U.S. patent application number 16/434689 was filed with the patent office on 2020-03-05 for rna-targeting fusion protein compositions and methods for use.
The applicant listed for this patent is Locana, Inc.. Invention is credited to Ranjan BATRA, David A. NELLES, Eugene YEO.
Application Number | 20200071718 16/434689 |
Document ID | / |
Family ID | 68769584 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200071718 |
Kind Code |
A1 |
NELLES; David A. ; et
al. |
March 5, 2020 |
RNA-TARGETING FUSION PROTEIN COMPOSITIONS AND METHODS FOR USE
Abstract
Disclosed are compositions comprising: (a) a sequence comprising
a guide RNA (gRNA) that specifically binds a target sequence within
an RNA molecule and (b) a sequence encoding a fusion protein, the
sequence comprising a sequence encoding a first RNA-binding
polypeptide and a sequence encoding a second RNA-binding
polypeptide, wherein neither the first RNA-binding polypeptide nor
the second RNA-binding polypeptide comprises a significant
DNA-nuclease activity, wherein the first RNA-binding polypeptide
and the second RNA-binding polypeptide are not identical, and
wherein the second RNA-binding polypeptide comprises an
RNA-nuclease activity. Methods of making and methods of using
compositions of the disclosure are also provided. For example,
compositions of the disclosure may be used in the treatment of a
disease or disorder in a subject. Exemplary disease or disorders of
the disclosure include genetic and epigenetic diseases or
disorders.
Inventors: |
NELLES; David A.; (San
Diego, CA) ; BATRA; Ranjan; (San Diego, CA) ;
YEO; Eugene; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Locana, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
68769584 |
Appl. No.: |
16/434689 |
Filed: |
June 7, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62682271 |
Jun 8, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/11 20130101;
C12N 15/85 20130101; C12N 15/907 20130101; C12N 15/1131 20130101;
C12N 15/67 20130101; C12N 9/22 20130101; C12N 15/63 20130101; C12N
2310/20 20170501; C07K 2319/85 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C12N 15/11 20060101 C12N015/11; C12N 9/22 20060101
C12N009/22; C12N 15/90 20060101 C12N015/90 |
Claims
1. A composition comprising a nucleic acid sequence encoding an
RNA-guided target RNA-binding fusion protein comprising (a) a first
RNA-binding polypeptide or portion thereof, and (b) a second
RNA-binding polypeptide, wherein the first RNA-binding polypeptide
binds a target RNA when guided by a gRNA sequence, and wherein the
second RNA-binding polypeptide comprises RNA-nuclease activity.
2. The composition of claim 1, wherein the first RNA-binding
polypeptide or portion thereof is a CRISPR/Cas polypeptide or
portion thereof.
3. The composition of claim 2, wherein the CRISPR/Cas polypeptide
or portion thereof is selected from the group consisting of Cas9,
Cpf1, Cas13a, Cas13b, Cas13c and CasRX/Cas13d, wherein the
CRISPR/Cas polypeptide has native, reduced or null activity.
4. The composition of claim 1, wherein the second RNA-binding
polypeptide binds RNA in a manner in which it associates with
RNA.
5. The composition of claim 4, wherein the second RNA-binding
polypeptide associates with RNA in a manner in which it cleaves
RNA.
6. The composition of claim 1, wherein the nucleic acid sequence
comprises a promoter.
7. The composition of claim 6, wherein the promoter is a
constitutive promoter or a tissue-specific promoter.
8. The composition of claim 1, wherein the nucleic acid sequence
further comprises a gRNA sequence, wherein the gRNA sequence
comprises a spacer sequence that specifically binds a target
sequence within an RNA molecule and a scaffold sequence that
specifically binds to the first RNA-binding polypeptide.
9. The composition of claim 8, wherein the spacer sequence
comprises a sequence comprising at least 1, 2, 3, 4, 5, 6, or 7
repeats of a sequence selected from the group consisting of: CUG
(SEQ ID NO: 18), CCUG (SEQ ID NO: 19), CAG (SEQ ID NO: 80), GGGGCC
(SEQ ID NO: 81), and a combination thereof.
