in the art will appreciate that treating or preventing a disorder associated with increased expression of a target polynucleotide sequence can be assessed by determining whether the levels and/or activity of the target polynucleotide sequence (or an expression product thereof) are decreased in a relevant cell after contacting a cell with a composition described herein. The skilled artisan will also appreciate that treating or preventing a disorder associated with decreased expression of a target polynucleotide sequence can be assessed by determining whether the levels and/or activity of the target polynucleotide sequence (or an expression product thereof) are increased in the relevant cell after contacting a cell with a composition described herein.

[0220] In some embodiments, the disorder is a genetic disorder. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the disorder is a multigenic disorder. In some embodiments, the disorder is a disorder associated with one or more SNPs. Exemplary disorders associated with one or more SNPs include a complex disease described in U.S. Pat. No. 7,627,436, Alzheimer's disease as described in PCT International Application Publication No. WO/2009/112882, inflammatory diseases as described in U.S. Patent Application Publication No. 2011/0039918, polycystic ovary syndrome as described in U.S. Patent Application Publication No. 2012/0309642, cardiovascular disease as described in U.S. Pat. No. 7,732,139, Huntington's disease as described in U.S. Patent Application Publication No. 2012/0136039, thromboembolic disease as described in European Patent Application Publication No. EP2535424, neurovascular diseases as described in PCT International Application Publication No. WO/2012/001613, psychosis as described in U.S. Patent Application Publication No. 2010/0292211, multiple sclerosis as described in U.S. Patent Application Publication No. 2011/0319288, schizophrenia, schizoaffective disorder, and bipolar disorder as described in PCT International Application Publication No. WO/2006/023719A2, bipolar disorder and other ailments as described in U.S. Patent Application Publication No. U.S. 2011/0104674, colorectal cancer as described in PCT International Application Publication No. WO/2006/104370A1, a disorder associated with a SNP adjacent to the AKT1 gene locus as described in U.S. Patent Application Publication No. U.S. 2006/0204969, an eating disorder as described in PCT International Application Publication No. WO/2003/012143A1, autoimmune disease as described in U.S. Patent Application Publication No. U.S. 2007/0269827, fibrostenosing disease in patients with Crohn's disease as described in U.S. Pat. No. 7,790,370, and Parkinson's disease as described in U.S. Pat. No. 8,187,811, each of which is incorporated herein by reference in its entirety. Other disorders associated with one or more SNPs which can be treated or prevented according to the methods of the present invention will be apparent to the skilled artisan.

[0221] In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immunodeficiency syndrome (AIDS).

[0222] The methods of the present invention are capable of altering target polynucleotide sequences in a variety of different cells. In some embodiments, the methods of the present invention are used to alter target polynucleotide sequences in cells ex vivo for subsequent introduction into a subject. In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a stem cell or a pluripotent cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a CD34+ mobilized peripheral blood cell. In some embodiments, the cell is a CD34+ cord blood cell. In some embodiments, the cell is a CD34+ bone marrow cell. In some embodiments, the cell is a CD34+CD38-Lineage-CD90+CD45RA- cell. In some embodiments, the cell is a CD4+ cell. In some embodiments, the cell is a CD4+ T cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a human pluripotent cell. In some embodiments, the cell is a primary human cell. In some embodiments, the cell is a primary CD34+ cell. In some embodiments, the cell is a primary CD34+ hematopoietic progenitor cell (HPC). In some embodiments, the cell is a primary CD4+ cell. In some embodiments, the cell is a primary CD4+ T cell. In some embodiments, the cell is an autologous primary cell. In some embodiments, the cell is an autologous primary somatic cell. In some embodiments, the cell is an allogeneic primary cell. In some embodiments, the cell is an allogeneic primary somatic cell. In some embodiments, the cell is a nucleated cell. In some embodiments, the cell is a non-transformed cell. In some embodiments, the cell is not a cancer cell. In some embodiments, the cell is not a tumor cell. In some embodiments, the cell is not a transformed cell.

[0223] In some aspects, the present invention provides a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0224] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

[0225] In some aspects, the present invention provides a method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%.

[0226] In some aspects, the present invention provides a method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences in a cell selected from the group consisting of a human pluripotent cell, a primary human cell, and a non-transformed human cell, with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 8% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

[0227] The present invention also provides compositions comprising Cas proteins of the present invention or functional portions thereof, nucleic acids encoding the Cas proteins or functional portions thereof, and ribonucleic acid sequences which direct Cas proteins to and hybridize to target motifs of target polynucleotides in a cell.

[0228] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1).

[0229] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1).

[0230] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2).

[0231] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2).

[0232] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2.

[0233] In some embodiments, at least one of the ribonucleic acids in the composition is a modified ribonucleic acid as described herein (e.g., a synthetic, modified ribonucleic acid, e.g., comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate, or any other modified nucleotides or modifications described herein).

[0234] In some embodiments, a composition of the present invention comprises a nucleic acid sequence encoding a Cas protein. In some embodiments, a composition of the present invention comprises nucleic acid sequence encoding Cas9 protein or a functional portion thereof.

[0235] In some embodiments, the nucleic acid encoding the Cas protein (e.g., Cas9) comprises a modified ribonucleic acid as described herein (e.g., a synthetic, modified mRNA described herein, e.g., comprising at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate or any other modified nucleotides or modifications described herein).

[0236] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid each having a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid each having a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acids each having a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acids each having a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 2).

[0237] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid sequences each of which are complementary to and/or hybridize to different sequences with single nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid sequences each of which are complementary to and/or hybridize to offset sequences with single nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 1-139 (FIG. 1).

[0238] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence comprising a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 2. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 1). In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two additional ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 140-297 (FIG. 1).

[0239] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303.

[0240] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least two additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 298-303.

[0241] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 299 and 303. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 300. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 299 and 300. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 303. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 299 and 301. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 299. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 301 and 303. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 302. In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and two guide ribonucleic acids comprising SEQ ID NOs: 298 and 301.

[0242] In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least two ribonucleic acids each having a sequence which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least two ribonucleic acids each having a sequence which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least one ribonucleic acid having a sequence which is complementary to and/or hybridizes to a sequence comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least two ribonucleic acids each having a sequence which is complementary to and/or hybridizes to a different sequence comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333. In some aspects, the present invention provides a composition comprising at least two ribonucleic acids each having a sequence which is complementary to and/or hybridizes to an offset sequence comprising at least one nucleotide mismatch compared to a sequence selected from the group consisting of SEQ ID NOs: 304-333.

[0243] In some aspects, the present invention provides a composition comprising a chimeric nucleic acid comprising a ribonucleic acid encoding a Cas protein and at least one additional ribonucleic acid having a sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2.

[0244] In some embodiments, a composition of the present invention comprises a nucleic acid sequence encoding a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein. In some embodiments, a composition of the present invention comprises a promoter operably linked to the chimeric nucleic acid. In some embodiments, the promoter is optimized for increased expression in human stem cells. In some embodiments, the promoter is optimized for increased expression in primary human cells. In some embodiments, the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter.

[0245] In some embodiments, the Cas protein comprises a Cas9 protein or a functional portion thereof.

[0246] The present invention also provides kits for practicing any of the methods of the present invention, as well as kits comprising the compositions of the present invention, and instructions for using the kits for altering target polynucleotide sequences in a cell.

[0247] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence selected from the group consisting of the ribonucleic acid sequences of FIG. 1, the ribonucleic acid sequences of FIG. 2, a sequence with a single nucleotide mismatch to a ribonucleic acid sequence of FIG. 1, and a sequence with a single nucleotide mismatch to a ribonucleic acid sequences of FIG. 2.

[0248] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 1-139 (FIG. 1).

[0249] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0250] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0251] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 1-139 (FIG. 1).

[0252] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0253] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0254] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0255] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 1-139 (FIG. 1).

[0256] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 140-297 (FIG. 2).

[0257] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0258] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least one ribonucleic acid sequence which is complementary to and/or hybridizes to a sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0259] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 140-297 (FIG. 2).

[0260] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0261] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0262] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0263] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 140-297 (FIG. 2).

[0264] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences selected from the group consisting of the ribonucleic acid sequences of SEQ ID NO: 298-303. In some embodiments, the at least two ribonucleic acid sequences of SEQ ID NO: 298-303 are complementary to and/or hybridize to offset target sequences.

[0265] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence selected from the group consisting of SEQ ID NO: 304-333.

[0266] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence selected from the group consisting of SEQ ID NO: 304-333.

[0267] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to a different sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 304-333.

[0268] In some aspects, the present invention comprises a kit for altering a target polynucleotide sequence in a cell comprising a Cas9 protein or a nucleic acid encoding the Cas9 protein, and at least two ribonucleic acid sequences each of which is complementary to and/or hybridizes to an offset sequence comprising at least one nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NO: 304-333.

[0269] In some embodiments, the kit comprises one or more cell lines, cultures, or populations selected from the group consisting of human pluripotent cells, primary human cells, and non-transformed cells. In some embodiments, the kit comprises a DNA repair template.

[0270] In some aspects, the invention provides a method of administering cells to a subject in need of such cells, the method comprising: (a) contacting a cell or population of cells ex vivo with a Cas protein and two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence is cleaved; and (b) administering the resulting cells from (a) to a subject in need of such cells.

[0271] In some aspects, the invention provides a method of administering cells to a subject in need of such cells, the method comprising: (a) contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, and (iii) at least two additional ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence in the cell or population of cells, wherein the target polynucleotide sequences are cleaved; and (b) administering the resulting cell or cells from (a) to a subject in need of such cells.

[0272] B2M is an accessory chain of the MHC class I proteins which is necessary for the expression of MHC class I proteins on the surface of cells. It is believed that engineering cells (e.g., mutant cells) devoid of surface MHC class I may reduce the likelihood that the engineered cells will be detected by cytotoxic T cells when the engineered cells are administered to a host. Accordingly, in some embodiments, cleavage of the target polynucleotide sequence encoding B2M in the cell or population of cells reduces the likelihood that the resulting cell or cells will trigger a host immune response when the cells are administered to the subject.

[0273] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with a Cas protein and two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M is cleaved, thereby reducing the likelihood that cells administered to the subject will trigger a host immune response in the subject; and (b) administering the resulting cells from (a) to a subject in need of such cells.

[0274] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M in the cell or population of cells is cleaved, thereby reducing the likelihood that the cell or population of cells will trigger a host immune response in the subject, and (iii) at least two additional ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence in the cell or population of cells, wherein the target polynucleotide sequence is cleaved; and (b) administering the resulting cell or cells from (a) to a subject in need of such cells.

[0275] It is contemplated that the methods of administering cells can be adapted for any purpose in which administering such cells is desirable. In some embodiments, the subject in need of administration of cells is suffering from a disorder. For example, the subject may be suffering from a disorder in which the particular cells are decreased in function or number, and it may be desirable to administer functional cells obtained from a healthy or normal individual in which the particular cells are functioning properly and to administer an adequate number of those healthy cells to the individual to restore the function provided by those cells (e.g., hormone producing cells which have decreased in cell number or function, immune cells which have decreased in cell number or function, etc.). In such instances, the healthy cells can be engineered to decrease the likelihood of host rejection of the healthy cells. In some embodiments, the disorder comprises a genetic disorder. In some embodiments, the disorder comprises an infection. In some embodiments, the disorder comprises HIV or AIDs. In some embodiments, the disorder comprises cancer.

[0276] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with a Cas protein and two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M is cleaved, thereby reducing the likelihood that cells administered to the subject will trigger a host immune response in the subject; and (b) administering the resulting cells from (a) to a subject in need of such cells.

[0277] In some aspects, the invention provides a method of reducing the likelihood that cells administered to a subject will trigger a host immune response in the subject, the method comprising: (a) contacting a cell or population of cells ex vivo with (i) a Cas protein, (ii) at least two ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence encoding B2M in the cell or population of cells, wherein the target polynucleotide sequence encoding B2M in the cell or population of cells is cleaved, thereby reducing the likelihood that the cell or population of cells will trigger a host immune response in the subject, and (iii) at least two additional ribonucleic acids which direct Cas protein to and hybridize to a target polynucleotide sequence in the cell or population of cells, wherein the target polynucleotide sequence is cleaved; and (b) administering the resulting cell or cells from (a) to a subject in need of such cells. As used herein "nucleic acid," in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides linked via a phosphodiester bond. Exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. They may also include RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc. in some embodiments, the nucleic acid encoding the Cas protein is an mRNA. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic, modified mRNA described herein).

[0278] The present invention contemplates the use of any nucleic acid modification available to the skilled artisan. The nucleic acids of the present invention can include any number of modifications. In some embodiments, the nucleic acid comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, I-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-meth'-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0279] Preparation of modified nucleosides and nucleotides used in the manufacture or synthesis of modified RNAs of the present invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.

[0280] The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0281] Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al. Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which are incorporated by reference in their entirety.

[0282] Modified nucleic acids (e.g., ribonucleic acids) need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

[0283] In some embodiments, at least one of the one to two ribonucleic acids is a modified ribonucleic acid. In some embodiments, each of the one to two ribonucleic acids is a modified ribonucleic acid. In some embodiments, at least one of the multiple ribonucleic acids is a modified ribonucleic acid. In some embodiments, a plurality of the multiple ribonucleic acids are modified. In some embodiments, each of the multiple ribonucleic acids are modified. Those skilled in the art will appreciate that the modified ribonucleic acids can include one or more of the nucleic acid modification described herein.

[0284] In some aspects, provided herein are synthetic, modified RNA molecules encoding polypeptides, where the synthetic, modified RNA molecules comprise one or more modifications, such that introducing the synthetic, modified RNA molecules to a cell results in a reduced innate immune response relative to a cell contacted with synthetic RNA molecules encoding the polypeptides not comprising the one or more modifications. In some embodiments, the Cas protein comprises a synthetic, modified RNA molecule encoding a Cas protein. In some embodiments, the Cas protein comprises a synthetic, modified RNA molecule encoding a Cas9 protein.

[0285] The synthetic, modified RNAs described herein include modifications to prevent rapid degradation by endo- and exo-nucleases and to avoid or reduce the cell's innate immune or interferon response to the RNA. Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. To the extent that such modifications interfere with translation (i.e., results in a reduction of 50% or more in translation relative to the lack of the modification--e.g., in a rabbit reticulocyte in vitro translation assay), the modification is not suitable for the methods and compositions described herein. Specific examples of synthetic, modified RNA compositions useful with the methods described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages. Synthetic, modified RNAs having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage. In other embodiments, the synthetic, modified RNA has a phosphorus atom in its internucleoside linkage(s).

[0286] Non-limiting examples of modified internucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

[0287] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is herein incorporated by reference in its entirety.

[0288] Modified internucleoside linkages that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0289] Representative U.S. patents that teach the preparation of modified oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety.

[0290] Some embodiments of the synthetic, modified RNAs described herein include nucleic acids with phosphorothioate internucleoside linkages and oligonucleosides with heteroatom internucleoside linkage, and in particular--CH2-NH--CH2-, --CH2-N(CH3)-O--CH2-[known as a methylene (methylimino) or MMI], --CH2-O--N(CH3)-CH2-, --CH2-N(CH3)-N(CH3)-CH2- and --N(CH3)-CH2-CH2-[wherein the native phosphodiester internucleoside linkage is represented as --O--P--O--CH2-] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240, both of which are herein incorporated by reference in their entirety. In some embodiments, the nucleic acid sequences featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506, herein incorporated by reference in its entirety.

[0291] Synthetic, modified RNAs described herein can also contain one or more substituted sugar moieties. The nucleic acids featured herein can include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O, S, or N-alkyl; O--, S--, or N-alkenyl; O--, 5- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments, synthetic, modified RNAs include one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of a synthetic, modified RNA, and other substituents having similar properties. In some embodiments, the modification includes a 2' methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chico. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2-O--CH2-N(CH2)2.

[0292] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleic acid sequence, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked nucleotides and the 5' position of 5' terminal nucleotide. A synthetic, modified RNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0293] As non-limiting examples, synthetic, modified RNAs described herein can include at least one modified nucleoside including a 2'-O-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof.

[0294] In some embodiments of this aspect and all other such aspects described herein, the at least one modified nucleoside is selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-O-methyluridine (Urn), 2' deoxyuridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G), and inosine (I).

[0295] Alternatively, a synthetic, modified RNA can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the nucleotide. At a minimum, a synthetic, modified RNA molecule comprising at least one modified nucleoside comprises a single nucleoside with a modification as described herein. It is not necessary for all positions in a given synthetic, modified RNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single synthetic, modified RNA or even at a single nucleoside within a synthetic, modified RNA. However, it is preferred, but not absolutely necessary, that each occurrence of a given nucleoside in a molecule is modified (e.g., each cytosine is a modified cytosine e.g., 5mC). However, it is also contemplated that different occurrences of the same nucleoside can be modified in a different way in a given synthetic, modified RNA molecule (e.g., some cytosines modified as 5mC, others modified as 2'-O-methylcytidine or other cytosine analog). The modifications need not be the same for each of a plurality of modified nucleosides in a synthetic, modified RNA. Furthermore, in some embodiments of the aspects described herein, a synthetic, modified RNA comprises at least two different modified nucleosides. In some such preferred embodiments of the aspects described herein, the at least two different modified nucleosides are 5-methylcytidine and pseudouridine. A synthetic, modified RNA can also contain a mixture of both modified and unmodified nucleosides.

[0296] As used herein, "unmodified" or "natural" nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). In some embodiments, a synthetic, modified RNA comprises at least one nucleoside ("base") modification or substitution. Modified nucleosides include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine, 2-(aminoalkyl)adenine, 2 (aminopropyl)adenine, 2 (methylthio) N6 (isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6 (methyl)adenine, N6,N6 (dimethyl)adenine, 2-(alkyl)guanine, 2 (propyl)guanine, 6-(alkyl)guanine, 6 (methyl)guanine, 7 (alkyl)guanine, 7 (methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine, 8-(alkenyl)guanine, 8 (alkynyl)guanine, 8-(amino)guanine, 8 (halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine, 8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5 (aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5 (methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine, 5 (trifluoromethyl)cytosine, 6-(azo)cytosine, N4 (acetyl)cytosine, 3 (3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2 (thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil, 4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5 (aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5 (1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5 (methoxycarbonylmethyl)-2-(thio)uracil, 5 (methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil, 5 (trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3 (methyl)uracil, 5-uracil (i.e., pseudouracil), 2 (thio)pseudouracil, 4 (thio)pseudouracil,2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil, 1 substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1 substituted 2,4-(dithio)pseudouracil, 1 (aminocarbonylethylenyl)-pseudouracil, 1 (aminocarbonylethylenyl)-2(thio)-pseudouracil, 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1) (aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole, 6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5 substituted pyrimidines, N2-substituted purines, N6-substituted purines, 06-substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Modified nucleosides also include natural bases that comprise conjugated moieties, e.g. a ligand. As discussed herein above, the RNA containing the modified nucleosides must be translatable in a host cell (i.e., does not prevent translation of the polypeptide encoded by the modified RNA). For example, transcripts containing s2U and m6A are translated poorly in rabbit reticulocyte lysates, while pseudouridine, m5U, and m5C are compatible with efficient translation. In addition, it is known in the art that 2'-fluoro-modified bases useful for increasing nuclease resistance of a transcript, leads to very inefficient translation. Translation can be assayed by one of ordinary skill in the art using e.g., a rabbit reticulocyte lysate translation assay.

[0297] Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Int. Appl. No. PCT/U.S. Ser. No. 09/038,425, filed Mar. 26, 2009; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0298] Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference in its entirety, and U.S. Pat. No. 5,750,692, also herein incorporated by reference in its entirety.

[0299] Another modification for use with the synthetic, modified RNAs described herein involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the RNA. The synthetic, modified RNAs described herein can further comprise a 5' cap. In some embodiments of the aspects described herein, the synthetic, modified RNAs comprise a 5' cap comprising a modified guanine nucleotide that is linked to the 5' end of an RNA molecule using a 5'-5' triphosphate linkage. As used herein, the term "5' cap" is also intended to encompass other 5' cap analogs including, e.g., 5' diguanosine cap, tetraphosphate cap analogs having a methylene-bis(phosphonate) moiety (see e.g., Rydzik, A M et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA 14(6):1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g., Grudzien-Nogalska, E. et al., (2007) RNA 13(10): 1745-1755), N7-benzylated dinucleoside tetraphosphate analogs (see e.g., Grudzien, E. et al., (2004) RNA 10(9):1479-1487), or anti-reverse cap analogs (see e.g., Jemielity, J. et al., (2003) RNA 9(9): 1108-1.122 and Stepinski, J. et al., (2001) RNA 7(10):1486-1495), In one such embodiment, the 5' cap analog is a 5' diguanosine cap. In some embodiments, the synthetic, modified RNA does not comprise a 5' triphosphate.

[0300] The 5' cap is important for recognition and attachment of an mRNA to a ribosome to initiate translation. The 5' cap also protects the synthetic, modified RNA from 5' exonuclease mediated degradation. It is not an absolute requirement that a synthetic, modified RNA comprise a 5' cap, and thus in other embodiments the synthetic, modified RNAs lack a 5' cap. However, due to the longer half-life of synthetic, modified RNAs comprising a 5' cap and the increased efficiency of translation, synthetic, modified RNAs comprising a 5' cap are preferred herein.

[0301] The synthetic, modified RNAs described herein can further comprise a 5' and/or 3' untranslated region (UTR). Untranslated regions are regions of the RNA before the start codon (5') and after the stop codon (3'), and are therefore not translated by the translation machinery. Modification of an RNA molecule with one or more untranslated regions can improve the stability of an mRNA, since the untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation. In addition, modification of an RNA with a 5' and/or 3' untranslated region can enhance translational efficiency by binding proteins that alter ribosome binding to an mRNA. Modification of an RNA with a 3' UTR can be used to maintain a cytoplasmic localization of the RNA, permitting translation to occur in the cytoplasm of the cell. In one embodiment, the synthetic, modified RNAs described herein do not comprise a 5' or 3' UTR. In another embodiment, the synthetic, modified RNAs comprise either a 5' or 3' UTR, In another embodiment, the synthetic, modified RNAs described herein comprise both a 5' and a 3' UTR. In one embodiment, the 5' and/or 3' UTR is selected from an mRNA known to have high stability in the cell (e.g., a murine alpha-globin 3' UTR). In some embodiments, the 5' UTR, the 3' UTR, or both comprise one or more modified nucleosides.

[0302] In some embodiments, the synthetic, modified RNAs described herein further comprise a Kozak sequence. The "Kozak sequence" refers to a sequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another `G`. The Kozak consensus sequence is recognized by the ribosome to initiate translation of a polypeptide. Typically, initiation occurs at the first AUG codon encountered by the translation machinery that is proximal to the 5' end of the transcript. However, in some cases, this AUG codon can be bypassed in a process called leaky scanning. The presence of a Kozak sequence near the AUG codon will strengthen that codon as the initiating site of translation, such that translation of the correct polypeptide occurs. Furthermore, addition of a Kozak sequence to a synthetic, modified RNA will promote more efficient translation, even if there is no ambiguity regarding the start codon. Thus, in some embodiments, the synthetic, modified RNAs described herein further comprise a Kozak consensus sequence at the desired site for initiation of translation to produce the correct length polypeptide. In some such embodiments, the Kozak sequence comprises one or more modified nucleosides.

[0303] In some embodiments, the synthetic, modified RNAs described herein further comprise a "poly (A) tail", which refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides). The inclusion of a 3' poly(A) tail can protect the synthetic, modified RNA from degradation in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency. In some embodiments, the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, 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 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more. In one embodiment, the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides.

[0304] It is contemplated that one or more modifications to the synthetic, modified RNAs described herein permit greater stability of the synthetic, modified RNA in a cell. To the extent that such modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the synthetic, modified RNA with the modification, such modifications are specifically contemplated for use herein. Generally, the greater the stability of a synthetic, modified RNA, the more protein can be produced from that synthetic, modified RNA. Typically, the presence of AU-rich regions in mammalian mRNAs tend to destabilize transcripts, as cellular proteins are recruited to AU-rich regions to stimulate removal of the poly(A) tail of the transcript. Loss of a poly(A) tail of a synthetic, modified RNA can result in increased RNA degradation. Thus, in one embodiment, a synthetic, modified RNA as described herein does not comprise an AU-rich region. In particular, it is preferred that the 3' UTR substantially lacks AUUUA sequence elements.

[0305] In one embodiment, a ligand alters the cellular uptake, intracellular targeting or half-life of a synthetic, modified RNA into which it is incorporated. In some embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, intracellular compartment, e.g., mitochondria, cytoplasm, peroxisome, lysosome, as, e.g., compared to a composition absent such a ligand. Preferred ligands do not interfere with expression of a polypeptide from the synthetic, modified RNA.

[0306] The ligand can be a substance, e.g., a drug, which can increase the uptake of the synthetic, modified RNA or a composition thereof into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0307] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a host cell. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up, for example, by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[0308] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0309] A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an .alpha.-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0310] The synthetic, modified RNAs described herein can be synthesized and/or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference in its entirety. Transcription methods are described further herein in the Examples.

[0311] In one embodiment of the aspects described herein, a template for a synthetic, modified RNA is synthesized using "splint-mediated ligation," which allows for the rapid synthesis of DNA constructs by controlled concatenation of long oligos and/or dsDNA PCR products and without the need to introduce restriction sites at the joining regions. It can be used to add generic untranslated regions (UTRs) to the coding sequences of genes during T7 template generation. Splint mediated ligation can also be used to add nuclear localization sequences to an open reading frame, and to make dominant-negative constructs with point mutations starting from a wild-type open reading frame. Briefly, single-stranded and/or denatured dsDNA components are annealed to splint oligos which bring the desired ends into conjunction, the ends are ligated by a thermostable DNA ligase and the desired constructs amplified by PCR. A synthetic, modified RNA is then synthesized from the template using an RNA polymerase in vitro. After synthesis of a synthetic, modified RNA is complete, the DNA template is removed from the transcription reaction prior to use with the methods described herein.

[0312] In some embodiments of these aspects, the synthetic, modified RNAs are further treated with an alkaline phosphatase.

[0313] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0314] The articles "a" and "an" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clause, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0315] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0316] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately", the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by "about" or "approximately", the invention includes an embodiment in which the value is prefaced by "about" or "approximately".

[0317] As used herein "A and/or B", where A and B are different claim terms, generally means at least one of A, B, or both A and B. For example, one sequence which is complementary to and/or hybridizes to another sequence includes (i) one sequence which is complementary to the other sequence even though the one sequence may not necessarily hybridize to the other sequence under all conditions, (ii) one sequence which hybridizes to the other sequence even if the one sequence is not perfectly complementary to the other sequence, and (iii) sequences which are both complementary to and hybridize to the other sequence.

[0318] "Approximately" or "about" generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered "isolated".

[0319] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0320] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0321] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

EXAMPLES

Example 1

[0322] Transcription activator-like effector nucleases (TALENs) bind as a pair around a genomic site, in which a double-strand break (DSB) is introduced by a dimer of FokI nuclease domains. The use of a TALEN genome-editing system to rapidly and efficiently generate mutant alleles of 15 different genes in human pluripotent stem cells (hPSCs) as a means of performing rigorous disease modeling was recently reported (Ding et al., Cell Stem Cell 12:238-251 (2013)); the proportions of clones bearing at least one mutant allele ranged from 2%-34%.

[0323] As described below, the relative efficacies of CRISPRs and TALENs targeting the same genomic sites in the same hPSC lines was assessed with the use of the same delivery platform described previously (Ding et al., Cell Stem Cell 12:238-251 (2013)). In the TALEN genome-editing system, the CAG promoter was used to co-translate (via a viral 2A peptide) each TALEN with green fluorescent protein (GFP) or red fluorescent protein (RFP). For CRISPRs, a human codon-optimized Cas9 gene was subcloned with a C-terminal nuclear localization signal (Mali et al., Science 339:823-826 (2013)) into the same CAG expression plasmid with GFP, and the guide RNA (gRNA) was separately expressed from a plasmid with the human U6 polymerase III promoter (Mali et al., Science 339:823-826 (2013)). The 20-nucleotide protospacer sequence for each gRNA was introduced using polymerase chain reaction (PCR)-based methods. Whether using TALENs or CRISPRs, equal amounts of the two plasmids were co-electroporated into hPSCs (either 25 .mu.g of each plasmid, or 12.5 .mu.g of each plasmid along with 25 .mu.g of a DNA repair template if attempting knock-in) followed by fluorescence-activated cell sorting (FACS) after 24-48 hours, clonal expansion of single cells, and screening for mutations at the genomic target site via PCR.

[0324] gRNAs were designed matching G(N)19NGG sequences in seven loci in six genes (AKT2, CELSR2, CIITA, GLUT4, LINC00116, and SORT1) previously successfully targeted with TALENs (Ding et al., Cell Stem Cell 12:238-251 (2013)) and one additional locus in LDLR. In this system, CRISPRs consistently and substantially outperformed TALENs across loci and hPSC lines (see Table S1). The TALENs yielded clones with at least one mutant allele at efficiencies of 0%-34%, but matched CRISPRs yielded mutant clones at efficiencies of 51%-79% (Table S1). Just as with TALENs, CRISPRs produced a variety of indels of sizes ranging from one nucleotide to several dozen nucleotides in size, centered on the predicted cleavage sites, suggesting that non-homologous end-joining mutagenesis occurs in the same way regardless of whether CRISPRs or TALENs are used. Moreover, CRISPRs readily generated homozygous mutant clones (7%-25% of all clones; Table S1) as discerned by sequencing.

[0325] Knock-in of E17K mutations into AKT2 was also attempted using a 67-nucleotide single-stranded DNA oligonucleotide as previously described (Ding et al., Cell Stem Cell 12:238-251 (2013)). Although the predicted. CRISPR cleavage site lay 11 and 13 nucleotides from the point mutations, respectively, the CRISPR yielded knock-in clones at a rate of 11%, whereas TALENs yielded only 1.6% (Table S1).

TABLE-US-00001 TABLE S1 Targeting Efficiency of CRISPRs Versus TALENs in Human Pluripotent Stem Chromosome: TALENs CRISPRs Position Efficiency Efficiency Efficiency (Start of Mutants/ (Mustants/ of Target Target Cell Clones Clones Homozygous Gene Sequence) Sequence.sup.a Line.sup.b Screened).sup.c Screened).sup.c Mustants AKT2 chr19: TCCCTTCCTGCCTCATTTCAGGTGA HUES 9 8.9% 40762982 ATACATCAAGACCTGGAGGCCA (17/192) AKT2 chr19: TCCCTTCCTGCC|TCATTTCAGGTG HUES 9 60.6% 12.7% 40762982 AATACATCAAGACCTGGAGGCCA (86/142) (18/142) CELSR2 chr1: TGCTGGCTCGGCTGCCCTGAGGTTG HUES 1 3.5% 109817568 CTCAATCAAGCACAGGTTTCAA (18/506) CELSR2 chr1: TGCTGGCTCGGCTGCCCTGAGGTTG HUES 1 66.2% 7.4% 109817568 CTCAATCAAG|CACAGGTTTCAA (45/68) (5/68) CIITA chr16: TAACAGCGATGCTGACCCCCTGTGC BJ-RiPS 12.7% 10989200 CTCTACCACTTCTATGACCAGA (37/292) CIITA chr16: CGATGCTGACCCCCTGTGCCTCTAC BJ-RiPS 78.7% 11.5% 10989206 CACTT|CTATGACCAGATGGACC (96/122) (14/122) GLUT4 chr17: TGGTCCTTGCTGTGTTCTCTGCGGT HUES 9 33.5% 7186601 GCTTGGCTCCCTGCAGTTTGGGTA (52/155) GLUT4 chr17: TGGTCCTTGCTGTGTTCT|CTGCGG HUES 9 66.5% 24.9% 7186631 TGCTTGGCTCCCTGCAGTTTGGGTA (123/185) (46/185) LDLR chr19: TGGGCGACAGATGCGAAAGAAACGA HUES 9 0% 11210899 GTTCCAGTGCCAAGACGGGAAA (90/568) LDLR chr19: GAAACGAGTTCCAGTGCCAAGACGG HUES 9 51.1% 8.0% 11210917 GAAATGCATCTCCTAC|AAGTGG (90/176) (14/176) LINC00116 chr2: TCAGAGAGGACACTGCAGTTGTCCG HUES 9 29.5% 110970093 TGCTAGTAGCCTTCGCTTCTGGA (26/88) LINC00116 chr2: TCAGAGAGGACACTGCAGTTGTCCG HUES 9 57.4% 8.6% 110970090 TGCTAGTAGCCTTCGC|TTCTGGA (93/162) (14/162) SORT1 exon 2 chr1: TGATGATCTCAGAGGCTCAGTATCC HUES 1 22.2% 109912203 TTGTCCTGGGTTGGAGATAGCA (128/576) SORT1 exon 2 chr1: TGATGATCTCAGAGGCTCAGTATCC HUES 1 68.5% 13.0% 109912203 TTG|TCCTGGGTTGGAGATAGCA (100/146) (19/146) SORT1 exon 3 chr1: TGGTAATTATGACTTTTGGACAGTC HUES 9 10.9% 109910969 CAAGCTATATCGAAGGTGAGATCA (21/192) SORT1 exon 3 chr1: TGGTAATTATGACTTTTGGACAGTC HUES 9 75.9% 10.3% 109910069 CAAGCTATAT|CGAAGGTGAGATCA (148/195) (20/195) AKT2 E17K chr19: TCCCTTCCTGCCTCATTTCAGGT A HUES 9 1.6% 40762982 TACATCAAGACCTGGAGGCCA (3/192) AKT2 E17K chr19: TCCCTTCCTGCCTCATTTCAGGTGA HUES 9 10.6% 1.1% 40762982 ATACATCAAGACCTGGAGGCCA (10/94).sup.d (1/94).sup.d AKT2 off-target chr5: CTATGCCCTGCCTCATTTCAGGTGA HUES 9 0% 0% 22683972 AGATGAAATCCCTGGAGCTTGG (0/142) (0/142) .sup.aFor TALENs, the binding sites are indicated with underlines, with the cleavage site predicted to be midway between the binding sites; for CRISPRs, the protospacer is underlined, the NGG motif is in bold (may be on the antisense strand), and the predicted cleavage site is indicated with "|"; for the AKT2 E17K target sequence, the sites of the knock-in mutations are indicated in bold/italics: for the AKT2 off-target site, the two mismatches in the protospacer are indicated in bold/italics .sup.bHUES 1 and HUES 9 are human embryonic stem cell lines; BJ-RiPS is an induced piuripotent stem cell line .sup.cMutants include single heterozygotes, compound hoterozygotes, and homozygous mutants; TALEN data is from Table 1 of Ding et al. (2013), with the exception of LDLR .sup.dSuccessfully inserted E17K knock-in mutations into an AKT2 allele(s) using single-stranded DNA oligonucleotide (refer to FIG. 3 of Ding et al., 2013)

[0326] It is worth noting that the requirement for a G(N)19NGG target sequence somewhat limits site selection. Because either DNA strand can be targeted, a target sequence occurs on average every 32 basepairs. This is no barrier for gene knockout, where any coding sequence can be targeted, but it may present difficulties when trying to knock in or correct a mutation at a specific location. However, the requirement for a G at the start of the protospacer is dictated by the use of the U6 promoter to express the gRNA, and alternative CRISPR/Cas systems can relieve this requirement (Cong et al., Science 339:819-823 (2013)). This allows for the use of (N)20NGG target sequences, which are found on average every 8 basepairs.

[0327] In addition, the extent of CRISPR off-target effects remains to be defined and is highly sequence-dependent. Previous analyses have suggested that one-nucleotide mismatches in the first half of the protospacer are better tolerated than mismatches in second half (Jinek et al., Science 337:816-821 (2012); Cong et al., Science 339:819-823 (2013)). For the AKT2 sequence, there is a two-mismatch sequence differing at nucleotides 1 and 3, in the more "tolerant" half of the protospacer. Zero clones were obtained with mutations at this potential off-target site, as compared to 61% at the on-target site (Table 51). For one of the SORT1 sequences, use of a different human pluripotent stem cell line in which a single nucleotide polymorphism results in a one-nucleotide mismatch at the target site yielded mutant clones at an efficiency of 42%, compared to 66% in the original cell line. Thus, judicious selection of target sites is necessary to minimize systematic off-target effects; target sites with perfect-match or single-nucleotide-mismatch sequences elsewhere in the genome should be avoided.

[0328] From a practical standpoint, CRISPRs are easier to implement than TALENs. Each TALEN pair must be constructed de novo, whereas for CRISPRs the Cas9 component is fixed and the gRNA requires only swapping of the 20-nucleotide protospacer. Given this consideration and the demonstration herein of substantially increased efficiency as a result of replacing TALENs with CRISPRs in an otherwise identical system, CRISPRs appear to be a very powerful and broadly applicable tool for genome editing, particularly in a therapeutic context.

Example 2

Efficient Targeting of Clinically Relevant Genes in Primary Somatic Cells

[0329] Work described herein shows for the first time that the CRISPR/Cas9 system can be used to edit the genome of somatic cells (e.g., primary) with high efficiency by using a double guide strategy. The inventors posit that this work will help bring genome editing in clinically relevant primary cells into reality.

[0330] The advent of genome editing tools that allow one to target any desired genomic site has greatly advanced the investigation of human biology and disease. In particular, the CRISPR/Cas9 system has become the gold standard in targeted genome editing technology, due to its flexibility and high efficacy. This system is constituted by the Cas9 nuclease from the microbial type II CRISPR/Cas system, which is targeted to specific genomic loci by a 20-nucleotide region in a synthetic guide RNA molecule. Similar to other targeted nucleases (ZFNs and TALENs), Cas9 induces double strand breaks (DSBs) that are repaired mainly by error-prone non-homologous end joining (NHEJ) (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013).

[0331] Implementation of the CRISPR/Cas9 system has made it possible to achieve unprecedentedly high targeting efficiencies in immortalized cell lines (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013), human pluripotent stem cells (Ding et al., 2013) and even zygotes of mice (Wang et al., 2013), rats (Li et al., 2013) and, most recently, monkeys (Niu et al., 2014), leading to the generation of knock-out or knock-in animals in very short periods of time when compared to classical strategies.

[0332] However, it remains to be proven whether CRISPR/Cas9 technology can be used to edit the genome of clinically relevant primary somatic cells with high efficiency, an essential step for the full realization of the promise of genome editing for regenerative medicine and transplantation therapies.

[0333] The inventors sought to test the amenability of the CRISPR/Cas9 system to edit clinically relevant genes in primary somatic cells. For this purpose the inventors chose to target two therapy-related genes: CCR5, a co-receptor for HIV, in CD34+ hematopoletic progenitor cells (HPCs), and B2M, the accessory chain of MHC class I molecules, in CD4+ T cells. The inventors found that a single guide strategy yielded very low to undetectable mutational rates in HPCs and T cells, despite high efficiencies in immortalized cell lines such 293T and K562. In contrast, surprisingly and unexpectedly a double guide strategy with a pair of gRNAs with different offsets targeting the locus of interest resulted in up to 40% homozygous deletion efficiency in HPCs and T cells. These results establish a novel approach through which the CRISPR/Cas9 system can be used to edit the genome in clinically relevant somatic cells with high efficiency.

[0334] Results

[0335] Efficient and Rapid Genome Editing Using the CRISPR/Cas9 System in Cell Lines

[0336] The inventors transfected HEK293T cells with Cas9 and a series of CRISPR guide RNAs targeting the B2M locus and measured cutting efficiency based on SURVEYOR assays (FIG. 4), as well as flow cytometry, taking advantage of the fact that B2M is a surface antigen. These experiments were performed only 72 h post-transfection, in order to account for the half-life of B2M on the cell membrane. Of note, B2M surface expression was abrogated in up to 60% of transfected HEK293T cells (FIG. 4). In addition, the inventors observed a wide variation of efficiency between individual guide RNAs, even if targeting the same exon. For instance, variation between single guide cutting efficiencies was several-fold amongst the seven guide RNAs binding within the 67 bp long protein coding portion of the first exon of B2M (FIG. 1X), strongly suggesting that CRISPR cutting efficiency is primarily guide sequence-dependent.

[0337] Primary Somatic Cells are Refractory to CRISPR/Cas9 Targeting

[0338] Next, the inventors tested the CRISPR/Cas9 system in primary cells. Two clinically relevant immune cell types were chosen: primary CD34.sup.+ hematopoietic progenitor cells (HPCs) and primary CD4.sup.+ T cells isolated from peripheral blood. Surprisingly, the same guide RNAs that resulted in up to 60% cutting efficiency in a cell line (B2M in 293T cells, FIG. 4) revealed ineffective in somatic cells (FIG. 4). The inventors speculate that such dramatic drop in targeting efficiency in primary cells is due to either a lower expression level of Cas9 nuclease in nucleofected cells, enhanced DNA repair mechanisms, or a combination of both.

[0339] Double Guide Strategy Dramatically Increases Targeting Efficiency in Primary Cells

[0340] The inventors sought to determine whether genome editing efficacy in clinically relevant primary cells using the CRISPR/Cas9 system could be improved, hoping to achieve targeting efficiencies high enough to be potentially used in therapy. The inventors devised a double guide strategy, where two CRISPR guide RNAs targeting the same locus were delivered to cells simultaneously.

[0341] Addition of another guide RNA targeting the HPRT locus almost invariably resulted in increased mutation efficiency compared with the first guide RNA alone. Cells deficient in HPRT were selected by resistance to 6-thioguanine (6-TG). The use of additional gRNAs invariably resulted in increased HPRT mutant frequency. In an embodiment, the target polynucleotide sequence comprises a HPRT gene sequence.

[0342] Different guide RNA pairs were tested for each locus, and the most active one was used for further studies with primary cells. FIGS. 4A-4E demonstrate that the single guide strategy achieves high efficiency genome editing in cell lines, but not in clinically relevant primary somatic cells. In the two systems used, the double guide strategy consistently and substantially outperformed the traditional single guide strategy in primary somatic cells. These results are demonstrated in FIGS. 5A-5E, which show that the double guide strategy achieves genome editing with high efficiency in clinically relevant cells.