10. The composition of claim 8, wherein the nucleic acid sequence
comprises a promoter which drives expression of the gRNA
sequence.
11. The composition of claim 9, wherein the promoter is a
polymerase III promoter.
12. The composition of claim 10, wherein the polymerase III
promoter is a U6 promoter.
13. The composition of claim 1, wherein the promoter is a tRNA
promoter.
14. The composition of claim 1, wherein the fusion protein
comprises an NLS, NES or tag.
15. A vector comprising the composition of claim 1.
16. The vector of claim 15, wherein the vector is selected from the
group consisting of: adeno-associated virus, retrovirus,
lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex,
polymersome, polyplex, and dendrimer.
17. A cell comprising the vector of claim 15.
18. The composition of claim 1, wherein the second RNA-binding
polypeptide is selected from the group consisting of: RNAse1,
RNAse4, RNAse6, RNAse7, RNAse8, RNAse2, RNAse6PL, RNAseL, RNAseT2,
RNAse11, RNAseT2-like, NOB 1, ENDOV, ENDOG, ENDOD1, hFEN1, hSLFN14,
hLACTB2, APEX2, ANG, HRSP12, ZC3H12A, RIDA, PDL6, NTHL, KIAA0391,
APEX1, AGO2, EXOG, ZC3H12D, ERN2, PELO, YBEY, CPSF4L, hCG_2002731,
ERCC1, RAC1, RAA1, RAB1, DNA2, FLJ35220, FLJ13173, ERCC4,
Rnase1(K41R), Rnase1(K41R, D121E), Rnase1(K41R, D121E, H119N),
Rnase1(H119N), Rnase1(R39D, N67D, N88A, G89D, R91D, H119N),
Rnase1(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E),
Rnase1(R39D, N67D, N88A, G89D, R91D), TENM1, TENM2, RNAseK, TALEN,
and ZNF638.
19. A composition comprising: (a) a guide RNA (gRNA) sequence
comprising a spacer sequence that specifically binds a target
sequence within an RNA molecule and a scaffold sequence that
specifically binds to the first RNA-binding polypeptide; (b) a
nucleic acid sequence encoding a fusion protein, the fusion protein
comprising a first RNA-binding polypeptide and a sequence encoding
a second RNA-binding polypeptide, wherein neither the first
RNA-binding polypeptide nor the second RNA-binding polypeptide
comprises a significant DNA-nuclease activity, wherein the first
RNA-binding polypeptide and the second RNA-binding polypeptide are
not identical, and wherein the second RNA-binding polypeptide
comprises an RNA-nuclease activity.
20. A method for modifying the level of expression of a target RNA
molecule or a protein encoded by the RNA molecule, the method
comprising contacting the composition of claim 19 and the RNA
molecule under conditions suitable for binding of the fusion
protein or a portion thereof to the RNA molecule.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent Application
No. 62/682,271, filed Jun. 8, 2018, the contents of which are
herein incorporated by reference in their entirety. The contents of
U.S. Patent Application No. 62/682,276, filed Jun. 8, 2018, are
herein incorporated by reference in their entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure is directed to molecular biology, and more,
specifically, to compositions and methods for modifying expression
and activity of RNA molecules.
INCORPORATION OF SEQUENCE LISTING
[0003] The contents of the text file named
"LOCN_002_001US_SeqList_ST25", which was created on Sep. 4, 2019
and is 773 KB in size, are hereby incorporated by reference in
their entirety.
BACKGROUND
[0004] There has been a long-felt but unmet need in the art for a
method of specifically binding target RNA molecules for
modification of expression or activity of the RNA molecule or a
protein encoded by the RNA molecule. The disclosure provides
compositions and methods for specifically targeting RNA molecules
in sequence-specific manner that further precludes modification of
DNA sequences.