[0343] Discussion

[0344] One of the major focuses in the field of CRISPR/Cas9 genome editing field is the search for parameters that modulate cutting efficiency by Cas9. The data described herein suggest that this phenomenon appears to be mostly determined by gRNA sequence, as gRNAs matching very close or even partially overlapping sequences within the same exon result in significantly different targeting efficiencies (FIG. 4).

[0345] In a previous report, an approach combining a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks has been used to drastically reduce CRISPR off-target effects without sacrificing on-target efficiency (Ran et al., 2013). In our hands, however, this strategy did not yield a significant mutation rate (Max & Pankaj). We thus combined WT Cas9 with pairs of gRNAs to increase cutting efficiency in cell types refractory to targeting--primary somatic cells.

[0346] B2M is an accessory chain of the MHC class I proteins, being necessary for their expression on the cells surface. Engineering cells devoid of surface MHC class I, hence invisible to cytotoxic T cells, is of utmost importance in transplantation and adoptive cell therapy.

[0347] Altogether, data shows that the CRISPR/Cas9 system can be used to edit the genome of clinically relevant primary somatic cells with significant efficiencies by using a double guide strategy. This strategy has the potential to be a general approach to target genes in somatic cells with a high enough efficiency that it becomes relevant for potential translation into therapeutics.

[0348] Some Experimental Procedures

[0349] Flow Cytometry.

[0350] Cells were stained with mouse monoclonal anti-B2M antibody 2M2 (Biolegend).

[0351] Primary Blood Cell Electroporation.

[0352] Primary CD4+ T cells were isolated from leukopacs (MGH) using RosetteSep CD4 T cell enrichment cocktail (Stem Cell Technologies) and electroporated with endotoxin-free DNA using Amaxa T cell nucleofection kit (Lonza).

[0353] 6-TG selection for HPRT deficiency. 5*10 6 cells were used per electroporation, with 25 ugCas9 and 12.5 ug of each gRNA. For the Cas9 control a non-cutting gRNA was used to keep the total DNA amount the same. FACS sorting ended up being relatively similar at 5-8% GFP 48 hours after EP. Cells were plated out at 40,000 per 10 cm plate per sample, and grown until colonies could clearly be seen. 30 uM 6-Thioguanine (6-TG) in mTESR (e.g., at a concentration of 30 .mu.m) and was used as selection medium for 8-9 days and colonies were counted again. The results are shown in Table 1 below.

TABLE-US-00002 TABLE 1 Starting Percentage - gRNA colonies Final colonies Percentage Cas9 background Cas9 105 17 0.161904762 0.00 1 121 55 0.454545455 0.29 3 118 67 0.56779661 0.41 5 124 76 0.612903226 0.45 7 125 27 0.216 0.05 9 131 29 0.221374046 0.06 11 93 63 0.677419355 0.52 1 + 5 64 43 0.671875 0.51 1 + 3 77 45 0.584415584 0.42 1 + 7 55 19 0.345454545 0.18 1 + 9 60 26 0.433333333 0.27 1 + 11 52 32 0.615384615 0.45 3 + 5 69 46 0.666666667 0.50 3 + 7 55 33 0.6 0.44 3 + 11 38 30 0.789473684 0.63 7 + 11 72 41 0.569444444 0.41

[0354] Table 2 below shows the results from Table 1 above ranked according to editing efficiency.

TABLE-US-00003 TABLE 2 gRNA Percentage 3 + 11 0.63 11 0.52 1 + 5 0.51 3 + 5 0.50 1 + 11 0.45 5 0.45 3 + 7 0.44 1 + 3 0.42 7 + 11 0.41 3 0.41 1 0.29 1 + 9 0.27 1 + 7 0.18 9 0.06 7 0.05 Cas9 0.00

[0355] gRNAs used in the experiments are shown below:

TABLE-US-00004 1- (SEQ ID NO: 298) gtcttgctcgagatgtgatg 3- (SEQ ID NO: 299) taaattctttgctgacctgc 5- (SEQ ID NO: 300) tagatccattcctatgactg 7- (SEQ ID NO: 301) cttcagtctgataaaatcta 9- (SEQ ID NO: 302) tttgatgtaatccagcaggt 11- (SEQ ID NO: 303) cacagagggctacaatgtga

Example 3

Modified Cas9 mRNA Functions to Efficiently Introduce On-Target Mutations

[0356] The inventors generated Figment (Fgm) knockout mice by CRIPSR/Cas9 gene editing utilizing a modified Cas9 mRNA. Fgm is a coding gene within the long non-coding RNA Lnc-Rap-5 (referred to herein as Fgm (Lnc-Rap-5; see Sun et al., "Long noncoding RNAs regulate adipogenesis," PNAS; 2013; 110(9):3387-3392, incorporated herein by reference in its entirety). The guide RNA (gRNA) sequence employed in this example was: 5' gaggegaaagccactagcac 3' (SEQ ID NO: 599). The modified Cas9 mRNA used in this example was made using an in vitro transcription reaction in which pseudouridine and 5-methyl-cytosine are reacted with unmodified nucleotides and randomly integrated into the resulting modified Cas9 mRNA. An exemplary protocol for generating Fgm knockout mice using CRISPR/Cas9 gene editing utilizing a modified Cas9mRNA is shown in FIG. 11A. As shown in FIG. 11A, 100 ng/.mu.l of the resulting modified Cas9 mRNA and 50 ng/.mu.l of guide RNA targeting Fgm (Lnc-Rap-5) (SEQ ID NO: 599) were injected into 250 C57BL/6 mouse zygotes that were subsequently transferred to pseudo-pregnant mice and after weening screened for mutations by PCR. As shown in the gel pictured in FIG. 11B, PCR screening revealed 63 mutant animals out of 65. These results indicate that modified Cas9 mRNA functions in vivo to efficiently (i.e., 97% efficiency) introduce on target mutations in mammals.

Example 4

Mutational Analysis of Genome Edited Hematopoietic Stem-Progenitor Cells (HSPCs) by Target Capture Deep Sequencing

[0357] CRISPR/Cas9 has previously been shown to generate off-target mutations to varying degrees depending upon experimental setting and cell type (Cho et al., 2014; Cradick et al., 2013; Fu et al., 2013; Fu et al., 2014; Hruscha et al., 2013; Lin et al., 2014). To examine this in primary CD34.sup.+ HSPCs we performed target capture sequencing, of CD34.sup.+ HSPCs-mPB subjected to CRISPR/Cas9 CCR5-editing. Experimental design included capture of each gRNA target site (n=6) and predicted off-target sites (n=172) with expanded capture intervals of 500 base pairs flanking each site to ensure accurate detection of any genetic lesion occurring at or near the selected sites (FIGS. 10A and 12). We have previously shown that this approach can also capture structural variation breakpoints, such as translocations and inversions, in proximity to the capture site (Talkowski et al., 2011). Sorted CD34.sup.+ HSPCs treated with Cas9 alone or in combination with multiple single gRNA (crCCR5_A, crCCR5_B, or crCCR5_C) or dual gRNA combinations (crCCR5_A+B, crCCR5_C+D, or crCCR5_D+Q) were sequenced to a mean target coverage of 3,390X across each 23 bp gRNA sequence and PAM (range 379.6X-7,969.5X) (FIG. 10B), Analysis of the resulting data revealed highly efficacious on-target mutagenesis with a diverse array of mutated sequence variants observed in both single-gRNA and dual-gRNA treatments (FIG. 10C). As expected we detected small InDels of up to 10 bp in addition to varying single nucleotide substitutions at the predicted target sites in the single-gRNA libraries. Strikingly, in each dual-gRNA library, no fewer than 15 alternate mutant alleles were observed at either one of the gRNA sites (FIGS. 13, 14 and 15). Notably, the extreme sequencing depth of our analysis permitted estimation of mutation frequency for each particular variant, including mutations that were observed in only a few hundredths of a percent of the sample sequenced (FIG. 16). Predicted deletions (i.e., deletions spanning between the two gRNA target sites) were the most common mutations observed (crCCR5_A+B: 19.95%; crCCR5_C+D: 20.45%; crCCR5_D+Q: 42.13%), while small InDels (crCCR5_A+B: 3.06%; crCCR5_C+D: 0.50%; crCCR5_D+Q: 2.95%) were also frequent (FIG. 10C). Interestingly, for two dual gRNA combinations (crCCR5_A+B and crCCR5_D+Q) we also observed inversions between the two predicted Cas9 cleavage sites (crCCR5_A+B: 3.06%; crCCR5_D+Q: 2.48%). The most efficacious dual gRNA combination crCCR5_D+Q led to mutations in approximately 48% of the captured sequence reads (FIG. 10C).

[0358] We next examined the capture sequence reads at predicted off-target sites in the genome (FIG. 12). An N-fold enrichment analysis was performed, wherein we compared the total number of non-reference sequencing reads at each predicted off-target site in gRNA treated and control (Cas9 only) samples. This analysis generated a ratio where 1.0 indicates an equivalent number of non-reference sequence reads in both treated and control samples, values less than 1.0 indicate fewer non-reference reads in treated samples, and values greater than 1.0 indicate a greater number of non-reference reads in treated samples (see supplementary materials for additional details) (FIG. 10D). Strikingly, this analysis showed that the mean enrichment of mutations at off-target sites in all the gRNA-treated samples compared to control closely conformed to the null hypothesis (i.e., 0.99-fold enrichment compared to controls) indicating that off-target mutation events were extremely rare. Indeed, statistical evaluation of all captured off-target sites yielded a single site (1/172; 0.6%) in the sample treated with gRNA crCCR8_B alone that passed multiple test correction for a statistically significant enrichment for off-target InDels in the gRNA crCCR5_B treated libraries versus control (p.ltoreq.7.6.times.10.sup.-11) (FIGS. 16 and 17). When we scrutinized the sequencing reads from the only statistically significant off-target site, which was located in the highly homologous CCR2 gene (FIG. 11A), we found that all sequence variants (36 out of 5,963 total reads) were one or two base InDels, (FIG. 11B). Of note, the other sample in which gRNA crCCR5_B was used (in combination with gRNA crCCR5_A) only 13 out of 5,339 reads supported mutation, however these events did not meet statistical significance above control or samples treated with other gRNAs (FIG. 11B, FIG. 16). Thus, off-target mutagenesis was exceedingly rare and moreover, the use of two gRNAs in combination did not increase the very low incidence of off-target mutagenesis. We also performed targeted analyses for structural variation at all sites and though we could easily detect on-target inversions in dual gRNA combination crCCR5_A+B and crCCR5_D+Q, there was no evidence for inversion or translocation at any off-target sites in any of the treatments. These data indicate that on-target mutagenesis efficiency was very high, and further that off-target mutagenesis was extremely infrequent for both single- and dual gRNA treatments.

[0359] Discussion

[0360] Our mutational analysis revealed highly efficacious mutagenesis of on-target sites in CD34.sup.+ HSPCs. Single gRNAs generated a range of mutations with the vast majority comprised of small InDels. In contrast, dual gRNA combinations largely led to predicted deletions through a diverse array of mutations including InDels and even inversions were detected. Importantly, we only identified one statistically significant off-target site in the highly homologous CCR2 gene, which occurred in one out of 6 experimental settings (gRNA crCCR5_B alone). Sequence analysis of gRNA crCCR5_B in comparison to the identified off-target site in CCR2 indicated that it perfectly matched in the seed region and contained 3 sequence mismatches at the 5' end of the gRNA sequence (positions 1, 4 and 6). This data is consistent with previous studies showing that mismatches in the 5' proximal end of the gRNA are well tolerated by Cas9 (Lin et al., 2014; Wu et al., 2014). Our data therefore supports the idea that judicious guide design is critical for minimizing off-target mutations. Of note, our very deep sequencing analysis enabled detection of the sole off-target event we describe, whereas sequence analysis performed at lower sequencing depth--such as 50.times. coverage that has been used in previous off-target analyses (Smith et al., 2014; Suzuki et al., 2014; Veres et al., 2014)--would have been unable to detect this event. Overall, our analysis of CRISPR/Cas9 mutational activity in CD34.sup.+ HSPCs revealed very high on-target mutation rates and extremely low incidence of off-target mutagenesis.

[0361] Off-Target Prediction and Capture Sequencing

[0362] Degenerate gRNA off-target sequences were predicted for each gRNA targeting CCR5 using the CRISPR Design off-target prediction tool (Hsu et al., 2013). Off-target sequences were further supplemented by alignment of each gRNA to the human genome using BOWTIE of which all results up to and including 3 mismatches were added to the total off-target list (Langmead et al., 2009). All instances of each predicted off-target sequence existent in the human genome reference build GRCh37v71 were recorded (FIG. 12). Each guide RNA target site (n=6) and predicted off-target site (n=172) was selected for capture sequencing using the Agilent SureSelectXT Target Enrichment System. Capture intervals were expanded by approximately 500 bp in both the 5' and 3' directions to ensure exhaustive capture of the targeted region and detection of any genetic lesion occurring at or near a predicted gRNA on- or off-target site, as we have previously shown accurate capability to detect translocations and inversions using targeted capture of probes in proximity to a rearrangement breakpoint using a CapBP procedure as described (Talkowski et al., 2011). Probes were tiled with 60-fold greater density over each predicted 23 bp on- or off-target gRNA binding site than the flanking kilobase of sequence. Isogenic CD34.sup.+ HSPCs-mPB were transfected with CRISPR/Cas9 plasmids (one Cas9 only-treated control group, three treatment groups transfected with a single gRNA, and three treatment groups transfected with dual gRNAs). Sorted CD34.sup.+ genome edited HSPCs were cultured for two weeks prior to DNA isolation. Capture libraries were prepared from DNA extracted from seven treatment groups. Capture libraries were sequenced as 101 bp paired-end reads on an Illumina HiSeq2000 platform.

[0363] NGS Data Processing and Computational Analysis

[0364] Read pairs were aligned to GRCh37v71 with Bwa-MFM v0.7.10-r789 (Li, arXiv 2013). Alignments were processed using PicardTools and SAMBLASTER (Faust and Hall, 2014). The Genome Analysis Toolkit (GATK) v3.1-1-g07a4bf8 was applied for base quality score recalibration, insertion/deletion (InDel) realignment, duplicate removal, and single nucleotide variant (SNV) and InDel discovery and genotyping per published best-practice protocols (McKenna et al, Genome Res 2010; DePristo et al, Nat Genet 2011; Van der Auwera et al, 2013). SNVs and InDels were annotated using ANNOVAR (Wang et al., 2010). Structural variants (SVs) were detected with LUMPY v0.2.5 considering both anomalous pair and split read evidence at a minimum call weight threshold of 7 and an evidence set score .ltoreq.0.05 (Layer et al., 2014). Candidate copy number variants (CNVs) were further statistically assessed by Student's t-test for a concomitant change in depth of coverage across the putative CNV. As a final exhaustive measure, each on- and off-target site was manually scrutinized in each capture library for evidence supporting predictable mutagenesis that is not detectable by the computational algorithms due to low levels of mosaicism in the sequenced population.

[0365] Evaluation of Off-Target Mutation Frequency

[0366] A statistical framework was developed to assess off-target mutational burden for each gRNA. For each off-target site (n=172), all reads with at least one nucleotide of overlap with that 23 bp off-target site were collected and their CIGAR information was tabulated into categories as follows: reads representing small InDels (CIGAR contains at least one "I" or "D"), reads potentially representative of other rearrangements (CIGAR contains at least one "S" or "H"), and reads reflecting reference sequence (CIGAR did not match either of the two former categories). Such counts were gathered at all 172 sites in all seven libraries and were further pooled to form comparison groups of "treatment" libraries (transfected gRNA matches corresponding off-target site gRNA) and "control" libraries (transfected gRNA does not match corresponding off-target site gRNA). Next, at each off-target site, relative n-fold enrichment of each read classification between treatment and control libraries was evaluated. Finally, a one-tailed Fisher's Exact Test was performed to assess the statistical significance of enrichment of variant reads in treatments versus controls at each off-target site, followed by Bonferroni correction to retain an experiment-wide significance threshold of .alpha.=0.05.