SUMMARY
[0005] The disclosure provides a composition comprising (a) a
sequence comprising a guide RNA (gRNA) that specifically binds a
target sequence within an RNA molecule and (b) a sequence encoding
a fusion protein, the sequence comprising a sequence encoding a
first RNA-binding polypeptide and a sequence encoding a second
RNA-binding polypeptide, wherein neither the first RNA-binding
polypeptide nor the second RNA-binding polypeptide comprises a
significant DNA-nuclease activity, wherein the first RNA-binding
polypeptide and the second RNA-binding polypeptide are not
identical, and wherein the second RNA-binding polypeptide comprises
an RNA-nuclease activity wherein the first RNA-binding polypeptide
and the second RNA-binding polypeptide are not identical, and
wherein the second RNA-binding polypeptide comprises an
RNA-nuclease activity.
[0006] The disclosure also provides a composition comprising a
sequence encoding an RNA-guided target RNA-binding fusion protein
comprising (a) a sequence encoding a first RNA-binding polypeptide
or portion thereof; and (b) a sequence encoding a second
RNA-binding polypeptide, wherein the first RNA-binding polypeptide
binds a target RNA guided by a gRNA sequence, and wherein the
second RNA-binding polypeptide comprises RNA-nuclease activity.
[0007] The disclosure additionally provides a composition
comprising a sequence encoding a target RNA-binding fusion protein
comprising (a) a sequence encoding a first RNA-binding polypeptide
or portion thereof, and (b) a sequence encoding a second
RNA-binding polypeptide, wherein the first RNA-binding polypeptide
binds a target RNA without a gRNA sequence, and wherein the second
RNA-binding polypeptide comprises RNA-nuclease activity.
[0008] In some embodiments of the compositions of the disclosure,
the target sequence comprises at least one repeated sequence.
[0009] In some embodiments of the compositions of the disclosure,
the sequence comprising the gRNA further comprises a sequence
encoding a promoter capable of expressing the gRNA in a eukaryotic
cell.
[0010] In some embodiments of the compositions of the disclosure,
the eukaryotic cell is an animal cell. In some embodiments, the
animal cell is a mammalian cell. In some embodiments, the animal
cell is a human cell.
[0011] In some embodiments of the compositions of the disclosure,
the promoter is a constitutively active promoter. In some
embodiments, the promoter sequence is isolated or derived from a
promoter capable of driving expression of an RNA polymerase. In
some embodiments, the promoter sequence is isolated or derived from
a U6 promoter. In some embodiments, the promoter is a sequence
isolated or derived from a promoter capable of driving expression
of a transfer RNA (tRNA). In some embodiments, the promoter is
isolated or derived from an alanine tRNA promoter, an arginine tRNA
promoter, an asparagine tRNA promoter, an aspartic acid tRNA
promoter, a cysteine tRNA promoter, a glutamine tRNA promoter, a
glutamic acid tRNA promoter, a glycine tRNA promoter, a histidine
tRNA promoter, an isoleucine tRNA promoter, a leucine tRNA
promoter, a lysine tRNA promoter, a methionine tRNA promoter, a
phenylalanine tRNA promoter, a proline tRNA promoter, a serine tRNA
promoter, a threonine tRNA promoter, a tryptophan tRNA promoter, a
tyrosine tRNA promoter, or a valine tRNA promoter. In some
embodiments, the promoter is isolated or derived from a valine tRNA
promoter.