REFERENCES

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Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 598 <210> SEQ ID NO 1 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 1 tgacatcaat tattatacat cgg 23 <210> SEQ ID NO 2 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 2 cctgcctccg ctctactcac tgg 23 <210> SEQ ID NO 3 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 3 tactcactgg tgttcatctt tgg 23 <210> SEQ ID NO 4 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 4 ggtgttcatc tttggttttg tgg 23 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 5 gtgttcatct ttggttttgt ggg 23 <210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 6 tggttttgtg ggcaacatgc tgg 23 <210> SEQ ID NO 7 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 7 tcatcctgat aaactgcaaa agg 23 <210> SEQ ID NO 8 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 8 tgacatctac ctgctcaacc tgg 23 <210> SEQ ID NO 9 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 9 tccttcttac tgtccccttc tgg 23 <210> SEQ ID NO 10 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 10 ccttcttact gtccccttct ggg 23 <210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 11 ctcactatgc tgccgcccag tgg 23 <210> SEQ ID NO 12 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 12 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 13 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 13 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 14 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 14 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 15 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 15 caatgtgtca actcttgaca ggg 23 <210> SEQ ID NO 16 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 16 ttgacagggc tctattttat agg 23 <210> SEQ ID NO 17 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 17 tattttatag gcttcttctc tgg 23 <210> SEQ ID NO 18 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 18 tcatcctcct gacaatcgat agg 23 <210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 19 cctgacaatc gataggtacc tgg 23 <210> SEQ ID NO 20 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 20 ctgtgtttgc tttaaaagcc agg 23 <210> SEQ ID NO 21 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 21 gtttgcttta aaagccagga cgg 23 <210> SEQ ID NO 22 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 22 aaagccagga cggtcacctt tgg 23 <210> SEQ ID NO 23 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 23 aagccaggac ggtcaccttt ggg 23 <210> SEQ ID NO 24 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 24 agccaggacg gtcacctttg ggg 23 <210> SEQ ID NO 25 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 25 caggacggtc acctttgggg tgg 23 <210> SEQ ID NO 26 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 26 tggtgacaag tgtgatcact tgg 23 <210> SEQ ID NO 27 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 27 ggtgacaagt gtgatcactt ggg 23 <210> SEQ ID NO 28 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 28 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 29 aagtgtgatc acttgggtgg tgg 23 <210> SEQ ID NO 30 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 30 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 31 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 31 tttaccagat ctcaaaaaga agg 23 <210> SEQ ID NO 32 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 32 catacagtca gtatcaattc tgg 23 <210> SEQ ID NO 33 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 33 gacattaaag atagtcatct tgg 23 <210> SEQ ID NO 34 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 34 acattaaaga tagtcatctt ggg 23 <210> SEQ ID NO 35 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 35 cattaaagat agtcatcttg ggg 23 <210> SEQ ID NO 36 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 36 aaagatagtc atcttggggc tgg 23 <210> SEQ ID NO 37 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 37 ggtcctgccg ctgcttgtca tgg 23 <210> SEQ ID NO 38 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 38 tgtcatggtc atctgctact cgg 23 <210> SEQ ID NO 39 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 39 gtcatggtca tctgctactc ggg 23 <210> SEQ ID NO 40 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 40 gaatcctaaa aactctgctt cgg 23 <210> SEQ ID NO 41 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 41 ggtgtcgaaa tgagaagaag agg 23 <210> SEQ ID NO 42 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 42 gaaatgagaa gaagaggcac agg 23 <210> SEQ ID NO 43 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 43 aaatgagaag aagaggcaca ggg 23 <210> SEQ ID NO 44 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 44 agaagaggca cagggctgtg agg 23 <210> SEQ ID NO 45 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 45 tgattgttta ttttctcttc tgg 23 <210> SEQ ID NO 46 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 46 gattgtttat tttctcttct ggg 23 <210> SEQ ID NO 47 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 47 ccttctcctg aacaccttcc agg 23 <210> SEQ ID NO 48 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 48 aacaccttcc aggaattctt tgg 23 <210> SEQ ID NO 49 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 49 ataattgcag tagctctaac agg 23 <210> SEQ ID NO 50 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 50 ttgcagtagc tctaacaggt tgg 23 <210> SEQ ID NO 51 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 51 caggttggac caagctatgc agg 23 <210> SEQ ID NO 52 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 52 atgcaggtga cagagactct tgg 23 <210> SEQ ID NO 53 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 53 tgcaggtgac agagactctt ggg 23 <210> SEQ ID NO 54 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 54 cccatcatct atgcctttgt cgg 23 <210> SEQ ID NO 55 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 55 ccatcatcta tgcctttgtc ggg 23 <210> SEQ ID NO 56 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 56 catcatctat gcctttgtcg ggg 23 <210> SEQ ID NO 57 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 57 ctgttctatt ttccagcaag agg 23 <210> SEQ ID NO 58 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 58 tcagtttaca cccgatccac tgg 23 <210> SEQ ID NO 59 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 59 cagtttacac ccgatccact ggg 23 <210> SEQ ID NO 60 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 60 agtttacacc cgatccactg ggg 23 <210> SEQ ID NO 61 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 61 cacccgatcc actggggagc agg 23 <210> SEQ ID NO 62 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 62 tggggagcag gaaatatctg tgg 23 <210> SEQ ID NO 63 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 63 ggggagcagg aaatatctgt ggg 23 <210> SEQ ID NO 64 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 64 taataattga tgtcatagat tgg 23 <210> SEQ ID NO 65 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 65 ttcacattga ttttttggca ggg 23 <210> SEQ ID NO 66 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 66 cttcacattg attttttggc agg 23 <210> SEQ ID NO 67 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 67 tttgcttcac attgattttt tgg 23 <210> SEQ ID NO 68 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 68 gtagagcgga ggcaggaggc ggg 23 <210> SEQ ID NO 69 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 69 agtagagcgg aggcaggagg cgg 23 <210> SEQ ID NO 70 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 70 gtgagtagag cggaggcagg agg 23 <210> SEQ ID NO 71 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 71 ccagtgagta gagcggaggc agg 23 <210> SEQ ID NO 72 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 72 aacaccagtg agtagagcgg agg 23 <210> SEQ ID NO 73 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 73 atgaacacca gtgagtagag cgg 23 <210> SEQ ID NO 74 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 74 ttttgcagtt tatcaggatg agg 23 <210> SEQ ID NO 75 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 75 tcagcctttt gcagtttatc agg 23 <210> SEQ ID NO 76 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR5 gRNA target site sequence <400> SEQUENCE: 76 cagagatggc caggttgagc agg 23 <210> SEQ ID NO 77 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 77 aaaacaggtc agagatggcc agg 23 <210> SEQ ID NO 78 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 78 aaggaaaaac aggtcagaga tgg 23 <210> SEQ ID NO 79 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 79 ggacagtaag aaggaaaaac agg 23 <210> SEQ ID NO 80 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 80 cccagaaggg gacagtaaga agg 23 <210> SEQ ID NO 81 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 81 cagcatagtg agcccagaag ggg 23 <210> SEQ ID NO 82 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 82 gcagcatagt gagcccagaa ggg 23 <210> SEQ ID NO 83 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 83 ggcagcatag tgagcccaga agg 23 <210> SEQ ID NO 84 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 84 atttccaaag tcccactggg cgg 23 <210> SEQ ID NO 85 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 85 tgtatttcca aagtcccact ggg 23 <210> SEQ ID NO 86 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 86 ttgtatttcc aaagtcccac tgg 23 <210> SEQ ID NO 87 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 87 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 88 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 88 ccaggtacct atcgattgtc agg 23 <210> SEQ ID NO 89 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 89 acacagcatg gacgacagcc agg 23 <210> SEQ ID NO 90 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 90 cttttaaagc aaacacagca tgg 23 <210> SEQ ID NO 91 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 91 caccccaaag gtgaccgtcc tgg 23 <210> SEQ ID NO 92 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 92 cacacttgtc accaccccaa agg 23 <210> SEQ ID NO 93 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 93 atctggtaaa gatgattcct ggg 23 <210> SEQ ID NO 94 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 94 gatctggtaa agatgattcc tgg 23 <210> SEQ ID NO 95 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 95 aagaccttct ttttgagatc tgg 23 <210> SEQ ID NO 96 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 96 gtatggaaaa tgagagctgc agg 23 <210> SEQ ID NO 97 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 97 cagaattgat actgactgta tgg 23 <210> SEQ ID NO 98 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 98 agatgactat ctttaatgtc tgg 23 <210> SEQ ID NO 99 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 99 tgaccatgac aagcagcggc agg 23 <210> SEQ ID NO 100 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 100 cagatgacca tgacaagcag cgg 23 <210> SEQ ID NO 101 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 101 gacaccgaag cagagttttt agg 23 <210> SEQ ID NO 102 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 102 gagaaaataa acaatcatga tgg 23 <210> SEQ ID NO 103 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 103 aggagaagga caatgttgta ggg 23 <210> SEQ ID NO 104 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 104 caggagaagg acaatgttgt agg 23 <210> SEQ ID NO 105 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 105 cctggaaggt gttcaggaga agg 23 <210> SEQ ID NO 106 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 106 agaattcctg gaaggtgttc agg 23 <210> SEQ ID NO 107 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 107 caggccaaag aattcctgga agg 23 <210> SEQ ID NO 108 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 108 tattcaggcc aaagaattcc tgg 23 <210> SEQ ID NO 109 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 109 tagagctact gcaattattc agg 23 <210> SEQ ID NO 110 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 110 tctctgtcac ctgcatagct tgg 23 <210> SEQ ID NO 111 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 111 cgacaaaggc atagatgatg ggg 23 <210> SEQ ID NO 112 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 112 ccgacaaagg catagatgat ggg 23 <210> SEQ ID NO 113 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 113 cccgacaaag gcatagatga tgg 23 <210> SEQ ID NO 114 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 114 tctgaacttc tccccgacaa agg 23 <210> SEQ ID NO 115 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 115 gcttttggaa gaagactaag agg 23 <210> SEQ ID NO 116 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 116 agcgtttggc aatgtgcttt tgg 23 <210> SEQ ID NO 117 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 117 acagcatttg cagaagcgtt tgg 23 <210> SEQ ID NO 118 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 118 ctcgctcggg agcctcttgc tgg 23 <210> SEQ ID NO 119 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 119 taaactgagc ttgctcgctc ggg 23 <210> SEQ ID NO 120 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 120 gtaaactgag cttgctcgct cgg 23 <210> SEQ ID NO 121 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 121 ttcctgctcc ccagtggatc ggg 23 <210> SEQ ID NO 122 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 122 tttcctgctc cccagtggat cgg 23 <210> SEQ ID NO 123 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 123 agatatttcc tgctccccag tgg 23 <210> SEQ ID NO 124 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 124 atcatcttta ccagatctca aaaagaag 28 <210> SEQ ID NO 125 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 125 aactctgctt cggtgtcgaa atgagaag 28 <210> SEQ ID NO 126 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 126 tctgcttcgg tgtcgaaatg agaagaag 28 <210> SEQ ID NO 127 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 127 catcatctat gcctttgtcg gggagaag 28 <210> SEQ ID NO 128 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 128 atgcctttgt cggggagaag ttcagaaa 28 <210> SEQ ID NO 129 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 129 agtgagccca gaaggggaca gtaagaag 28 <210> SEQ ID NO 130 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 130 ctgggcggca gcatagtgag cccagaag 28 <210> SEQ ID NO 131 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 131 gatgatgaag aagattccag agaagaag 28 <210> SEQ ID NO 132 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 132 gaggatgatg aagaagattc cagagaag 28 <210> SEQ ID NO 133 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 133 atcgattgtc aggaggatga tgaagaag 28 <210> SEQ ID NO 134 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 134 caatgttgta gggagcccag aagagaaa 28 <210> SEQ ID NO 135 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 135 aaggacaatg ttgtagggag cccagaag 28 <210> SEQ ID NO 136 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 136 agaattcctg gaaggtgttc aggagaag 28 <210> SEQ ID NO 137 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 137 gcgtttggca atgtgctttt ggaagaag 28 <210> SEQ ID NO 138 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 138 ctggaaaata gaacagcatt tgcagaag 28 <210> SEQ ID NO 139 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 139 tcgggagcct cttgctggaa aatagaac 28 <210> SEQ ID NO 140 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 140 acttgaagac tcagactcag tgg 23 <210> SEQ ID NO 141 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 141 atgtccacct cgctttcctt tgg 23 <210> SEQ ID NO 142 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 142 cacctcgctt tcctttggag agg 23 <210> SEQ ID NO 143 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 143 ttcctttgga gaggatcttg agg 23 <210> SEQ ID NO 144 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 144 tttggagagg atcttgaggc tgg 23 <210> SEQ ID NO 145 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 145 gctggaccct ctgctcacag agg 23 <210> SEQ ID NO 146 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 146 tcacagaggt gagtgcgtgc tgg 23 <210> SEQ ID NO 147 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 147 cacagaggtg agtgcgtgct ggg 23 <210> SEQ ID NO 148 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 148 ggtgagtgcg tgctgggcag agg 23 <210> SEQ ID NO 149 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 149 ctgggcagag gttttaaatt tgg 23 <210> SEQ ID NO 150 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 150 aggttttaaa tttggctcca agg 23 <210> SEQ ID NO 151 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 151 tggctccaag gaaagcatag agg 23 <210> SEQ ID NO 152 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 152 tccaaggaaa gcatagagga tgg 23 <210> SEQ ID NO 153 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 153 ccaaggaaag catagaggat ggg 23 <210> SEQ ID NO 154 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 154 caaggaaagc atagaggatg ggg 23 <210> SEQ ID NO 155 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 155 ggatggggtt cagacaacag tgg 23 <210> SEQ ID NO 156 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 156 gacaacagtg gaagaaagct agg 23 <210> SEQ ID NO 157 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 157 acaacagtgg aagaaagcta ggg 23 <210> SEQ ID NO 158 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 158 gtggaagaaa gctagggcct cgg 23 <210> SEQ ID NO 159 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 159 gaaagctagg gcctcggtga tgg 23 <210> SEQ ID NO 160 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 160 acccttgctt gatgatttcc agg 23 <210> SEQ ID NO 161 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 161 cttgcttgat gatttccagg agg 23 <210> SEQ ID NO 162 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 162 gatgatttcc aggaggatga agg 23 <210> SEQ ID NO 163 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 163 atgctgatcc caatgtagta agg 23 <210> SEQ ID NO 164 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 164 aatgtagtaa ggcagccaac agg 23 <210> SEQ ID NO 165 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 165 gccaacaggc gaagaaagcc agg 23 <210> SEQ ID NO 166 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 166 aggcgaagaa agccaggatg agg 23 <210> SEQ ID NO 167 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 167 agccaggatg aggatgactg tgg 23 <210> SEQ ID NO 168 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 168 tgaggatgac tgtggtcttg agg 23 <210> SEQ ID NO 169 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 169 gaggatgact gtggtcttga ggg 23 <210> SEQ ID NO 170 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 170 tcttgagggc cttgcgcttc tgg 23 <210> SEQ ID NO 171 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 171 tgagggcctt gcgcttctgg tgg 23 <210> SEQ ID NO 172 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 172 cttgcgcttc tggtggccct tgg 23 <210> SEQ ID NO 173 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 173 gcccttggag tgtgacagct tgg 23 <210> SEQ ID NO 174 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 174 ggagatgata atgcaatagc agg 23 <210> SEQ ID NO 175 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 175 tgataatgca atagcaggac agg 23 <210> SEQ ID NO 176 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 176 caggacagga tgacaatacc agg 23 <210> SEQ ID NO 177 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 177 acaggatgac aataccaggc agg 23 <210> SEQ ID NO 178 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 178 tgacaatacc aggcaggata agg 23 <210> SEQ ID NO 179 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 179 caaccatgat gtgctgaaac tgg 23 <210> SEQ ID NO 180 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 180 cacaaccacc cacaagtcat tgg 23 <210> SEQ ID NO 181 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 181 acaaccaccc acaagtcatt ggg 23 <210> SEQ ID NO 182 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 182 caaccaccca caagtcattg ggg 23 <210> SEQ ID NO 183 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 183 acaagtcatt ggggtagaag cgg 23 <210> SEQ ID NO 184 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 184 gtcatctgcc tcactgacgt tgg 23 <210> SEQ ID NO 185 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 185 gacgttggca aagatgaagt cgg 23 <210> SEQ ID NO 186 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 186 acgttggcaa agatgaagtc ggg 23 <210> SEQ ID NO 187 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 187 tgaagtcggg aatagtcagc agg 23 <210> SEQ ID NO 188 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 188 agtcgggaat agtcagcagg agg 23 <210> SEQ ID NO 189 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 189 gtcgggaata gtcagcagga ggg 23 <210> SEQ ID NO 190 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 190 ggaatagtca gcaggagggc agg 23 <210> SEQ ID NO 191 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 191 gaatagtcag caggagggca ggg 23 <210> SEQ ID NO 192 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 192 ttttcagcca acagcttcct tgg 23 <210> SEQ ID NO 193 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 193 cttccttggc ctctgactgt tgg 23 <210> SEQ ID NO 194 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 194 ccttggcctc tgactgttgg tgg 23 <210> SEQ ID NO 195 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 195 gcctctgact gttggtggcg tgg 23 <210> SEQ ID NO 196 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 196 actgttggtg gcgtggacga tgg 23 <210> SEQ ID NO 197 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 197 tggtggcgtg gacgatggcc agg 23 <210> SEQ ID NO 198 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 198 cgtggacgat ggccaggtag cgg 23 <210> SEQ ID NO 199 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 199 gtagcggtcc agactgatga agg 23 <210> SEQ ID NO 200 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 200 ggtccagact gatgaaggcc agg 23 <210> SEQ ID NO 201 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 201 gactgatgaa ggccaggatg agg 23 <210> SEQ ID NO 202 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 202 ggatgaggac actgctgtag agg 23 <210> SEQ ID NO 203 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 203 ggttgactgt gtagatgaca tgg 23 <210> SEQ ID NO 204 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 204 tgacatggac tgccttgcat agg 23 <210> SEQ ID NO 205 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 205 cccaaagtac cagtttgcca cgg 23 <210> SEQ ID NO 206 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 206 cacggcatca actgcccaga agg 23 <210> SEQ ID NO 207 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 207 acggcatcaa ctgcccagaa ggg 23 <210> SEQ ID NO 208 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 208 agggaagcgt gatgacaaag agg 23 <210> SEQ ID NO 209 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 209 gaagcgtgat gacaaagagg agg 23 <210> SEQ ID NO 210 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 210 cgtgatgaca aagaggaggt cgg 23 <210> SEQ ID NO 211 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 211 agaggaggtc ggccactgac agg 23 <210> SEQ ID NO 212 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 212 tcatgcttct cagtttcttc tgg 23 <210> SEQ ID NO 213 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 213 tcttctggta acccatgacc agg 23 <210> SEQ ID NO 214 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 214 aatgccagtt aagaagatga tgg 23 <210> SEQ ID NO 215 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 215 taagaagatg atggagtaga tgg 23 <210> SEQ ID NO 216 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 216 gaagatgatg gagtagatgg tgg 23 <210> SEQ ID NO 217 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 217 aagatgatgg agtagatggt ggg 23 <210> SEQ ID NO 218 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 218 tgatggagta gatggtgggc agg 23 <210> SEQ ID NO 219 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 219 tgaaattagc attttcttca cgg 23 <210> SEQ ID NO 220 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 220 agcattttct tcacggaaac agg 23 <210> SEQ ID NO 221 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 221 gcattttctt cacggaaaca ggg 23 <210> SEQ ID NO 222 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 222 acggaaacag ggttccttca tgg 23 <210> SEQ ID NO 223 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 223 gtcccctgag cccatttcct cgg 23 <210> SEQ ID NO 224 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 224 gaagtgtata tctgcaaaag agg 23 <210> SEQ ID NO 225 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 225 tatatctgca aaagaggcaa agg 23 <210> SEQ ID NO 226 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 226 ctgcaaaaga ggcaaaggaa tgg 23 <210> SEQ ID NO 227 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 227 ctctccaaag gaaagcgagg tgg 23 <210> SEQ ID NO 228 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 228 atcctctcca aaggaaagcg agg 23 <210> SEQ ID NO 229 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 229 agcctcaaga tcctctccaa agg 23 <210> SEQ ID NO 230 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 230 cactcacctc tgtgagcaga ggg 23 <210> SEQ ID NO 231 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 231 gcactcacct ctgtgagcag agg 23 <210> SEQ ID NO 232 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 232 cccatcctct atgctttcct tgg 23 <210> SEQ ID NO 233 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 233 caagtggatt tccatcaccg agg 23 <210> SEQ ID NO 234 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 234 ttgagaacac tgtgcacaag tgg 23 <210> SEQ ID NO 235 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 235 tcctggaaat catcaagcaa ggg 23 <210> SEQ ID NO 236 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 236 ctcctggaaa tcatcaagca agg 23 <210> SEQ ID NO 237 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 237 catcgactcc ttcatcctcc tgg 23 <210> SEQ ID NO 238 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 238 gttggctgcc ttactacatt ggg 23 <210> SEQ ID NO 239 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 239 tgttggctgc cttactacat tgg 23 <210> SEQ ID NO 240 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 240 tcctggcttt cttcgcctgt tgg 23 <210> SEQ ID NO 241 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 241 gaccacagtc atcctcatcc tgg 23 <210> SEQ ID NO 242 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 242 caagggccac cagaagcgca agg 23 <210> SEQ ID NO 243 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 243 tccaagctgt cacactccaa ggg 23 <210> SEQ ID NO 244 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 244 ctccaagctg tcacactcca agg 23 <210> SEQ ID NO 245 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 245 atggttggcc ttatcctgcc tgg 23 <210> SEQ ID NO 246 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 246 cagtttcagc acatcatggt tgg 23 <210> SEQ ID NO 247 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 247 gttccagttt cagcacatca tgg 23 <210> SEQ ID NO 248 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 248 ctaccccaat gacttgtggg tgg 23 <210> SEQ ID NO 249 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 249 cttctacccc aatgacttgt ggg 23 <210> SEQ ID NO 250 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 250 gcttctaccc caatgacttg tgg 23 <210> SEQ ID NO 251 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 251 catctttgcc aacgtcagtg agg 23 <210> SEQ ID NO 252 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 252 aggtggtcta tgttggcgtc tgg 23 <210> SEQ ID NO 253 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 253 gctgaaaagg tggtctatgt tgg 23 <210> SEQ ID NO 254 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 254 gaagctgttg gctgaaaagg tgg 23 <210> SEQ ID NO 255 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 255 aaggaagctg ttggctgaaa agg 23 <210> SEQ ID NO 256 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 256 tcagaggcca aggaagctgt tgg 23 <210> SEQ ID NO 257 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 257 ccaccaacag tcagaggcca agg 23 <210> SEQ ID NO 258 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 258 tccacgccac caacagtcag agg 23 <210> SEQ ID NO 259 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 259 catcagtctg gaccgctacc tgg 23 <210> SEQ ID NO 260 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 260 catcctggcc ttcatcagtc tgg 23 <210> SEQ ID NO 261 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 261 ctacagcagt gtcctcatcc tgg 23 <210> SEQ ID NO 262 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 262 ctttgggaac ttcctatgca agg 23 <210> SEQ ID NO 263 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 263 ccgtggcaaa ctggtacttt ggg 23 <210> SEQ ID NO 264 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 264 gccgtggcaa actggtactt tgg 23 <210> SEQ ID NO 265 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 265 cagttgatgc cgtggcaaac tgg 23 <210> SEQ ID NO 266 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 266 cttctgggca gttgatgccg tgg 23 <210> SEQ ID NO 267 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 267 tgtcatcacg cttcccttct ggg 23 <210> SEQ ID NO 268 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 268 ttgtcatcac gcttcccttc tgg 23 <210> SEQ ID NO 269 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 269 gtacaggctg cacctgtcag tgg 23 <210> SEQ ID NO 270 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 270 gaagcatgac ggacaagtac agg 23 <210> SEQ ID NO 271 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 271 gaagaaactg agaagcatga cgg 23 <210> SEQ ID NO 272 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 272 ggattggtca tcctggtcat ggg 23 <210> SEQ ID NO 273 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 273 tggattggtc atcctggtca tgg 23 <210> SEQ ID NO 274 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 274 gggcaatgga ttggtcatcc tgg 23 <210> SEQ ID NO 275 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 275 tggcattgtg ggcaatggat tgg 23 <210> SEQ ID NO 276 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 276 ttaactggca ttgtgggcaa tgg 23 <210> SEQ ID NO 277 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 277 atcttcttaa ctggcattgt ggg 23 <210> SEQ ID NO 278 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 278 catcttctta actggcattg tgg 23 <210> SEQ ID NO 279 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 279 tactccatca tcttcttaac tgg 23 <210> SEQ ID NO 280 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 280 aggggactat gactccatga agg 23 <210> SEQ ID NO 281 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 281 caccgaggaa atgggctcag ggg 23 <210> SEQ ID NO 282 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 282 acaccgagga aatgggctca ggg 23 <210> SEQ ID NO 283 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 283 tacaccgagg aaatgggctc agg 23 <210> SEQ ID NO 284 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 284 gataactaca ccgaggaaat ggg 23 <210> SEQ ID NO 285 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 285 agataactac accgaggaaa tgg 23 <210> SEQ ID NO 286 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 286 cacttcagat aactacaccg agg 23 <210> SEQ ID NO 287 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 287 gatggggttc agacaacagt ggaagaaa 28 <210> SEQ ID NO 288 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 288 gtagtaaggc agccaacagg cgaagaaa 28 <210> SEQ ID NO 289 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 289 aaccacccac aagtcattgg ggtagaag 28 <210> SEQ ID NO 290 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 290 gtttgccacg gcatcaactg cccagaag 28 <210> SEQ ID NO 291 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 291 tccattgccc acaatgccag ttaagaag 28 <210> SEQ ID NO 292 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 292 catcaagcaa gggtgtgagt ttgagaac 28 <210> SEQ ID NO 293 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 293 gctgtcacac tccaagggcc accagaag 28 <210> SEQ ID NO 294 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 294 tcatgggtta ccagaagaaa ctgagaag 28 <210> SEQ ID NO 295 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 295 catcctggtc atgggttacc agaagaaa 28 <210> SEQ ID NO 296 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 296 ggtcatcctg gtcatgggtt accagaag 28 <210> SEQ ID NO 297 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 297 atgaaggaac cctgtttccg tgaagaaa 28 <210> SEQ ID NO 298 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: CAS protein <400> SEQUENCE: 298 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 299 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 299 taaattcttt gctgacctgc 20 <210> SEQ ID NO 300 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 300 tagatccatt cctatgactg 20 <210> SEQ ID NO 301 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 301 cttcagtctg ataaaatcta 20 <210> SEQ ID NO 302 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 302 tttgatgtaa tccagcaggt 20 <210> SEQ ID NO 303 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 303 cacagagggc tacaatgtga 20 <210> SEQ ID NO 304 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 304 gtagagcgga ggcaggaggc ggg 23 <210> SEQ ID NO 305 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 305 gtgagtagag cggaggcagg agg 23 <210> SEQ ID NO 306 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 306 ggtgttcatc tttggttttg tgg 23 <210> SEQ ID NO 307 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 307 gtgttcatct ttggttttgt ggg 23 <210> SEQ ID NO 308 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 308 ggacagtaag aaggaaaaac agg 23 <210> SEQ ID NO 309 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 309 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 310 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 310 gcagcatagt gagcccagaa ggg 23 <210> SEQ ID NO 311 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 311 ggcagcatag tgagcccaga agg 23 <210> SEQ ID NO 312 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 312 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 313 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 313 gtttgcttta aaagccagga cgg 23 <210> SEQ ID NO 314 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 314 ggtgacaagt gtgatcactt ggg 23 <210> SEQ ID NO 315 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 315 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 316 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 316 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 317 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 317 gatctggtaa agatgattcc tgg 23 <210> SEQ ID NO 318 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 318 gtatggaaaa tgagagctgc agg 23 <210> SEQ ID NO 319 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 319 gacattaaag atagtcatct tgg 23 <210> SEQ ID NO 320 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 320 ggtcctgccg ctgcttgtca tgg 23 <210> SEQ ID NO 321 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 321 gtcatggtca tctgctactc ggg 23 <210> SEQ ID NO 322 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 322 gaatcctaaa aactctgctt cgg 23 <210> SEQ ID NO 323 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 323 ggtgtcgaaa tgagaagaag agg 23 <210> SEQ ID NO 324 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 324 gacaccgaag cagagttttt agg 23 <210> SEQ ID NO 325 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 325 gaaatgagaa gaagaggcac agg 23 <210> SEQ ID NO 326 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 326 gattgtttat tttctcttct ggg 23 <210> SEQ ID NO 327 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 327 gagaaaataa acaatcatga tgg 23 <210> SEQ ID NO 328 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 328 gcttttggaa gaagactaag agg 23 <210> SEQ ID NO 329 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 329 gtaaactgag cttgctcgct cgg 23 <210> SEQ ID NO 330 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 330 ggggagcagg aaatatctgt ggg 23 <210> SEQ ID NO 331 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 331 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 332 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 332 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 333 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 333 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 334 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 334 gtcttgctcg agatgtgatg 20 <210> SEQ ID NO 335 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 335 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 336 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 336 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 337 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 337 tgctggctcg gctgccctga ggttgctcaa tcaagcacag gtttcaa 47 <210> SEQ ID NO 338 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 338 tgctggctcg gctgccctga ggttgctcaa tcaagcacag gtttcaa 47 <210> SEQ ID NO 339 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 339 taacagcgat gctgaccccc tgtgcctcta ccacttctat gaccaga 47 <210> SEQ ID NO 340 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 340 cgatgctgac cccctgtgcc tctaccactt ctatgaccag atggacc 47 <210> SEQ ID NO 341 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 341 tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggta 49 <210> SEQ ID NO 342 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 342 tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggta 49 <210> SEQ ID NO 343 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 343 tgggcgacag atgcgaaaga aacgagttcc agtgccaaga cgggaaa 47 <210> SEQ ID NO 344 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 344 gaaacgagtt ccagtgccaa gacgggaaat gcatctccta caagtgg 47 <210> SEQ ID NO 345 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 345 tcagagagga cactgcagtt gtccgtgcta gtagccttcg cttctgga 48 <210> SEQ ID NO 346 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 346 tcagagagga cactgcagtt gtccgtgcta gtagccttcg cttctgga 48 <210> SEQ ID NO 347 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 347 tgatgatctc agaggctcag tatccttgtc ctgggttgga gatagca 47 <210> SEQ ID NO 348 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 348 tgatgatctc agaggctcag tatccttgtc ctgggttgga gatagca 47 <210> SEQ ID NO 349 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 349 tggtaattat gacttttgga cagtccaagc tatatcgaag gtgagatca 49 <210> SEQ ID NO 350 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 350 tggtaattat gacttttgga cagtccaagc tatatcgaag gtgagatca 49 <210> SEQ ID NO 351 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 351 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 352 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 352 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 353 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 353 ctatgccctg cctcatttca ggtgaagatg aaatccctgg agcttgg 47 <210> SEQ ID NO 354 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 354 ctccctgggc tgcaccatag tga 23 <210> SEQ ID NO 355 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 355 gaccttgtga agatgattcc tgg 23 <210> SEQ ID NO 356 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 356 gatctgggga agaggattcc agg 23 <210> SEQ ID NO 357 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 357 agattgtgta tactcttgac tag 23 <210> SEQ ID NO 358 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 358 ccaggaatca tctctacgat atc 23 <210> SEQ ID NO 359 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 359 cctggaattc tctttactag atc 23 <210> SEQ ID NO 360 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 360 ccggggagag gcgcggacac agc 23 <210> SEQ ID NO 361 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 361 ccagggagag acggaaccaa aac 23 <210> SEQ ID NO 362 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 362 gatctgatac agatgattca tgg 23 <210> SEQ ID NO 363 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 363 ctgggaatca tctttacaag atg 23 <210> SEQ ID NO 364 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 364 tgaatatcct gtcgcccagt cag 23 <210> SEQ ID NO 365 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 365 ccacccaagt gatcactctt cct 23 <210> SEQ ID NO 366 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 366 aggaagagtg atcacttggg tgg 23 <210> SEQ ID NO 367 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 367 tctgtgtttt tgtctctccc cag 23 <210> SEQ ID NO 368 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 368 aggaagagtg atcacttggg tgg 23 <210> SEQ ID NO 369 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 369 ccacccaagt gatcactctt cct 23 <210> SEQ ID NO 370 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 370 ctaggaatca tcttccccag atg 23 <210> SEQ ID NO 371 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 371 tctgtggaaa agatgattcc aag 23 <210> SEQ ID NO 372 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 372 gttcttgtta agatgattcc tgg 23 <210> SEQ ID NO 373 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 373 ggcatgtttc atcacttggg ggg 23 <210> SEQ ID NO 374 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 374 gggctggtaa agatgtttcc agg 23 <210> SEQ ID NO 375 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 375 gacaagtgtg atcacctggt tgg 23 <210> SEQ ID NO 376 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 376 gctgtgtttg cttctgtccc agg 23 <210> SEQ ID NO 377 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 377 ccaggaatca tctttactaa atg 23 <210> SEQ ID NO 378 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 378 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 379 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 379 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 380 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 380 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 381 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 381 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 382 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 382 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 383 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 383 ccaggaatca tctttaccag atc 23 <210> SEQ ID NO 384 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 384 acaaggggtc aactctggac aag 23 <210> SEQ ID NO 385 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 385 cccactgggc tgcagcatcc tgg 23 <210> SEQ ID NO 386 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 386 ccagcgcgtg acgcaaacac agc 23 <210> SEQ ID NO 387 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 387 acaatgtgtc accttttaac tgg 23 <210> SEQ ID NO 388 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 388 acaatatttc aacacttgac aag 23 <210> SEQ ID NO 389 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 389 cttggagtca tctttgccac atc 23 <210> SEQ ID NO 390 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 390 gacaagcgtg atcccatggg gag 23 <210> SEQ ID NO 391 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 391 cctcccaagt aatcaccctt ttc 23 <210> SEQ ID NO 392 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 392 cccaagtccc actggcaggt ggc 23 <210> SEQ ID NO 393 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 393 gctatgtgtt aactcttgac aag 23 <210> SEQ ID NO 394 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 394 ccactatggt gcctcccagt cag 23 <210> SEQ ID NO 395 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 395 tcactctgct gctgtccagt ggg 23 <210> SEQ ID NO 396 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 396 actctgtcct gccgcccagt gag 23 <210> SEQ ID NO 397 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 397 cttggaatca tctctacctg att 23 <210> SEQ ID NO 398 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 398 cccaagtccc actgggtgac atg 23 <210> SEQ ID NO 399 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 399 ctaaggagag aggcacacac agc 23 <210> SEQ ID NO 400 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 400 ccagggaggg acgcaaaccc agc 23 <210> SEQ ID NO 401 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 401 cttctcaagt gatcccactg gtc 23 <210> SEQ ID NO 402 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 402 cctgagagag aagcaaacac aga 23 <210> SEQ ID NO 403 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 403 cttaggaaag acgcaaacat agc 23 <210> SEQ ID NO 404 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 404 acattgatta aactcttgac tag 23 <210> SEQ ID NO 405 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 405 acaaagttta aactcttgag cag 23 <210> SEQ ID NO 406 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 406 cttcccaagt gattacactt tat 23 <210> SEQ ID NO 407 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 407 cccactgggg cgcagcctag tga 23 <210> SEQ ID NO 408 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 408 cttgtcaaga tttgccacat tat 23 <210> SEQ ID NO 409 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 409 ccttggtgag aggcaaacac agc 23 <210> SEQ ID NO 410 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 410 cctactgggc gtcagcactg tgt 23 <210> SEQ ID NO 411 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 411 tcactatgca gcaccccagt ggg 23 <210> SEQ ID NO 412 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 412 ccaggcatct tctttaccag ctc 23 <210> SEQ ID NO 413 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 413 ctcactgggc tgcagcattg ggg 23 <210> SEQ ID NO 414 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 414 ccagggagag acgcagaaac aac 23 <210> SEQ ID NO 415 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 415 tacatgagta atcacttggg gag 23 <210> SEQ ID NO 416 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 416 ctccccaagt gattactcat gta 23 <210> SEQ ID NO 417 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 417 cagaggtgtg atcacttggg cag 23 <210> SEQ ID NO 418 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 418 cttcccatgt gataacactt gtc 23 <210> SEQ ID NO 419 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 419 cctgagagag aggcaaacac atc 23 <210> SEQ ID NO 420 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 420 ccacgaatcc tctttaccaa atc 23 <210> SEQ ID NO 421 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 421 cctgagggtg cgtctctccc ggg 23 <210> SEQ ID NO 422 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 422 gctgtgtgct cgtctctccc tgg 23 <210> SEQ ID NO 423 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 423 gctgcggccc agcgggacct ggg 23 <210> SEQ ID NO 424 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 424 gctgtacttg ggtctctccc cag 23 <210> SEQ ID NO 425 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 425 gccgtgtttt cctctctccc aag 23 <210> SEQ ID NO 426 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 426 cccactgggc tgcagaatac aga 23 <210> SEQ ID NO 427 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 427 ccccccagga gatcacattt gtc 23 <210> SEQ ID NO 428 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 428 gctgccggac agtgggacct ggg 23 <210> SEQ ID NO 429 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 429 ctgactggac ggcaccttag tga 23 <210> SEQ ID NO 430 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 430 gctgtctttt cctctctccc tgg 23 <210> SEQ ID NO 431 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 431 ccagaaataa tctttaccag ctc 23 <210> SEQ ID NO 432 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 432 gctgtgtttg tgcctccccc agg 23 <210> SEQ ID NO 433 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 433 acaatgtgtt ggctcttgac tag 23 <210> SEQ ID NO 434 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 434 gacaagtctg atcattttgg ggg 23 <210> SEQ ID NO 435 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 435 ccacggagaa acacaaacac agc 23 <210> SEQ ID NO 436 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 436 aaaatgtgtc aactcttgat tag 23 <210> SEQ ID NO 437 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 437 tctgtgtttg cctctctctc agg 23 <210> SEQ ID NO 438 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 438 acaatgtgcc agctctggac tag 23 <210> SEQ ID NO 439 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 439 gacaagtatg agcacttggt aag 23 <210> SEQ ID NO 440 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 440 gactagtgtg ttctcttggg aag 23 <210> SEQ ID NO 441 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 441 gacatgtgta aacacttggg aag 23 <210> SEQ ID NO 442 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 442 ccaaagtccc actgggtgac atc 23 <210> SEQ ID NO 443 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 443 cctgtgagag gctcaaacac agc 23 <210> SEQ ID NO 444 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 444 ccgcagtccc actgtgcggc acc 23 <210> SEQ ID NO 445 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 445 gacaagtgtg ttcacttctg cag 23 <210> SEQ ID NO 446 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 446 gatttgggaa agatcattcc agg 23 <210> SEQ ID NO 447 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 447 gctttggtaa agttgattcc tag 23 <210> SEQ ID NO 448 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 448 aaactgtgat aactcttgac tgg 23 <210> SEQ ID NO 449 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 449 cctaagtccc actggccgaa agt 23 <210> SEQ ID NO 450 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 450 gttctggtca agatgactcc tgg 23 <210> SEQ ID NO 451 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 451 cccaggaccc actgggcagc agc 23 <210> SEQ ID NO 452 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 452 aaaatctgtc aactcttgaa tag 23 <210> SEQ ID NO 453 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 453 ctcatcagga gtggacacat tgt 23 <210> SEQ ID NO 454 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 454 ccagggggac acgcaaacac tgc 23 <210> SEQ ID NO 455 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 455 tcactttgct gccagccagt tgg 23 <210> SEQ ID NO 456 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 456 ctgaagtccc actgggtggg tgt 23 <210> SEQ ID NO 457 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 457 gcaccagccc agtgggactt cag 23 <210> SEQ ID NO 458 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 458 ccagggaaag aggcaagcac agc 23 <210> SEQ ID NO 459 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 459 ccactataca cccgcccagt cag 23 <210> SEQ ID NO 460 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 460 ccattgtgct gccgcccagc cag 23 <210> SEQ ID NO 461 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 461 gatctggaag agatgattcc aag 23 <210> SEQ ID NO 462 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 462 cccgggaggg aggcaaaaac agc 23 <210> SEQ ID NO 463 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 463 gctgagtctg ggtctctccc cag 23 <210> SEQ ID NO 464 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 464 cctggaatgt tctttcccag atc 23 <210> SEQ ID NO 465 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 465 gacaggtgtg agcactttgg gag 23 <210> SEQ ID NO 466 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 466 gacacttgtg atgacttggg tag 23 <210> SEQ ID NO 467 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 467 ctttggagag acgcagacac tgc 23 <210> SEQ ID NO 468 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 468 ctacccaagt gttcatattt gtc 23 <210> SEQ ID NO 469 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 469 ccagaaatca tgtttaccag ctc 23 <210> SEQ ID NO 470 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 470 ctggagtccc actgcgcggc agc 23 <210> SEQ ID NO 471 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 471 cctgggaaag gcgcaaacac agc 23 <210> SEQ ID NO 472 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 472 gatctgataa aggtgagtcc agg 23 <210> SEQ ID NO 473 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 473 gcaagttgct gccgcccagt ggg 23 <210> SEQ ID NO 474 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 474 gcagtgtgtg ggtctctccc agg 23 <210> SEQ ID NO 475 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 475 ccaaagtcgc actggcctgc agc 23 <210> SEQ ID NO 476 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 476 gatctgggag agaagattcc agg 23 <210> SEQ ID NO 477 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 477 ccagaaaaga gttgacacat agt 23 <210> SEQ ID NO 478 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 478 cctgccaagg gttgacacat ggt 23 <210> SEQ ID NO 479 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 479 gacaagtgtc ataactttgg aag 23 <210> SEQ ID NO 480 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 480 ataatgtgtc aaccctggac cag 23 <210> SEQ ID NO 481 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 481 ccaaagaccc actggacggc agc 23 <210> SEQ ID NO 482 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 482 ccaactggcc ggcagcctgg tga 23 <210> SEQ ID NO 483 <400> SEQUENCE: 483 000 <210> SEQ ID NO 484 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 484 tatgctgccg cccagtggga ctttggaaat acaatgtgtc aactc 45 <210> SEQ ID NO 485 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 485 ggctgtgttt gcgtctctcc caggaatcat ctttaccaga tctca 45 <210> SEQ ID NO 486 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 486 tatgctgccg cccagtggga 20 <210> SEQ ID NO 487 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 487 atcatcttta ccagatctca aaaag 25 <210> SEQ ID NO 488 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 488 gagttgacac attgtatttc caaag 25 <210> SEQ ID NO 489 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 489 atcatcttta ccagatctca 20 <210> SEQ ID NO 490 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 490 tcctgggaga gacgcaaaca cagcc 25 <210> SEQ ID NO 491 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 491 tggctgtgtt tgcgtctctc ccagg 25 <210> SEQ ID NO 492 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 492 catcatcttt accagatctc aaaaa 25 <210> SEQ ID NO 493 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 493 tttggaaata caatgtgtca actct 25 <210> SEQ ID NO 494 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 494 tcatctttac cagatctcaa aaaga 25 <210> SEQ ID NO 495 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 495 gctgtgtttg cgtctctccc aggaa 25 <210> SEQ ID NO 496 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 496 actttggaaa tacaatgtgt caact 25 <210> SEQ ID NO 497 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 497 tggaaataca atgtgtcaac tcttg 25 <210> SEQ ID NO 498 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 498 gtggctgtgt ttgcgtctct cccag 25 <210> SEQ ID NO 499 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 499 tcctttggaa atacaatgtg tcaac 25 <210> SEQ ID NO 500 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 500 atctttacca gatctcaaaa agaag 25 <210> SEQ ID NO 501 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 501 tggtggctgt gtttgcgtct ctccc 25 <210> SEQ ID NO 502 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 502 aatcatcttt accagatctc aaaaa 25 <210> SEQ ID NO 503 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 503 tcactatgct gccgcccagt gggaa 25 <210> SEQ ID NO 504 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 504 aatgtgtcaa ctcttgacag ggctc 25 <210> SEQ ID NO 505 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 505 ttggaaatac aatgtgtcaa ctctt 25 <210> SEQ ID NO 506 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 506 gagttacatg atcccccatg ttgtg 25 <210> SEQ ID NO 507 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 507 gtggtggctg tgtttgcgtc tcccc 25 <210> SEQ ID NO 508 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 508 ggctgtgttt gcgtctctcc caggtatcat ctttaccaga tctca 45 <210> SEQ ID NO 509 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 509 catctttacc agatctcaaa aagaa 25 <210> SEQ ID NO 510 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 510 aatacaatgt gtcaactctt gacag 25 <210> SEQ ID NO 511 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 511 atacaatgtg tcaactcttg acagg 25 <210> SEQ ID NO 512 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 512 tatgctgccg cccagtggga ttttggaaat acaatgtttc aactc 45 <210> SEQ ID NO 513 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 513 atacaatgtg tcaactcttg acagggctct attttatagg cttct 45 <210> SEQ ID NO 514 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 514 ggctcactat gctgccgccc agtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 515 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 515 ggctcactat gctgccgccc 20 <210> SEQ ID NO 516 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 516 gacagggctc tattttatag gcttc 25 <210> SEQ ID NO 517 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 517 tgacagggct ctattttata ggctt 25 <210> SEQ ID NO 518 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 518 ttgacagggc tctattttat aggct 25 <210> SEQ ID NO 519 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 519 aatacaatgt gtcaactct 19 <210> SEQ ID NO 520 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 520 gacagggctc tattttatag gcttct 26 <210> SEQ ID NO 521 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 521 ggctcactat gctgccgccc agggggactt tggaaataca atgtg 45 <210> SEQ ID NO 522 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 522 gggactttgg aaatacaatg tgtca 25 <210> SEQ ID NO 523 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 523 ccggcaaaca aaccaccgc 19 <210> SEQ ID NO 524 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 524 gaaatacaat gtgtcaact 19 <210> SEQ ID NO 525 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 525 tcactataca atgtgtcaag ac 22 <210> SEQ ID NO 526 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 526 agggctctat tttataggct tct 23 <210> SEQ ID NO 527 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 527 aaatacaatg tgtcaactc 19 <210> SEQ ID NO 528 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 528 aacagggctc tattttatag gcttc 25 <210> SEQ ID NO 529 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 529 atacaatgtg tcaactcttg acaaggctct attttatagg cttct 45 <210> SEQ ID NO 530 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 530 cccagtggga ctttggaaaa 20 <210> SEQ ID NO 531 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 531 acagggctct attttatagg cttct 25 <210> SEQ ID NO 532 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 532 atacaatgtg tcaactcct 19 <210> SEQ ID NO 533 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 533 atacaatgtg tcaactctc 19 <210> SEQ ID NO 534 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 534 ggctcactat gctgccgccc agtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 535 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 535 gggtggtggc tgtgtttgcg tctctcccag gaatcatctt tacca 45 <210> SEQ ID NO 536 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 536 ttctgggctc actatgctgc cgccc 25 <210> SEQ ID NO 537 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 537 cccaggaatc atctttacca 20 <210> SEQ ID NO 538 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 538 ggctcactat gctgccgccc 20 <210> SEQ ID NO 539 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 539 cccaggaatc atctttacca gatct 25 <210> SEQ ID NO 540 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 540 tcccaggaat catctttacc agatc 25 <210> SEQ ID NO 541 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 541 tctgggctca ctatgctgcc gccct 25 <210> SEQ ID NO 542 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 542 agagacgcaa acacagccac caccc 25 <210> SEQ ID NO 543 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 543 cacattgtat ttccaaagtc ccact 25 <210> SEQ ID NO 544 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 544 aatgtgtcaa ctcttgacag ggctc 25 <210> SEQ ID NO 545 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 545 acattgtatt tccaaagtcc cactt 25 <210> SEQ ID NO 546 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 546 tagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 547 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 547 ggtggtggct gtgtttgcgt ctctt 25 <210> SEQ ID NO 548 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 548 cttctgggct cactatgctg ccgcc 25 <210> SEQ ID NO 549 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 549 gctgtttcct gtgtgaaatt gttat 25 <210> SEQ ID NO 550 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 550 cccttctggg ctcactatgc tgccg 25 <210> SEQ ID NO 551 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 551 aggaatcatc tttaccagat ctcaa 25 <210> SEQ ID NO 552 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 552 ccaggaatca tctttaccag atctc 25 <210> SEQ ID NO 553 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 553 ttactgtcgt ccatgctgtg tttgc 25 <210> SEQ ID NO 554 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 554 tcccaggaat catctttacc a 21 <210> SEQ ID NO 555 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 555 ttaccagatc tcaaaaagaa ggtct 25 <210> SEQ ID NO 556 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 556 tgtataccgt cgacctctag ctaga 25 <210> SEQ ID NO 557 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 557 cagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 558 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 558 tgggactttg gaaatacaat gtgtc 25 <210> SEQ ID NO 559 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 559 aatacaatgt gtcaactctt gacag 25 <210> SEQ ID NO 560 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 560 tctcccagga atcatcttta ccaga 25 <210> SEQ ID NO 561 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 561 tgggctcact atgctgccgc cctct 25 <210> SEQ ID NO 562 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 562 tactgtcccc ttctgggctc actat 25 <210> SEQ ID NO 563 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 563 tgggtggtgg ctgtgtttgc gtctt 25 <210> SEQ ID NO 564 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 564 acttgggtgg tggctgtgtt tgcgt 25 <210> SEQ ID NO 565 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 565 aagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 566 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 566 gtgggacttt ggaaatacaa tgtgt 25 <210> SEQ ID NO 567 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 567 tcttcatcat cctcctgaca atcga 25 <210> SEQ ID NO 568 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 568 ttataggctt cttctctgga atctt 25 <210> SEQ ID NO 569 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 569 ttcttcgatc agtctaaaaa tggct 25 <210> SEQ ID NO 570 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 570 catctttacc agatctcaaa aagaa 25 <210> SEQ ID NO 571 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 571 aataggcttc ttctctggaa tcttc 25 <210> SEQ ID NO 572 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 572 tttggggtgg tgacaagtgt gatca 25 <210> SEQ ID NO 573 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 573 ttaaaagcca ggacggtcac ctttg 25 <210> SEQ ID NO 574 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 574 tgtttgcgtc tctcccagga atcat 25 <210> SEQ ID NO 575 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 575 agtgtgatca cttgggtggt ggctg 25 <210> SEQ ID NO 576 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 576 tctggaatct tcttcatcat cctcc 25 <210> SEQ ID NO 577 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 577 ggctcactat gctgccgccc tgtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 578 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 578 tttggaaata caatgtgtca actct 25 <210> SEQ ID NO 579 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 579 cccaagaatc atctttacca gatct 25 <210> SEQ ID NO 580 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 580 ccttctgggc tcactatgct gccgc 25 <210> SEQ ID NO 581 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 581 tgggtggtgg ctgtgtttgc gtctc 25 <210> SEQ ID NO 582 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 582 acaatgtgtc aactcttgac agggc 25 <210> SEQ ID NO 583 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 583 gtgggacttt ggaaattcaa tgtgt 25 <210> SEQ ID NO 584 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 584 gcccaggaat catctttacc agatc 25 <210> SEQ ID NO 585 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 585 ggctgtgttt gcgtctctcc aggaa 25 <210> SEQ ID NO 586 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 586 tcttactgtc cccttctgcg tctct 25 <210> SEQ ID NO 587 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 587 ggtggtggct gtgtttgcgt ctctc 25 <210> SEQ ID NO 588 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 588 ccaggaatca tctttactaa atg 23 <210> SEQ ID NO 589 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 589 ccaggcatct tctttaccag ctc 23 <210> SEQ ID NO 590 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 590 aaaatgtgtc aactcttgat tag 23 <210> SEQ ID NO 591 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 591 cccactgggc tgcagaatac aga 23 <210> SEQ ID NO 592 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 592 tctgtgtttg cctctctctc agg 23 <210> SEQ ID NO 593 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 593 cccgggaggg aggcaaaaac agc 23 <210> SEQ ID NO 594 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 594 actgtctccc tgtagaaaac tgg 23 <210> SEQ ID NO 595 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 595 catttagtaa agatgattcc tgg 23 <210> SEQ ID NO 596 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 596 gcattttctg ttctctgaag t 21 <210> SEQ ID NO 597 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 597 tcactaggca tgctgccaga gc 22 <210> SEQ ID NO 598 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 598 gctgtgtttg cttctgtccc agg 23