[0012] In some embodiments of the compositions of the disclosure,
the sequence comprising the gRNA further comprises a spacer
sequence that specifically binds to the target RNA sequence. In
some embodiments, the spacer sequence has at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in
between of complementarity to the target RNA sequence. In some
embodiments, the spacer sequence has 100% complementarity to the
target RNA sequence. In some embodiments, the spacer sequence
comprises or consists of 20 nucleotides. In some embodiments, the
spacer sequence comprises or consists of 21 nucleotides. In some
embodiments, the spacer sequence comprises or consists of the
sequence
TABLE-US-00001 (SEQ ID NO: 1) UGGAGCGAGCAUCCCCCAAA, (SEQ ID NO: 2)
GUUUGGGGGAUGCUCGCUCCA, (SEQ ID NO: 3) CCCUCACUGCUGGGGAGUCC, (SEQ ID
NO: 4) GGACUCCCCAGCAGUGAGGG, (SEQ ID NO: 5) GCAACUGGAUCAAUUUGCUG,
(SEQ ID NO: 6) GCAGCAAAUUGAUCCAGUUGC, (SEQ ID NO: 7)
GCAUUCUUAUCUGGUCAGUGC, (SEQ ID NO: 8) GCACUGACCAGAUAAGAAUG, (SEQ ID
NO: 9) GAGCAGCAGCAGCAGCAGCAG, (SEQ ID NO: 10)
GCAGGCAGGCAGGCAGGCAGG, (SEQ ID NO: 11) GCCCCGGCCCCGGCCCCGGC, or
(SEQ ID NO: 12) GCTGCTGCTGCTGCTGCTGC, (SEQ ID NO: 74)
GGGGCCGGGGCCGGGGCCGG, (SEQ ID NO: 75) GGGCCGGGGCCGGGGCCGGG, (SEQ ID
NO: 76) GGCCGGGGCCGGGGCCGGGG, (SEQ ID NO: 77) GCCGGGGCCGGGGCCGGGGC,
(SEQ ID NO: 78) CCGGGGCCGGGGCCGGGGCC, or (SEQ ID NO: 79)
CGGGGCCGGGGCCGGGGCCG.
[0013] In some embodiments of the compositions of the disclosure,
the sequence comprising the gRNA further comprises a spacer
sequence that specifically binds to the target RNA sequence. In
some embodiments, the spacer sequence has at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in
between of complementarity to the target RNA sequence.
[0014] In some embodiments, the spacer sequence has 100%
complementarity to the target RNA sequence. In some embodiments,
the spacer sequence comprises or consists of 20 nucleotides. In
some embodiments, the spacer sequence comprises or consists of 21
nucleotides. In some embodiments, the spacer sequence comprises or
consists of the sequence
TABLE-US-00002 (SEQ ID NO: 14) GUGAUAAGUGGAAUGCCAUG, (SEQ ID NO:
15) CUGGUGAACUUCCGAUAGUG, or (SEQ ID NO: 16)
GAGATATAGCCTGGTGGTTC.
[0015] In some embodiments of the compositions of the disclosure,
the sequence comprising the gRNA further comprises a spacer
sequence that specifically binds to the target RNA sequence. In
some embodiments, the spacer sequence has at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in
between of complementarity to the target RNA sequence. In some
embodiments, the spacer sequence has 100% complementarity to the
target RNA sequence. In some embodiments, the spacer sequence
comprises or consists of 20 nucleotides. In some embodiments, the
spacer sequence comprises or consists of 21 nucleotides. In some
embodiments, the spacer sequence comprises or consists of a
sequence comprising at least 1, 2, 3, 4, 5, 6, or 7 repeats of the
sequence CUG (SEQ ID NO: 18), CCUG (SEQ ID NO: 19), CAG (SEQ ID NO:
80), GGGGCC (SEQ ID NO: 81) or any combination thereof.
[0016] In some embodiments of the compositions of the disclosure,
the sequence comprising the gRNA further comprises a scaffold
sequence that specifically binds to the first RNA binding protein.
In some embodiments, the scaffold sequence comprises a stem-loop
structure. In some embodiments, the scaffold sequence comprises or
consists of 90 nucleotides. In some embodiments, the scaffold
sequence comprises or consists of 93 nucleotides. In some
embodiments, the scaffold sequence comprises or consists of the
sequence
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUU
[0017] AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 13). In
some embodiments, the scaffold sequence comprises or consists of
the sequence
GGACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG
[0018] CACCGAGUCGGUGCUUUUU (SEQ ID NO: 17). In some embodiments,
the scaffold sequence comprises or consists of the sequence
TABLE-US-00003 (SEQ ID NO: 82)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU or (SEQ ID NO: 83)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.
[0019] In some embodiments of the compositions of the disclosure,
the gRNA does not bind or does not selectively bind to a second
sequence within the RNA molecule.
[0020] In some embodiments of the compositions of the disclosure,
an RNA genome or an RNA transcriptome comprises the RNA
molecule.
[0021] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises a CRISPR-Cas protein. In
some embodiments, the CRISPR-Cas protein is a Type II CRISPR-Cas
protein. In some embodiments, the first RNA binding protein
comprises a Cas9 polypeptide or an RNA-binding portion thereof. In
some embodiments, the CRISPR-Cas protein comprises a native RNA
nuclease activity. In some embodiments, the native RNA nuclease
activity is reduced or inhibited. In some embodiments, the native
RNA nuclease activity is increased or induced. In some embodiments,
the CRISPR-Cas protein comprises a native DNA nuclease activity and
the native DNA nuclease activity is inhibited. In some embodiments,
the CRISPR-Cas protein comprises a mutation. In some embodiments, a
nuclease domain of the CRISPR-Cas protein comprises the mutation.
In some embodiments, the mutation occurs in a nucleic acid encoding
the CRISPR-Cas protein. In some embodiments, the mutation occurs in
an amino acid encoding the CRISPR-Cas protein. In some embodiments,
the mutation comprises a substitution, an insertion, a deletion, a
frameshift, an inversion, or a transposition. In some embodiments,
the mutation comprises a deletion of a nuclease domain, a binding
site within the nuclease domain, an active site within the nuclease
domain, or at least one essential amino acid residue within the
nuclease domain.
[0022] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises a CRISPR-Cas protein. In
some embodiments, the CRISPR-Cas protein is a Type V CRISPR-Cas
protein. In some embodiments, the first RNA binding protein
comprises a Cpf1 polypeptide or an RNA-binding portion thereof. In
some embodiments, the CRISPR-Cas protein comprises a native RNA
nuclease activity. In some embodiments, the native RNA nuclease
activity is reduced or inhibited. In some embodiments, the native
RNA nuclease activity is increased or induced. In some embodiments,
the CRISPR-Cas protein comprises a native DNA nuclease activity and
the native DNA nuclease activity is inhibited. In some embodiments,
the CRISPR-Cas protein comprises a mutation. In some embodiments, a
nuclease domain of the CRISPR-Cas protein comprises the mutation.
In some embodiments, the mutation occurs in a nucleic acid encoding
the CRISPR-Cas protein. In some embodiments, the mutation occurs in
an amino acid encoding the CRISPR-Cas protein. In some embodiments,
the mutation comprises a substitution, an insertion, a deletion, a
frameshift, an inversion, or a transposition. In some embodiments,
the mutation comprises a deletion of a nuclease domain, a binding
site within the nuclease domain, an active site within the nuclease
domain, or at least one essential amino acid residue within the
nuclease domain.
[0023] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises a CRISPR-Cas protein. In
some embodiments, the CRISPR-Cas protein is a Type VI CRISPR-Cas
protein. In some embodiments, the first RNA binding protein
comprises a Cas13 polypeptide or an RNA-binding portion thereof. In
some embodiments, the first RNA binding protein comprises a
CasRx/Cas13d polypeptide or an RNA-binding portion thereof. In some
embodiments, the CRISPR-Cas protein comprises a native RNA nuclease
activity. In some embodiments, the native RNA nuclease activity is
reduced or inhibited. In some embodiments, the native RNA nuclease
activity is increased or induced. In some embodiments, the
CRISPR-Cas protein comprises a native DNA nuclease activity and the
native DNA nuclease activity is inhibited. In some embodiments, the
CRISPR-Cas protein comprises a mutation. In some embodiments, a
nuclease domain of the CRISPR-Cas protein comprises the mutation.
In some embodiments, the mutation occurs in a nucleic acid encoding
the CRISPR-Cas protein. In some embodiments, the mutation occurs in
an amino acid encoding the CRISPR-Cas protein. In some embodiments,
the mutation comprises a substitution, an insertion, a deletion, a
frameshift, an inversion, or a transposition. In some embodiments,
the mutation comprises a deletion of a nuclease domain, a binding
site within the nuclease domain, an active site within the nuclease
domain, or at least one essential amino acid residue within the
nuclease domain.
[0024] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises a Pumilio and FBF (PUF)
protein or an RNA binding portion thereof. In some embodiments, the
first RNA binding protein comprises a Pumilio-based assembly
(PUMBY) protein or an RNA binding portion thereof.
[0025] In some embodiments of the compositions of the disclosure,
the first RNA binding protein does not require multimerization for
RNA-binding activity. In some embodiments, the first RNA binding
protein is not a monomer of a multimer complex. In some
embodiments, a multimer protein complex does not comprise the first
RNA binding protein.
[0026] In some embodiments of the compositions of the disclosure,
the first RNA binding protein selectively binds to a target
sequence within the RNA molecule. In some embodiments, the first
RNA binding protein does not comprise an affinity for a second
sequence within the RNA molecule. In some embodiments, the first
RNA binding protein does not comprise a high affinity for or
selectively bind a second sequence within the RNA molecule.
[0027] In some embodiments of the compositions of the disclosure,
an RNA genome or an RNA transcriptome comprises the RNA
molecule.
[0028] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises between 2 and 1300 amino
acids, inclusive of the endpoints.
[0029] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein further
comprises a sequence encoding a nuclear localization signal (NLS),
a nuclear export signal (NES) or tag. In some embodiments, the
sequence encoding a nuclear localization signal (NLS) is positioned
3' to the sequence encoding the first RNA binding protein. In some
embodiments, the first RNA binding protein comprises an NLS at a
C-terminus of the protein.
[0030] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein further
comprises a first sequence encoding a first NLS and a second
sequence encoding a second NLS. In some embodiments, the sequence
encoding the first NLS or the second NLS is positioned 3' to the
sequence encoding the first RNA binding protein. In some
embodiments, the first RNA binding protein comprises the first NLS
or the second NLS at a C-terminus of the protein.
[0031] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a nuclease
domain. In some embodiments, the second RNA binding protein binds
RNA in a manner in which it associates with RNA. In some
embodiments, the second RNA binding protein associates with RNA in
a manner in which it cleaves RNA.
[0032] In some embodiments of the compositions of the disclosure,
the sequence encoding the second RNA binding protein comprises or
consists of an RNAse. In some embodiments, the second RNA binding
protein comprises or consists of an RNAse1. In some embodiments,
the RNAse1 comprises or consists of SEQ ID NO: 20. In some
embodiments, the second RNA binding protein comprises or consists
of an RNAse4. In some embodiments, the RNAse4 comprises or consists
of SEQ ID NO: 21. In some embodiments, the second RNA binding
protein comprises or consists of an RNAse6. In some embodiments,
the RNAse6 comprises or consists of SEQ ID NO: 22. In some
embodiments, the second RNA binding protein comprises or consists
of an RNAse7. In some embodiments, the RNAse7 comprises or consists
of SEQ ID NO: 23. In some embodiments, the second RNA binding
protein comprises or consists of an RNAse8. In some embodiments,
the RNAse8 protein comprises or consists of SEQ ID NO: 24. In some
embodiments, the second RNA binding protein comprises or consists
of an RNAse2. In some embodiments, the RNAse2 protein comprises or
consists of SEQ ID NO: 25. In some embodiments, the second RNA
binding protein comprises or consists of an RNAse6PL. In some
embodiments, the RNAse6PL protein comprises or consists of SEQ ID
NO: 26. In some embodiments, the second RNA binding protein
comprises or consists of an RNAseL. In some embodiments the RNAseL
protein comprises or consists of SEQ ID NO: 27. In some
embodiments, the second RNA binding protein comprises or consists
of an RNAseT2. In some embodiments, the RNAseT2 protein comprises
or consists of SEQ ID NO: 28. In some embodiments, the second RNA
binding protein comprises or consists of an RNAse11. In some
embodiments, the RNAse11 protein comprises or consists of SEQ ID
NO: 29. In some embodiments, the second RNA binding protein
comprises or consists of an RNAseT2-like. In some embodiments, the
RNAseT2-like protein comprises or consists of SEQ ID NO: 30.