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 598 <210> SEQ ID NO 1 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 1 tgacatcaat tattatacat cgg 23 <210> SEQ ID NO 2 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 2 cctgcctccg ctctactcac tgg 23 <210> SEQ ID NO 3 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 3 tactcactgg tgttcatctt tgg 23 <210> SEQ ID NO 4 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 4 ggtgttcatc tttggttttg tgg 23 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 5 gtgttcatct ttggttttgt ggg 23 <210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 6 tggttttgtg ggcaacatgc tgg 23 <210> SEQ ID NO 7 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 7 tcatcctgat aaactgcaaa agg 23 <210> SEQ ID NO 8 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 8 tgacatctac ctgctcaacc tgg 23 <210> SEQ ID NO 9 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 9 tccttcttac tgtccccttc tgg 23 <210> SEQ ID NO 10 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 10 ccttcttact gtccccttct ggg 23 <210> SEQ ID NO 11 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 11 ctcactatgc tgccgcccag tgg 23 <210> SEQ ID NO 12 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 12 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 13 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 13 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 14 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 14 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 15 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 15 caatgtgtca actcttgaca ggg 23 <210> SEQ ID NO 16 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 16 ttgacagggc tctattttat agg 23 <210> SEQ ID NO 17 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 17 tattttatag gcttcttctc tgg 23 <210> SEQ ID NO 18 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 18 tcatcctcct gacaatcgat agg 23 <210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 19 cctgacaatc gataggtacc tgg 23 <210> SEQ ID NO 20 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 20 ctgtgtttgc tttaaaagcc agg 23 <210> SEQ ID NO 21 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial

<220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 21 gtttgcttta aaagccagga cgg 23 <210> SEQ ID NO 22 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 22 aaagccagga cggtcacctt tgg 23 <210> SEQ ID NO 23 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 23 aagccaggac ggtcaccttt ggg 23 <210> SEQ ID NO 24 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 24 agccaggacg gtcacctttg ggg 23 <210> SEQ ID NO 25 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 25 caggacggtc acctttgggg tgg 23 <210> SEQ ID NO 26 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 26 tggtgacaag tgtgatcact tgg 23 <210> SEQ ID NO 27 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 27 ggtgacaagt gtgatcactt ggg 23 <210> SEQ ID NO 28 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 28 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 29 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 29 aagtgtgatc acttgggtgg tgg 23 <210> SEQ ID NO 30 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 30 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 31 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 31 tttaccagat ctcaaaaaga agg 23 <210> SEQ ID NO 32 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 32 catacagtca gtatcaattc tgg 23 <210> SEQ ID NO 33 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 33 gacattaaag atagtcatct tgg 23 <210> SEQ ID NO 34 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 34 acattaaaga tagtcatctt ggg 23 <210> SEQ ID NO 35 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 35 cattaaagat agtcatcttg ggg 23 <210> SEQ ID NO 36 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 36 aaagatagtc atcttggggc tgg 23 <210> SEQ ID NO 37 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 37 ggtcctgccg ctgcttgtca tgg 23 <210> SEQ ID NO 38 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 38 tgtcatggtc atctgctact cgg 23 <210> SEQ ID NO 39 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 39 gtcatggtca tctgctactc ggg 23 <210> SEQ ID NO 40 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 40 gaatcctaaa aactctgctt cgg 23 <210> SEQ ID NO 41 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 41 ggtgtcgaaa tgagaagaag agg 23 <210> SEQ ID NO 42 <211> LENGTH: 23 <212> TYPE: DNA

<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 42 gaaatgagaa gaagaggcac agg 23 <210> SEQ ID NO 43 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 43 aaatgagaag aagaggcaca ggg 23 <210> SEQ ID NO 44 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 44 agaagaggca cagggctgtg agg 23 <210> SEQ ID NO 45 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 45 tgattgttta ttttctcttc tgg 23 <210> SEQ ID NO 46 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 46 gattgtttat tttctcttct ggg 23 <210> SEQ ID NO 47 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 47 ccttctcctg aacaccttcc agg 23 <210> SEQ ID NO 48 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 48 aacaccttcc aggaattctt tgg 23 <210> SEQ ID NO 49 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 49 ataattgcag tagctctaac agg 23 <210> SEQ ID NO 50 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 50 ttgcagtagc tctaacaggt tgg 23 <210> SEQ ID NO 51 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 51 caggttggac caagctatgc agg 23 <210> SEQ ID NO 52 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 52 atgcaggtga cagagactct tgg 23 <210> SEQ ID NO 53 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 53 tgcaggtgac agagactctt ggg 23 <210> SEQ ID NO 54 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 54 cccatcatct atgcctttgt cgg 23 <210> SEQ ID NO 55 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 55 ccatcatcta tgcctttgtc ggg 23 <210> SEQ ID NO 56 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 56 catcatctat gcctttgtcg ggg 23 <210> SEQ ID NO 57 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 57 ctgttctatt ttccagcaag agg 23 <210> SEQ ID NO 58 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 58 tcagtttaca cccgatccac tgg 23 <210> SEQ ID NO 59 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 59 cagtttacac ccgatccact ggg 23 <210> SEQ ID NO 60 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 60 agtttacacc cgatccactg ggg 23 <210> SEQ ID NO 61 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 61 cacccgatcc actggggagc agg 23 <210> SEQ ID NO 62 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 62 tggggagcag gaaatatctg tgg 23 <210> SEQ ID NO 63 <211> LENGTH: 23

<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 63 ggggagcagg aaatatctgt ggg 23 <210> SEQ ID NO 64 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 64 taataattga tgtcatagat tgg 23 <210> SEQ ID NO 65 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 65 ttcacattga ttttttggca ggg 23 <210> SEQ ID NO 66 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 66 cttcacattg attttttggc agg 23 <210> SEQ ID NO 67 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 67 tttgcttcac attgattttt tgg 23 <210> SEQ ID NO 68 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 68 gtagagcgga ggcaggaggc ggg 23 <210> SEQ ID NO 69 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 69 agtagagcgg aggcaggagg cgg 23 <210> SEQ ID NO 70 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 70 gtgagtagag cggaggcagg agg 23 <210> SEQ ID NO 71 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 71 ccagtgagta gagcggaggc agg 23 <210> SEQ ID NO 72 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 72 aacaccagtg agtagagcgg agg 23 <210> SEQ ID NO 73 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 73 atgaacacca gtgagtagag cgg 23 <210> SEQ ID NO 74 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 74 ttttgcagtt tatcaggatg agg 23 <210> SEQ ID NO 75 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 75 tcagcctttt gcagtttatc agg 23 <210> SEQ ID NO 76 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR5 gRNA target site sequence <400> SEQUENCE: 76 cagagatggc caggttgagc agg 23 <210> SEQ ID NO 77 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 77 aaaacaggtc agagatggcc agg 23 <210> SEQ ID NO 78 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 78 aaggaaaaac aggtcagaga tgg 23 <210> SEQ ID NO 79 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 79 ggacagtaag aaggaaaaac agg 23 <210> SEQ ID NO 80 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 80 cccagaaggg gacagtaaga agg 23 <210> SEQ ID NO 81 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 81 cagcatagtg agcccagaag ggg 23 <210> SEQ ID NO 82 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 82 gcagcatagt gagcccagaa ggg 23 <210> SEQ ID NO 83 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 83 ggcagcatag tgagcccaga agg 23 <210> SEQ ID NO 84

<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 84 atttccaaag tcccactggg cgg 23 <210> SEQ ID NO 85 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 85 tgtatttcca aagtcccact ggg 23 <210> SEQ ID NO 86 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 86 ttgtatttcc aaagtcccac tgg 23 <210> SEQ ID NO 87 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 87 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 88 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 88 ccaggtacct atcgattgtc agg 23 <210> SEQ ID NO 89 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 89 acacagcatg gacgacagcc agg 23 <210> SEQ ID NO 90 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 90 cttttaaagc aaacacagca tgg 23 <210> SEQ ID NO 91 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 91 caccccaaag gtgaccgtcc tgg 23 <210> SEQ ID NO 92 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 92 cacacttgtc accaccccaa agg 23 <210> SEQ ID NO 93 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 93 atctggtaaa gatgattcct ggg 23 <210> SEQ ID NO 94 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 94 gatctggtaa agatgattcc tgg 23 <210> SEQ ID NO 95 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 95 aagaccttct ttttgagatc tgg 23 <210> SEQ ID NO 96 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 96 gtatggaaaa tgagagctgc agg 23 <210> SEQ ID NO 97 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 97 cagaattgat actgactgta tgg 23 <210> SEQ ID NO 98 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 98 agatgactat ctttaatgtc tgg 23 <210> SEQ ID NO 99 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 99 tgaccatgac aagcagcggc agg 23 <210> SEQ ID NO 100 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 100 cagatgacca tgacaagcag cgg 23 <210> SEQ ID NO 101 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 101 gacaccgaag cagagttttt agg 23 <210> SEQ ID NO 102 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 102 gagaaaataa acaatcatga tgg 23 <210> SEQ ID NO 103 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 103 aggagaagga caatgttgta ggg 23 <210> SEQ ID NO 104 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 104 caggagaagg acaatgttgt agg 23

<210> SEQ ID NO 105 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 105 cctggaaggt gttcaggaga agg 23 <210> SEQ ID NO 106 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 106 agaattcctg gaaggtgttc agg 23 <210> SEQ ID NO 107 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 107 caggccaaag aattcctgga agg 23 <210> SEQ ID NO 108 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 108 tattcaggcc aaagaattcc tgg 23 <210> SEQ ID NO 109 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 109 tagagctact gcaattattc agg 23 <210> SEQ ID NO 110 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 110 tctctgtcac ctgcatagct tgg 23 <210> SEQ ID NO 111 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 111 cgacaaaggc atagatgatg ggg 23 <210> SEQ ID NO 112 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 112 ccgacaaagg catagatgat ggg 23 <210> SEQ ID NO 113 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 113 cccgacaaag gcatagatga tgg 23 <210> SEQ ID NO 114 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 114 tctgaacttc tccccgacaa agg 23 <210> SEQ ID NO 115 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 115 gcttttggaa gaagactaag agg 23 <210> SEQ ID NO 116 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 116 agcgtttggc aatgtgcttt tgg 23 <210> SEQ ID NO 117 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 117 acagcatttg cagaagcgtt tgg 23 <210> SEQ ID NO 118 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 118 ctcgctcggg agcctcttgc tgg 23 <210> SEQ ID NO 119 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 119 taaactgagc ttgctcgctc ggg 23 <210> SEQ ID NO 120 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 120 gtaaactgag cttgctcgct cgg 23 <210> SEQ ID NO 121 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 121 ttcctgctcc ccagtggatc ggg 23 <210> SEQ ID NO 122 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 122 tttcctgctc cccagtggat cgg 23 <210> SEQ ID NO 123 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 123 agatatttcc tgctccccag tgg 23 <210> SEQ ID NO 124 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 124 atcatcttta ccagatctca aaaagaag 28 <210> SEQ ID NO 125 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 125 aactctgctt cggtgtcgaa atgagaag 28

<210> SEQ ID NO 126 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 126 tctgcttcgg tgtcgaaatg agaagaag 28 <210> SEQ ID NO 127 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 127 catcatctat gcctttgtcg gggagaag 28 <210> SEQ ID NO 128 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 128 atgcctttgt cggggagaag ttcagaaa 28 <210> SEQ ID NO 129 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 129 agtgagccca gaaggggaca gtaagaag 28 <210> SEQ ID NO 130 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 130 ctgggcggca gcatagtgag cccagaag 28 <210> SEQ ID NO 131 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 131 gatgatgaag aagattccag agaagaag 28 <210> SEQ ID NO 132 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 132 gaggatgatg aagaagattc cagagaag 28 <210> SEQ ID NO 133 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 133 atcgattgtc aggaggatga tgaagaag 28 <210> SEQ ID NO 134 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 134 caatgttgta gggagcccag aagagaaa 28 <210> SEQ ID NO 135 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 135 aaggacaatg ttgtagggag cccagaag 28 <210> SEQ ID NO 136 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 136 agaattcctg gaaggtgttc aggagaag 28 <210> SEQ ID NO 137 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 137 gcgtttggca atgtgctttt ggaagaag 28 <210> SEQ ID NO 138 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 138 ctggaaaata gaacagcatt tgcagaag 28 <210> SEQ ID NO 139 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CCR5 gRNA target site sequence <400> SEQUENCE: 139 tcgggagcct cttgctggaa aatagaac 28 <210> SEQ ID NO 140 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 140 acttgaagac tcagactcag tgg 23 <210> SEQ ID NO 141 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 141 atgtccacct cgctttcctt tgg 23 <210> SEQ ID NO 142 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 142 cacctcgctt tcctttggag agg 23 <210> SEQ ID NO 143 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 143 ttcctttgga gaggatcttg agg 23 <210> SEQ ID NO 144 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 144 tttggagagg atcttgaggc tgg 23 <210> SEQ ID NO 145 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 145 gctggaccct ctgctcacag agg 23 <210> SEQ ID NO 146 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 146 tcacagaggt gagtgcgtgc tgg 23