[0033] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a mutated
RNAse. In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(K41R))
polypeptide. In some embodiments, the Rnase1 (K41R) polypeptide
comprises or consists of SEQ ID NO: 116. In some embodiments, the
second RNA binding protein comprises or consists of a mutated
Rnase1 (Rnase1(K41R, D121E)) polypeptide. In some embodiments, the
Rnase1 (Rnase1(K41R, D121E)) polypeptide comprises or consists of
SEQ ID NO: 66. In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E,
H119N)) polypeptide. In some embodiments, the Rnase1 (Rnase1(K41R,
D121E, H119N)) polypeptide comprises or consists of SEQ ID NO: 118.
In some embodiments, the second RNA binding protein comprises or
consists of a mutated Rnase1. In some embodiments, the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(H119N)) polypeptide. In some embodiments, the Rnase1
(Rnase1(H119N)) polypeptide comprises or consists SEQ ID NO: 119.
In some embodiments, the second RNA binding protein comprises or
consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D,
H119N)) polypeptide. In some embodiments, the Rnase1 (Rnase1(R39D,
N67D, N88A, G89D, R91D, H119N)) polypeptide comprises or consists
of SEQ ID NO: 120. In some embodiments, the second RNA binding
protein comprises or consists of a mutated Rnase1 (Rnase1(R39D,
N67D, N88A, G89D, R91D, H119N)) polypeptide. In some embodiments,
the Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N, K41R,
D121E)) polypeptide comprises or consists of SEQ ID NO: 121. In
some embodiments, the second RNA binding protein comprises or
consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D,
H119N)) polypeptide. In some embodiments, the Rnase1 (Rnase1(R39D,
N67D, N88A, G89D, R91D)) polypeptide comprises or consists of SEQ
ID NO: 122.
[0034] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a NOB 1
polypeptide. In some embodiments, the NOB 1 polypeptide comprises
or consists of SEQ ID NO: 31.
[0035] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
endonuclease. In some embodiments, the second RNA binding protein
comprises or consists of an endonuclease V (ENDOV). In some
embodiments, the ENDOV protein comprises or consists of SEQ ID NO:
32. In some embodiments, the second RNA binding protein comprises
or consists of an endonuclease G (ENDOG). In some embodiments, the
ENDOG protein comprises or consists of SEQ ID NO: 33. In some
embodiments, the second RNA binding protein comprises or consists
of an endonuclease D1 (ENDOD1). In some embodiments, the ENDOD1
protein comprises or consists of SEQ ID NO: 34. In some
embodiments, the second RNA binding protein comprises or consists
of a Human flap endonuclease-1 (hFEN1). In some embodiments, the
hFEN1 protein comprises or consists of SEQ ID NO: 35. In some
embodiments, the second RNA binding protein comprises or consists
of a DNA repair endonuclease XPF (ERCC4) polypeptide. In some
embodiments, the ERCC4 protein comprises or consists of SEQ ID NO:
64.
[0036] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
Endonuclease III-like protein 1 (NTHL) polypeptide. In some
embodiments, the NTHL polypeptide comprises or consists of SEQ ID
NO: 123.
[0037] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a human
Schlafen 14 (hSLFN14) polypeptide. In some embodiments, the hSLFN14
polypeptide comprises or consists of SEQ ID NO: 36.
[0038] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a human
beta-lactamase-like protein 2 (hLACTB2) polypeptide. In some
embodiments, the hLACTB2 polypeptide comprises or consists of SEQ
ID NO: 37.