<210> SEQ ID NO 147 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 147 cacagaggtg agtgcgtgct ggg 23 <210> SEQ ID NO 148 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 148 ggtgagtgcg tgctgggcag agg 23 <210> SEQ ID NO 149 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 149 ctgggcagag gttttaaatt tgg 23 <210> SEQ ID NO 150 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 150 aggttttaaa tttggctcca agg 23 <210> SEQ ID NO 151 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 151 tggctccaag gaaagcatag agg 23 <210> SEQ ID NO 152 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 152 tccaaggaaa gcatagagga tgg 23 <210> SEQ ID NO 153 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 153 ccaaggaaag catagaggat ggg 23 <210> SEQ ID NO 154 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 154 caaggaaagc atagaggatg ggg 23 <210> SEQ ID NO 155 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 155 ggatggggtt cagacaacag tgg 23 <210> SEQ ID NO 156 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 156 gacaacagtg gaagaaagct agg 23 <210> SEQ ID NO 157 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 157 acaacagtgg aagaaagcta ggg 23 <210> SEQ ID NO 158 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 158 gtggaagaaa gctagggcct cgg 23 <210> SEQ ID NO 159 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 159 gaaagctagg gcctcggtga tgg 23 <210> SEQ ID NO 160 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 160 acccttgctt gatgatttcc agg 23 <210> SEQ ID NO 161 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 161 cttgcttgat gatttccagg agg 23 <210> SEQ ID NO 162 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 162 gatgatttcc aggaggatga agg 23 <210> SEQ ID NO 163 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 163 atgctgatcc caatgtagta agg 23 <210> SEQ ID NO 164 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 164 aatgtagtaa ggcagccaac agg 23 <210> SEQ ID NO 165 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 165 gccaacaggc gaagaaagcc agg 23 <210> SEQ ID NO 166 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 166 aggcgaagaa agccaggatg agg 23 <210> SEQ ID NO 167 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 167

agccaggatg aggatgactg tgg 23 <210> SEQ ID NO 168 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 168 tgaggatgac tgtggtcttg agg 23 <210> SEQ ID NO 169 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 169 gaggatgact gtggtcttga ggg 23 <210> SEQ ID NO 170 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 170 tcttgagggc cttgcgcttc tgg 23 <210> SEQ ID NO 171 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 171 tgagggcctt gcgcttctgg tgg 23 <210> SEQ ID NO 172 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 172 cttgcgcttc tggtggccct tgg 23 <210> SEQ ID NO 173 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 173 gcccttggag tgtgacagct tgg 23 <210> SEQ ID NO 174 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 174 ggagatgata atgcaatagc agg 23 <210> SEQ ID NO 175 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 175 tgataatgca atagcaggac agg 23 <210> SEQ ID NO 176 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 176 caggacagga tgacaatacc agg 23 <210> SEQ ID NO 177 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 177 acaggatgac aataccaggc agg 23 <210> SEQ ID NO 178 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 178 tgacaatacc aggcaggata agg 23 <210> SEQ ID NO 179 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 179 caaccatgat gtgctgaaac tgg 23 <210> SEQ ID NO 180 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 180 cacaaccacc cacaagtcat tgg 23 <210> SEQ ID NO 181 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 181 acaaccaccc acaagtcatt ggg 23 <210> SEQ ID NO 182 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 182 caaccaccca caagtcattg ggg 23 <210> SEQ ID NO 183 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 183 acaagtcatt ggggtagaag cgg 23 <210> SEQ ID NO 184 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 184 gtcatctgcc tcactgacgt tgg 23 <210> SEQ ID NO 185 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 185 gacgttggca aagatgaagt cgg 23 <210> SEQ ID NO 186 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 186 acgttggcaa agatgaagtc ggg 23 <210> SEQ ID NO 187 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 187 tgaagtcggg aatagtcagc agg 23 <210> SEQ ID NO 188 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 188

agtcgggaat agtcagcagg agg 23 <210> SEQ ID NO 189 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 189 gtcgggaata gtcagcagga ggg 23 <210> SEQ ID NO 190 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 190 ggaatagtca gcaggagggc agg 23 <210> SEQ ID NO 191 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 191 gaatagtcag caggagggca ggg 23 <210> SEQ ID NO 192 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 192 ttttcagcca acagcttcct tgg 23 <210> SEQ ID NO 193 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 193 cttccttggc ctctgactgt tgg 23 <210> SEQ ID NO 194 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 194 ccttggcctc tgactgttgg tgg 23 <210> SEQ ID NO 195 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 195 gcctctgact gttggtggcg tgg 23 <210> SEQ ID NO 196 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 196 actgttggtg gcgtggacga tgg 23 <210> SEQ ID NO 197 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 197 tggtggcgtg gacgatggcc agg 23 <210> SEQ ID NO 198 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 198 cgtggacgat ggccaggtag cgg 23 <210> SEQ ID NO 199 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 199 gtagcggtcc agactgatga agg 23 <210> SEQ ID NO 200 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 200 ggtccagact gatgaaggcc agg 23 <210> SEQ ID NO 201 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 201 gactgatgaa ggccaggatg agg 23 <210> SEQ ID NO 202 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 202 ggatgaggac actgctgtag agg 23 <210> SEQ ID NO 203 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 203 ggttgactgt gtagatgaca tgg 23 <210> SEQ ID NO 204 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 204 tgacatggac tgccttgcat agg 23 <210> SEQ ID NO 205 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 205 cccaaagtac cagtttgcca cgg 23 <210> SEQ ID NO 206 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 206 cacggcatca actgcccaga agg 23 <210> SEQ ID NO 207 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 207 acggcatcaa ctgcccagaa ggg 23 <210> SEQ ID NO 208 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 208 agggaagcgt gatgacaaag agg 23 <210> SEQ ID NO 209 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence

<400> SEQUENCE: 209 gaagcgtgat gacaaagagg agg 23 <210> SEQ ID NO 210 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 210 cgtgatgaca aagaggaggt cgg 23 <210> SEQ ID NO 211 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 211 agaggaggtc ggccactgac agg 23 <210> SEQ ID NO 212 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 212 tcatgcttct cagtttcttc tgg 23 <210> SEQ ID NO 213 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 213 tcttctggta acccatgacc agg 23 <210> SEQ ID NO 214 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 214 aatgccagtt aagaagatga tgg 23 <210> SEQ ID NO 215 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 215 taagaagatg atggagtaga tgg 23 <210> SEQ ID NO 216 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 216 gaagatgatg gagtagatgg tgg 23 <210> SEQ ID NO 217 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 217 aagatgatgg agtagatggt ggg 23 <210> SEQ ID NO 218 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 218 tgatggagta gatggtgggc agg 23 <210> SEQ ID NO 219 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 219 tgaaattagc attttcttca cgg 23 <210> SEQ ID NO 220 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 220 agcattttct tcacggaaac agg 23 <210> SEQ ID NO 221 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 221 gcattttctt cacggaaaca ggg 23 <210> SEQ ID NO 222 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 222 acggaaacag ggttccttca tgg 23 <210> SEQ ID NO 223 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 223 gtcccctgag cccatttcct cgg 23 <210> SEQ ID NO 224 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 224 gaagtgtata tctgcaaaag agg 23 <210> SEQ ID NO 225 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 225 tatatctgca aaagaggcaa agg 23 <210> SEQ ID NO 226 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 226 ctgcaaaaga ggcaaaggaa tgg 23 <210> SEQ ID NO 227 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 227 ctctccaaag gaaagcgagg tgg 23 <210> SEQ ID NO 228 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 228 atcctctcca aaggaaagcg agg 23 <210> SEQ ID NO 229 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 229 agcctcaaga tcctctccaa agg 23 <210> SEQ ID NO 230 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence

<400> SEQUENCE: 230 cactcacctc tgtgagcaga ggg 23 <210> SEQ ID NO 231 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 231 gcactcacct ctgtgagcag agg 23 <210> SEQ ID NO 232 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 232 cccatcctct atgctttcct tgg 23 <210> SEQ ID NO 233 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 233 caagtggatt tccatcaccg agg 23 <210> SEQ ID NO 234 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 234 ttgagaacac tgtgcacaag tgg 23 <210> SEQ ID NO 235 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 235 tcctggaaat catcaagcaa ggg 23 <210> SEQ ID NO 236 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 236 ctcctggaaa tcatcaagca agg 23 <210> SEQ ID NO 237 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 237 catcgactcc ttcatcctcc tgg 23 <210> SEQ ID NO 238 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 238 gttggctgcc ttactacatt ggg 23 <210> SEQ ID NO 239 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 239 tgttggctgc cttactacat tgg 23 <210> SEQ ID NO 240 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 240 tcctggcttt cttcgcctgt tgg 23 <210> SEQ ID NO 241 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 241 gaccacagtc atcctcatcc tgg 23 <210> SEQ ID NO 242 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 242 caagggccac cagaagcgca agg 23 <210> SEQ ID NO 243 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 243 tccaagctgt cacactccaa ggg 23 <210> SEQ ID NO 244 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 244 ctccaagctg tcacactcca agg 23 <210> SEQ ID NO 245 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 245 atggttggcc ttatcctgcc tgg 23 <210> SEQ ID NO 246 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 246 cagtttcagc acatcatggt tgg 23 <210> SEQ ID NO 247 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 247 gttccagttt cagcacatca tgg 23 <210> SEQ ID NO 248 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 248 ctaccccaat gacttgtggg tgg 23 <210> SEQ ID NO 249 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 249 cttctacccc aatgacttgt ggg 23 <210> SEQ ID NO 250 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 250 gcttctaccc caatgacttg tgg 23 <210> SEQ ID NO 251 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:

<223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 251 catctttgcc aacgtcagtg agg 23 <210> SEQ ID NO 252 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 252 aggtggtcta tgttggcgtc tgg 23 <210> SEQ ID NO 253 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 253 gctgaaaagg tggtctatgt tgg 23 <210> SEQ ID NO 254 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 254 gaagctgttg gctgaaaagg tgg 23 <210> SEQ ID NO 255 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 255 aaggaagctg ttggctgaaa agg 23 <210> SEQ ID NO 256 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 256 tcagaggcca aggaagctgt tgg 23 <210> SEQ ID NO 257 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 257 ccaccaacag tcagaggcca agg 23 <210> SEQ ID NO 258 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 258 tccacgccac caacagtcag agg 23 <210> SEQ ID NO 259 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 259 catcagtctg gaccgctacc tgg 23 <210> SEQ ID NO 260 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 260 catcctggcc ttcatcagtc tgg 23 <210> SEQ ID NO 261 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 261 ctacagcagt gtcctcatcc tgg 23 <210> SEQ ID NO 262 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 262 ctttgggaac ttcctatgca agg 23 <210> SEQ ID NO 263 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 263 ccgtggcaaa ctggtacttt ggg 23 <210> SEQ ID NO 264 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 264 gccgtggcaa actggtactt tgg 23 <210> SEQ ID NO 265 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 265 cagttgatgc cgtggcaaac tgg 23 <210> SEQ ID NO 266 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 266 cttctgggca gttgatgccg tgg 23 <210> SEQ ID NO 267 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 267 tgtcatcacg cttcccttct ggg 23 <210> SEQ ID NO 268 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 268 ttgtcatcac gcttcccttc tgg 23 <210> SEQ ID NO 269 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 269 gtacaggctg cacctgtcag tgg 23 <210> SEQ ID NO 270 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 270 gaagcatgac ggacaagtac agg 23 <210> SEQ ID NO 271 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 271 gaagaaactg agaagcatga cgg 23 <210> SEQ ID NO 272 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial

<220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 272 ggattggtca tcctggtcat ggg 23 <210> SEQ ID NO 273 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 273 tggattggtc atcctggtca tgg 23 <210> SEQ ID NO 274 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 274 gggcaatgga ttggtcatcc tgg 23 <210> SEQ ID NO 275 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 275 tggcattgtg ggcaatggat tgg 23 <210> SEQ ID NO 276 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 276 ttaactggca ttgtgggcaa tgg 23 <210> SEQ ID NO 277 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 277 atcttcttaa ctggcattgt ggg 23 <210> SEQ ID NO 278 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 278 catcttctta actggcattg tgg 23 <210> SEQ ID NO 279 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 279 tactccatca tcttcttaac tgg 23 <210> SEQ ID NO 280 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 280 aggggactat gactccatga agg 23 <210> SEQ ID NO 281 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 281 caccgaggaa atgggctcag ggg 23 <210> SEQ ID NO 282 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 282 acaccgagga aatgggctca ggg 23 <210> SEQ ID NO 283 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 283 tacaccgagg aaatgggctc agg 23 <210> SEQ ID NO 284 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 284 gataactaca ccgaggaaat ggg 23 <210> SEQ ID NO 285 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 285 agataactac accgaggaaa tgg 23 <210> SEQ ID NO 286 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 286 cacttcagat aactacaccg agg 23 <210> SEQ ID NO 287 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 287 gatggggttc agacaacagt ggaagaaa 28 <210> SEQ ID NO 288 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 288 gtagtaaggc agccaacagg cgaagaaa 28 <210> SEQ ID NO 289 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 289 aaccacccac aagtcattgg ggtagaag 28 <210> SEQ ID NO 290 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 290 gtttgccacg gcatcaactg cccagaag 28 <210> SEQ ID NO 291 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 291 tccattgccc acaatgccag ttaagaag 28 <210> SEQ ID NO 292 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 292 catcaagcaa gggtgtgagt ttgagaac 28 <210> SEQ ID NO 293 <211> LENGTH: 28 <212> TYPE: DNA

<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 293 gctgtcacac tccaagggcc accagaag 28 <210> SEQ ID NO 294 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 294 tcatgggtta ccagaagaaa ctgagaag 28 <210> SEQ ID NO 295 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 295 catcctggtc atgggttacc agaagaaa 28 <210> SEQ ID NO 296 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 296 ggtcatcctg gtcatgggtt accagaag 28 <210> SEQ ID NO 297 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: CXCR4 gRNA target site sequence <400> SEQUENCE: 297 atgaaggaac cctgtttccg tgaagaaa 28 <210> SEQ ID NO 298 <211> LENGTH: 1368 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: CAS protein <400> SEQUENCE: 298 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 299 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 299 taaattcttt gctgacctgc 20 <210> SEQ ID NO 300 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 300 tagatccatt cctatgactg 20 <210> SEQ ID NO 301 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 301 cttcagtctg ataaaatcta 20 <210> SEQ ID NO 302 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 302 tttgatgtaa tccagcaggt 20 <210> SEQ ID NO 303 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: gRNA <400> SEQUENCE: 303 cacagagggc tacaatgtga 20 <210> SEQ ID NO 304 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 304 gtagagcgga ggcaggaggc ggg 23 <210> SEQ ID NO 305 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 305 gtgagtagag cggaggcagg agg 23 <210> SEQ ID NO 306 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 306 ggtgttcatc tttggttttg tgg 23 <210> SEQ ID NO 307 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 307 gtgttcatct ttggttttgt ggg 23 <210> SEQ ID NO 308 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 308 ggacagtaag aaggaaaaac agg 23 <210> SEQ ID NO 309 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 309 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 310 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 310 gcagcatagt gagcccagaa ggg 23 <210> SEQ ID NO 311 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 311 ggcagcatag tgagcccaga agg 23 <210> SEQ ID NO 312 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 312 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 313 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG

<400> SEQUENCE: 313 gtttgcttta aaagccagga cgg 23 <210> SEQ ID NO 314 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 314 ggtgacaagt gtgatcactt ggg 23 <210> SEQ ID NO 315 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 315 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 316 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 316 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 317 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 317 gatctggtaa agatgattcc tgg 23 <210> SEQ ID NO 318 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 318 gtatggaaaa tgagagctgc agg 23 <210> SEQ ID NO 319 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 319 gacattaaag atagtcatct tgg 23 <210> SEQ ID NO 320 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 320 ggtcctgccg ctgcttgtca tgg 23 <210> SEQ ID NO 321 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 321 gtcatggtca tctgctactc ggg 23 <210> SEQ ID NO 322 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 322 gaatcctaaa aactctgctt cgg 23 <210> SEQ ID NO 323 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 323 ggtgtcgaaa tgagaagaag agg 23 <210> SEQ ID NO 324 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 324 gacaccgaag cagagttttt agg 23 <210> SEQ ID NO 325 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 325 gaaatgagaa gaagaggcac agg 23 <210> SEQ ID NO 326 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 326 gattgtttat tttctcttct ggg 23 <210> SEQ ID NO 327 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 327 gagaaaataa acaatcatga tgg 23 <210> SEQ ID NO 328 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 328 gcttttggaa gaagactaag agg 23 <210> SEQ ID NO 329 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 329 gtaaactgag cttgctcgct cgg 23 <210> SEQ ID NO 330 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 330 ggggagcagg aaatatctgt ggg 23 <210> SEQ ID NO 331 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 331 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 332 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 332 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 333 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 333 ggtacctatc gattgtcagg agg 23 <210> SEQ ID NO 334 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:

<223> OTHER INFORMATION: Target site sequence with NGG <400> SEQUENCE: 334 gtcttgctcg agatgtgatg 20 <210> SEQ ID NO 335 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 335 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 336 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 336 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 337 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 337 tgctggctcg gctgccctga ggttgctcaa tcaagcacag gtttcaa 47 <210> SEQ ID NO 338 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 338 tgctggctcg gctgccctga ggttgctcaa tcaagcacag gtttcaa 47 <210> SEQ ID NO 339 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 339 taacagcgat gctgaccccc tgtgcctcta ccacttctat gaccaga 47 <210> SEQ ID NO 340 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 340 cgatgctgac cccctgtgcc tctaccactt ctatgaccag atggacc 47 <210> SEQ ID NO 341 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 341 tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggta 49 <210> SEQ ID NO 342 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 342 tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggta 49 <210> SEQ ID NO 343 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 343 tgggcgacag atgcgaaaga aacgagttcc agtgccaaga cgggaaa 47 <210> SEQ ID NO 344 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 344 gaaacgagtt ccagtgccaa gacgggaaat gcatctccta caagtgg 47 <210> SEQ ID NO 345 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 345 tcagagagga cactgcagtt gtccgtgcta gtagccttcg cttctgga 48 <210> SEQ ID NO 346 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 346 tcagagagga cactgcagtt gtccgtgcta gtagccttcg cttctgga 48 <210> SEQ ID NO 347 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 347 tgatgatctc agaggctcag tatccttgtc ctgggttgga gatagca 47 <210> SEQ ID NO 348 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 348 tgatgatctc agaggctcag tatccttgtc ctgggttgga gatagca 47 <210> SEQ ID NO 349 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 349 tggtaattat gacttttgga cagtccaagc tatatcgaag gtgagatca 49 <210> SEQ ID NO 350 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 350 tggtaattat gacttttgga cagtccaagc tatatcgaag gtgagatca 49 <210> SEQ ID NO 351 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 351 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 352 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 352 tcccttcctg cctcatttca ggtgaataca tcaagacctg gaggcca 47 <210> SEQ ID NO 353 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 353 ctatgccctg cctcatttca ggtgaagatg aaatccctgg agcttgg 47 <210> SEQ ID NO 354 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 354 ctccctgggc tgcaccatag tga 23 <210> SEQ ID NO 355 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial

<220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 355 gaccttgtga agatgattcc tgg 23 <210> SEQ ID NO 356 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 356 gatctgggga agaggattcc agg 23 <210> SEQ ID NO 357 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 357 agattgtgta tactcttgac tag 23 <210> SEQ ID NO 358 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 358 ccaggaatca tctctacgat atc 23 <210> SEQ ID NO 359 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 359 cctggaattc tctttactag atc 23 <210> SEQ ID NO 360 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 360 ccggggagag gcgcggacac agc 23 <210> SEQ ID NO 361 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 361 ccagggagag acggaaccaa aac 23 <210> SEQ ID NO 362 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 362 gatctgatac agatgattca tgg 23 <210> SEQ ID NO 363 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 363 ctgggaatca tctttacaag atg 23 <210> SEQ ID NO 364 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 364 tgaatatcct gtcgcccagt cag 23 <210> SEQ ID NO 365 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 365 ccacccaagt gatcactctt cct 23 <210> SEQ ID NO 366 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 366 aggaagagtg atcacttggg tgg 23 <210> SEQ ID NO 367 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 367 tctgtgtttt tgtctctccc cag 23 <210> SEQ ID NO 368 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 368 aggaagagtg atcacttggg tgg 23 <210> SEQ ID NO 369 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 369 ccacccaagt gatcactctt cct 23 <210> SEQ ID NO 370 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 370 ctaggaatca tcttccccag atg 23 <210> SEQ ID NO 371 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 371 tctgtggaaa agatgattcc aag 23 <210> SEQ ID NO 372 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 372 gttcttgtta agatgattcc tgg 23 <210> SEQ ID NO 373 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 373 ggcatgtttc atcacttggg ggg 23 <210> SEQ ID NO 374 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 374 gggctggtaa agatgtttcc agg 23 <210> SEQ ID NO 375 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 375 gacaagtgtg atcacctggt tgg 23 <210> SEQ ID NO 376 <211> LENGTH: 23 <212> TYPE: DNA

<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 376 gctgtgtttg cttctgtccc agg 23 <210> SEQ ID NO 377 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 377 ccaggaatca tctttactaa atg 23 <210> SEQ ID NO 378 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 378 tcactatgct gccgcccagt ggg 23 <210> SEQ ID NO 379 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 379 gctgccgccc agtgggactt tgg 23 <210> SEQ ID NO 380 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 380 acaatgtgtc aactcttgac agg 23 <210> SEQ ID NO 381 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 381 gacaagtgtg atcacttggg tgg 23 <210> SEQ ID NO 382 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 382 gctgtgtttg cgtctctccc agg 23 <210> SEQ ID NO 383 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 383 ccaggaatca tctttaccag atc 23 <210> SEQ ID NO 384 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 384 acaaggggtc aactctggac aag 23 <210> SEQ ID NO 385 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 385 cccactgggc tgcagcatcc tgg 23 <210> SEQ ID NO 386 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 386 ccagcgcgtg acgcaaacac agc 23 <210> SEQ ID NO 387 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 387 acaatgtgtc accttttaac tgg 23 <210> SEQ ID NO 388 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 388 acaatatttc aacacttgac aag 23 <210> SEQ ID NO 389 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 389 cttggagtca tctttgccac atc 23 <210> SEQ ID NO 390 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 390 gacaagcgtg atcccatggg gag 23 <210> SEQ ID NO 391 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 391 cctcccaagt aatcaccctt ttc 23 <210> SEQ ID NO 392 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 392 cccaagtccc actggcaggt ggc 23 <210> SEQ ID NO 393 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 393 gctatgtgtt aactcttgac aag 23 <210> SEQ ID NO 394 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 394 ccactatggt gcctcccagt cag 23 <210> SEQ ID NO 395 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 395 tcactctgct gctgtccagt ggg 23 <210> SEQ ID NO 396 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 396 actctgtcct gccgcccagt gag 23 <210> SEQ ID NO 397 <211> LENGTH: 23

<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 397 cttggaatca tctctacctg att 23 <210> SEQ ID NO 398 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 398 cccaagtccc actgggtgac atg 23 <210> SEQ ID NO 399 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 399 ctaaggagag aggcacacac agc 23 <210> SEQ ID NO 400 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 400 ccagggaggg acgcaaaccc agc 23 <210> SEQ ID NO 401 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 401 cttctcaagt gatcccactg gtc 23 <210> SEQ ID NO 402 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 402 cctgagagag aagcaaacac aga 23 <210> SEQ ID NO 403 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 403 cttaggaaag acgcaaacat agc 23 <210> SEQ ID NO 404 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 404 acattgatta aactcttgac tag 23 <210> SEQ ID NO 405 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 405 acaaagttta aactcttgag cag 23 <210> SEQ ID NO 406 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 406 cttcccaagt gattacactt tat 23 <210> SEQ ID NO 407 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 407 cccactgggg cgcagcctag tga 23 <210> SEQ ID NO 408 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 408 cttgtcaaga tttgccacat tat 23 <210> SEQ ID NO 409 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 409 ccttggtgag aggcaaacac agc 23 <210> SEQ ID NO 410 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 410 cctactgggc gtcagcactg tgt 23 <210> SEQ ID NO 411 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 411 tcactatgca gcaccccagt ggg 23 <210> SEQ ID NO 412 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 412 ccaggcatct tctttaccag ctc 23 <210> SEQ ID NO 413 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 413 ctcactgggc tgcagcattg ggg 23 <210> SEQ ID NO 414 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 414 ccagggagag acgcagaaac aac 23 <210> SEQ ID NO 415 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 415 tacatgagta atcacttggg gag 23 <210> SEQ ID NO 416 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 416 ctccccaagt gattactcat gta 23 <210> SEQ ID NO 417 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 417 cagaggtgtg atcacttggg cag 23 <210> SEQ ID NO 418

<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 418 cttcccatgt gataacactt gtc 23 <210> SEQ ID NO 419 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 419 cctgagagag aggcaaacac atc 23 <210> SEQ ID NO 420 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 420 ccacgaatcc tctttaccaa atc 23 <210> SEQ ID NO 421 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 421 cctgagggtg cgtctctccc ggg 23 <210> SEQ ID NO 422 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 422 gctgtgtgct cgtctctccc tgg 23 <210> SEQ ID NO 423 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 423 gctgcggccc agcgggacct ggg 23 <210> SEQ ID NO 424 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 424 gctgtacttg ggtctctccc cag 23 <210> SEQ ID NO 425 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 425 gccgtgtttt cctctctccc aag 23 <210> SEQ ID NO 426 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 426 cccactgggc tgcagaatac aga 23 <210> SEQ ID NO 427 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 427 ccccccagga gatcacattt gtc 23 <210> SEQ ID NO 428 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 428 gctgccggac agtgggacct ggg 23 <210> SEQ ID NO 429 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 429 ctgactggac ggcaccttag tga 23 <210> SEQ ID NO 430 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 430 gctgtctttt cctctctccc tgg 23 <210> SEQ ID NO 431 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 431 ccagaaataa tctttaccag ctc 23 <210> SEQ ID NO 432 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 432 gctgtgtttg tgcctccccc agg 23 <210> SEQ ID NO 433 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 433 acaatgtgtt ggctcttgac tag 23 <210> SEQ ID NO 434 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 434 gacaagtctg atcattttgg ggg 23 <210> SEQ ID NO 435 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 435 ccacggagaa acacaaacac agc 23 <210> SEQ ID NO 436 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 436 aaaatgtgtc aactcttgat tag 23 <210> SEQ ID NO 437 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 437 tctgtgtttg cctctctctc agg 23 <210> SEQ ID NO 438 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 438 acaatgtgcc agctctggac tag 23

<210> SEQ ID NO 439 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 439 gacaagtatg agcacttggt aag 23 <210> SEQ ID NO 440 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 440 gactagtgtg ttctcttggg aag 23 <210> SEQ ID NO 441 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 441 gacatgtgta aacacttggg aag 23 <210> SEQ ID NO 442 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 442 ccaaagtccc actgggtgac atc 23 <210> SEQ ID NO 443 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 443 cctgtgagag gctcaaacac agc 23 <210> SEQ ID NO 444 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 444 ccgcagtccc actgtgcggc acc 23 <210> SEQ ID NO 445 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 445 gacaagtgtg ttcacttctg cag 23 <210> SEQ ID NO 446 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 446 gatttgggaa agatcattcc agg 23 <210> SEQ ID NO 447 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 447 gctttggtaa agttgattcc tag 23 <210> SEQ ID NO 448 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 448 aaactgtgat aactcttgac tgg 23 <210> SEQ ID NO 449 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 449 cctaagtccc actggccgaa agt 23 <210> SEQ ID NO 450 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 450 gttctggtca agatgactcc tgg 23 <210> SEQ ID NO 451 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 451 cccaggaccc actgggcagc agc 23 <210> SEQ ID NO 452 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 452 aaaatctgtc aactcttgaa tag 23 <210> SEQ ID NO 453 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 453 ctcatcagga gtggacacat tgt 23 <210> SEQ ID NO 454 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 454 ccagggggac acgcaaacac tgc 23 <210> SEQ ID NO 455 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 455 tcactttgct gccagccagt tgg 23 <210> SEQ ID NO 456 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 456 ctgaagtccc actgggtggg tgt 23 <210> SEQ ID NO 457 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 457 gcaccagccc agtgggactt cag 23 <210> SEQ ID NO 458 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 458 ccagggaaag aggcaagcac agc 23 <210> SEQ ID NO 459 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 459 ccactataca cccgcccagt cag 23

<210> SEQ ID NO 460 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 460 ccattgtgct gccgcccagc cag 23 <210> SEQ ID NO 461 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 461 gatctggaag agatgattcc aag 23 <210> SEQ ID NO 462 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 462 cccgggaggg aggcaaaaac agc 23 <210> SEQ ID NO 463 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 463 gctgagtctg ggtctctccc cag 23 <210> SEQ ID NO 464 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 464 cctggaatgt tctttcccag atc 23 <210> SEQ ID NO 465 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 465 gacaggtgtg agcactttgg gag 23 <210> SEQ ID NO 466 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 466 gacacttgtg atgacttggg tag 23 <210> SEQ ID NO 467 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 467 ctttggagag acgcagacac tgc 23 <210> SEQ ID NO 468 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 468 ctacccaagt gttcatattt gtc 23 <210> SEQ ID NO 469 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 469 ccagaaatca tgtttaccag ctc 23 <210> SEQ ID NO 470 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 470 ctggagtccc actgcgcggc agc 23 <210> SEQ ID NO 471 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 471 cctgggaaag gcgcaaacac agc 23 <210> SEQ ID NO 472 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 472 gatctgataa aggtgagtcc agg 23 <210> SEQ ID NO 473 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 473 gcaagttgct gccgcccagt ggg 23 <210> SEQ ID NO 474 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 474 gcagtgtgtg ggtctctccc agg 23 <210> SEQ ID NO 475 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 475 ccaaagtcgc actggcctgc agc 23 <210> SEQ ID NO 476 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 476 gatctgggag agaagattcc agg 23 <210> SEQ ID NO 477 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 477 ccagaaaaga gttgacacat agt 23 <210> SEQ ID NO 478 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 478 cctgccaagg gttgacacat ggt 23 <210> SEQ ID NO 479 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 479 gacaagtgtc ataactttgg aag 23 <210> SEQ ID NO 480 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 480 ataatgtgtc aaccctggac cag 23

<210> SEQ ID NO 481 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 481 ccaaagaccc actggacggc agc 23 <210> SEQ ID NO 482 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 482 ccaactggcc ggcagcctgg tga 23 <210> SEQ ID NO 483 <400> SEQUENCE: 483 000 <210> SEQ ID NO 484 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 484 tatgctgccg cccagtggga ctttggaaat acaatgtgtc aactc 45 <210> SEQ ID NO 485 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 485 ggctgtgttt gcgtctctcc caggaatcat ctttaccaga tctca 45 <210> SEQ ID NO 486 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 486 tatgctgccg cccagtggga 20 <210> SEQ ID NO 487 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 487 atcatcttta ccagatctca aaaag 25 <210> SEQ ID NO 488 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 488 gagttgacac attgtatttc caaag 25 <210> SEQ ID NO 489 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 489 atcatcttta ccagatctca 20 <210> SEQ ID NO 490 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 490 tcctgggaga gacgcaaaca cagcc 25 <210> SEQ ID NO 491 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 491 tggctgtgtt tgcgtctctc ccagg 25 <210> SEQ ID NO 492 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 492 catcatcttt accagatctc aaaaa 25 <210> SEQ ID NO 493 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 493 tttggaaata caatgtgtca actct 25 <210> SEQ ID NO 494 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 494 tcatctttac cagatctcaa aaaga 25 <210> SEQ ID NO 495 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 495 gctgtgtttg cgtctctccc aggaa 25 <210> SEQ ID NO 496 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 496 actttggaaa tacaatgtgt caact 25 <210> SEQ ID NO 497 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 497 tggaaataca atgtgtcaac tcttg 25 <210> SEQ ID NO 498 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 498 gtggctgtgt ttgcgtctct cccag 25 <210> SEQ ID NO 499 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 499 tcctttggaa atacaatgtg tcaac 25 <210> SEQ ID NO 500 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 500 atctttacca gatctcaaaa agaag 25 <210> SEQ ID NO 501 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 501 tggtggctgt gtttgcgtct ctccc 25 <210> SEQ ID NO 502 <211> LENGTH: 25

<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 502 aatcatcttt accagatctc aaaaa 25 <210> SEQ ID NO 503 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 503 tcactatgct gccgcccagt gggaa 25 <210> SEQ ID NO 504 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 504 aatgtgtcaa ctcttgacag ggctc 25 <210> SEQ ID NO 505 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 505 ttggaaatac aatgtgtcaa ctctt 25 <210> SEQ ID NO 506 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 506 gagttacatg atcccccatg ttgtg 25 <210> SEQ ID NO 507 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 507 gtggtggctg tgtttgcgtc tcccc 25 <210> SEQ ID NO 508 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 508 ggctgtgttt gcgtctctcc caggtatcat ctttaccaga tctca 45 <210> SEQ ID NO 509 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 509 catctttacc agatctcaaa aagaa 25 <210> SEQ ID NO 510 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 510 aatacaatgt gtcaactctt gacag 25 <210> SEQ ID NO 511 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 511 atacaatgtg tcaactcttg acagg 25 <210> SEQ ID NO 512 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 512 tatgctgccg cccagtggga ttttggaaat acaatgtttc aactc 45 <210> SEQ ID NO 513 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 513 atacaatgtg tcaactcttg acagggctct attttatagg cttct 45 <210> SEQ ID NO 514 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 514 ggctcactat gctgccgccc agtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 515 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 515 ggctcactat gctgccgccc 20 <210> SEQ ID NO 516 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 516 gacagggctc tattttatag gcttc 25 <210> SEQ ID NO 517 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 517 tgacagggct ctattttata ggctt 25 <210> SEQ ID NO 518 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 518 ttgacagggc tctattttat aggct 25 <210> SEQ ID NO 519 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 519 aatacaatgt gtcaactct 19 <210> SEQ ID NO 520 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 520 gacagggctc tattttatag gcttct 26 <210> SEQ ID NO 521 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 521 ggctcactat gctgccgccc agggggactt tggaaataca atgtg 45 <210> SEQ ID NO 522 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 522 gggactttgg aaatacaatg tgtca 25 <210> SEQ ID NO 523

<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 523 ccggcaaaca aaccaccgc 19 <210> SEQ ID NO 524 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 524 gaaatacaat gtgtcaact 19 <210> SEQ ID NO 525 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 525 tcactataca atgtgtcaag ac 22 <210> SEQ ID NO 526 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 526 agggctctat tttataggct tct 23 <210> SEQ ID NO 527 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 527 aaatacaatg tgtcaactc 19 <210> SEQ ID NO 528 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 528 aacagggctc tattttatag gcttc 25 <210> SEQ ID NO 529 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 529 atacaatgtg tcaactcttg acaaggctct attttatagg cttct 45 <210> SEQ ID NO 530 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 530 cccagtggga ctttggaaaa 20 <210> SEQ ID NO 531 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 531 acagggctct attttatagg cttct 25 <210> SEQ ID NO 532 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 532 atacaatgtg tcaactcct 19 <210> SEQ ID NO 533 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 533 atacaatgtg tcaactctc 19 <210> SEQ ID NO 534 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 534 ggctcactat gctgccgccc agtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 535 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 535 gggtggtggc tgtgtttgcg tctctcccag gaatcatctt tacca 45 <210> SEQ ID NO 536 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 536 ttctgggctc actatgctgc cgccc 25 <210> SEQ ID NO 537 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 537 cccaggaatc atctttacca 20 <210> SEQ ID NO 538 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 538 ggctcactat gctgccgccc 20 <210> SEQ ID NO 539 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 539 cccaggaatc atctttacca gatct 25 <210> SEQ ID NO 540 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 540 tcccaggaat catctttacc agatc 25 <210> SEQ ID NO 541 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 541 tctgggctca ctatgctgcc gccct 25 <210> SEQ ID NO 542 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 542 agagacgcaa acacagccac caccc 25 <210> SEQ ID NO 543 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 543 cacattgtat ttccaaagtc ccact 25

<210> SEQ ID NO 544 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 544 aatgtgtcaa ctcttgacag ggctc 25 <210> SEQ ID NO 545 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 545 acattgtatt tccaaagtcc cactt 25 <210> SEQ ID NO 546 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 546 tagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 547 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 547 ggtggtggct gtgtttgcgt ctctt 25 <210> SEQ ID NO 548 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 548 cttctgggct cactatgctg ccgcc 25 <210> SEQ ID NO 549 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 549 gctgtttcct gtgtgaaatt gttat 25 <210> SEQ ID NO 550 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 550 cccttctggg ctcactatgc tgccg 25 <210> SEQ ID NO 551 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 551 aggaatcatc tttaccagat ctcaa 25 <210> SEQ ID NO 552 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 552 ccaggaatca tctttaccag atctc 25 <210> SEQ ID NO 553 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 553 ttactgtcgt ccatgctgtg tttgc 25 <210> SEQ ID NO 554 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 554 tcccaggaat catctttacc a 21 <210> SEQ ID NO 555 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 555 ttaccagatc tcaaaaagaa ggtct 25 <210> SEQ ID NO 556 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 556 tgtataccgt cgacctctag ctaga 25 <210> SEQ ID NO 557 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 557 cagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 558 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 558 tgggactttg gaaatacaat gtgtc 25 <210> SEQ ID NO 559 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 559 aatacaatgt gtcaactctt gacag 25 <210> SEQ ID NO 560 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 560 tctcccagga atcatcttta ccaga 25 <210> SEQ ID NO 561 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 561 tgggctcact atgctgccgc cctct 25 <210> SEQ ID NO 562 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 562 tactgtcccc ttctgggctc actat 25 <210> SEQ ID NO 563 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 563 tgggtggtgg ctgtgtttgc gtctt 25 <210> SEQ ID NO 564 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 564 acttgggtgg tggctgtgtt tgcgt 25

<210> SEQ ID NO 565 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 565 aagtgggact ttggaaatac aatgt 25 <210> SEQ ID NO 566 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 566 gtgggacttt ggaaatacaa tgtgt 25 <210> SEQ ID NO 567 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 567 tcttcatcat cctcctgaca atcga 25 <210> SEQ ID NO 568 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 568 ttataggctt cttctctgga atctt 25 <210> SEQ ID NO 569 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 569 ttcttcgatc agtctaaaaa tggct 25 <210> SEQ ID NO 570 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 570 catctttacc agatctcaaa aagaa 25 <210> SEQ ID NO 571 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 571 aataggcttc ttctctggaa tcttc 25 <210> SEQ ID NO 572 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 572 tttggggtgg tgacaagtgt gatca 25 <210> SEQ ID NO 573 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 573 ttaaaagcca ggacggtcac ctttg 25 <210> SEQ ID NO 574 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 574 tgtttgcgtc tctcccagga atcat 25 <210> SEQ ID NO 575 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 575 agtgtgatca cttgggtggt ggctg 25 <210> SEQ ID NO 576 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 576 tctggaatct tcttcatcat cctcc 25 <210> SEQ ID NO 577 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 577 ggctcactat gctgccgccc tgtgggactt tggaaataca atgtg 45 <210> SEQ ID NO 578 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 578 tttggaaata caatgtgtca actct 25 <210> SEQ ID NO 579 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 579 cccaagaatc atctttacca gatct 25 <210> SEQ ID NO 580 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 580 ccttctgggc tcactatgct gccgc 25 <210> SEQ ID NO 581 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 581 tgggtggtgg ctgtgtttgc gtctc 25 <210> SEQ ID NO 582 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 582 acaatgtgtc aactcttgac agggc 25 <210> SEQ ID NO 583 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 583 gtgggacttt ggaaattcaa tgtgt 25 <210> SEQ ID NO 584 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 584 gcccaggaat catctttacc agatc 25 <210> SEQ ID NO 585 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 585 ggctgtgttt gcgtctctcc aggaa 25

<210> SEQ ID NO 586 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 586 tcttactgtc cccttctgcg tctct 25 <210> SEQ ID NO 587 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 587 ggtggtggct gtgtttgcgt ctctc 25 <210> SEQ ID NO 588 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 588 ccaggaatca tctttactaa atg 23 <210> SEQ ID NO 589 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 589 ccaggcatct tctttaccag ctc 23 <210> SEQ ID NO 590 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 590 aaaatgtgtc aactcttgat tag 23 <210> SEQ ID NO 591 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 591 cccactgggc tgcagaatac aga 23 <210> SEQ ID NO 592 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 592 tctgtgtttg cctctctctc agg 23 <210> SEQ ID NO 593 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 593 cccgggaggg aggcaaaaac agc 23 <210> SEQ ID NO 594 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 594 actgtctccc tgtagaaaac tgg 23 <210> SEQ ID NO 595 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 595 catttagtaa agatgattcc tgg 23 <210> SEQ ID NO 596 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 596 gcattttctg ttctctgaag t 21 <210> SEQ ID NO 597 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 597 tcactaggca tgctgccaga gc 22 <210> SEQ ID NO 598 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: target sequence <400> SEQUENCE: 598 gctgtgtttg cttctgtccc agg 23

Claims

1. A method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

2. A method for treating or preventing a disorder associated with expression of a polynucleotide sequence in a subject, the method comprising (a) altering a target polynucleotide sequence in a cell ex vivo by contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequence.

3. A method for simultaneously altering multiple target polynucleotide sequences in a cell comprising contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%.

4. A method for treating or preventing a disorder associated with expression of polynucleotide sequences in a subject, the method comprising (a) altering target polynucleotide sequences in a cell ex vivo by contacting the polynucleotide sequences with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and multiple ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to target motifs of the target polynucleotide sequences, wherein the target polynucleotide sequences are cleaved, and wherein the efficiency of alteration of cells that express Cas protein is from about 50% to about 80%, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the polynucleotide sequences.

5. A method according to claim 1, wherein the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof.

6. The method according to claim 5, wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

7. (canceled)

8. A method according to claim 1, wherein the Cas protein is Cas9 protein from any bacterial species or functional portion thereof.

9. The method according to claim 8, wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

10-12. (canceled)

13. A method according to claim 1, wherein the target motif is a 20-nucleotide DNA sequence.

14. (canceled)

15. A method according to claim 1, wherein the target motif is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein.

16. (canceled)

17. A method according to claim 1, wherein the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein.

18. (canceled)

19. A method according to claim 1, wherein the target motif is G(N).sub.19NGG.

20. (canceled)

21. A method according to claim 1, wherein the target motif is (N).sub.20NGG.

22-43. (canceled)

44. A method according to claim 1, wherein the cell is selected from the group consisting of a peripheral blood cell, a stem cell, a pluripotent cell, a hematopoietic stem cell, a CD34+ cell, a CD34+ mobilized peripheral blood cell, a CD34+ cord blood cell, a CD34+ bone marrow cell, a CD34+CD38-Lineage-CD90.sup.+CD45RA.sup.- cell, and a hepatocyte.

45-52. (canceled)

53. A method according to claim 1, wherein the target polynucleotide sequence is CCR5.

54-55. (canceled)

56. A method according to claim 1, wherein the target polynucleotide sequence is CXCR4.

57-67. (canceled)

68. A method according to claim 2, wherein the disorder is selected from the group consisting of a genetic disorder, a monogenic disorder, human HIV infection, and AIDS.

69-86. (canceled)

87. A method according to claim 1, wherein the Cas protein is encoded by a modified nucleic acid.

88. (canceled)

89. A method according to claim 1, wherein at least one of the ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

90. A method according to claim 1, wherein any of the Cas protein or the ribonucleic acids are expressed from a plasmid.

91-122. (canceled)

123. A method according to claim 1, wherein the cell comprises a primary cell.

124-126. (canceled)

127. A method according to claim 1, wherein the target polynucleotide sequence is B2M.

128-133. (canceled)

134. A method according to claim 1, wherein the one to two ribonucleic acids comprise two guide ribonucleic acid sequences.

135. A method according to claim 134, wherein the target polynucleotide sequence comprises CCR5.

136-144. (canceled)

145. A method according to claim 134, wherein the target polynucleotide sequence comprises CXCR4.

146-149. (canceled)

150. A method according to claim 134, wherein the target polynucleotide sequence comprises B2M.

151-361. (canceled)