[0039] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
apurinic/apyrimidinic (AP) endodeoxyribonuclease (APEX)
polypeptide. In some embodiments, the second RNA binding protein
comprises or consists of an apurinic/apyrimidinic (AP)
endodeoxyribonuclease (APEX2) polypeptide. In some embodiments, the
APEX2 polypeptide comprises or consists of SEQ ID NO: 38. In some
embodiments, the APEX2 polypeptide comprises or consists of SEQ ID
NO: 39. In some embodiments, the second RNA binding protein
comprises or consists of an apurinic or apyrimidinic site lyase
(APEX1) polypeptide. In some embodiments, the APEX1 polypeptide
comprises or consists of SEQ ID NO: 125.
[0040] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
angiogenin (ANG) polypeptide. In some embodiments, the ANG
polypeptide comprises or consists SEQ ID NO: 40.
[0041] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a heat
responsive protein 12 (HRSP12) polypeptide. In some embodiments,
the HRSP12 polypeptide comprises or consists of SEQ ID NO: 41.
[0042] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Zinc
Finger CCCH-Type Containing 12A (ZC3H12A) polypeptide. In some
embodiments, the ZC3H12A polypeptide comprises or consists of SEQ
ID NO: 42. In some embodiments, the ZC3H12A polypeptide comprises
or consists of SEQ ID NO: 43.
[0043] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Reactive
Intermediate Imine Deaminase A (RIDA) polypeptide. In some
embodiments, the RIDA polypeptide comprises or consists of SEQ ID
NO: 44.
[0044] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Phospholipase D Family Member 6 (PDL6) polypeptide. In some
embodiments, the PDL6 polypeptide comprises or consists of SEQ ID
NO: 126.
[0045] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
mitochondrial ribonuclease P catalytic subunit (KIAA0391)
polypeptide. In some embodiments, the KIAA0391 polypeptide
comprises or consists of SEQ ID NO: 127.
[0046] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
argonaute 2 (AGO2) polypeptide.
In some embodiments of the compositions of the disclosure, the AGO2
polypeptide comprises or consists of SEQ ID NO: 128.
[0047] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
mitochondrial nuclease EXOG (EXOG) polypeptide. In some
embodiments, the EXOG polypeptide comprises or consists of SEQ ID
NO: 129.
[0048] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Zinc
Finger CCCH-Type Containing 12D (ZC3H12D) polypeptide. In some
embodiments, the ZC3H12D polypeptide comprises or consists of SEQ
ID NO: 130.
[0049] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
endoplasmic reticulum to nucleus signaling 2 (ERN2) polypeptide. In
some embodiments, the ERN2 polypeptide comprises or consists of SEQ
ID NO: 131.
[0050] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a pelota
mRNA surveillance and ribosome rescue factor (PELO) polypeptide. In
some embodiments, the PELO polypeptide comprises or consists of SEQ
ID NO: 132.
[0051] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a YBEY
metallopeptidase (YBEY) polypeptide. In some embodiments, the YBEY
polypeptide comprises or consists of SEQ ID NO: 133.
[0052] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a cleavage
and polyadenylation specific factor 4 like (CPSF4L) polypeptide. In
some embodiments, the CPSF4L polypeptide comprises or consists of
SEQ ID NO: 134.
[0053] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
hCG_200273 ipolypeptide. In some embodiments, the hCG_2002731
comprises or consists of SEQ ID NO: 135. In some embodiments, the
hCG_2002731 polypeptide comprises or consists of SEQ ID NO:
136.
[0054] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an Excision
Repair Cross-Complementation Group 1 (ERCC1) polypeptide. In some
embodiments, the ERCC1 polypeptide comprises or consists of SEQ ID
NO: 137.
[0055] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
ras-related C3 botulinum toxin substrate 1 isoform (RAC1)
polypeptide. In some embodiments, the RAC1 polypeptide comprises or
consists of SEQ ID NO: 138.
[0056] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Ribonuclease A A1 (RAA1) polypeptide. In some embodiments, the RAA1
polypeptide comprises or consists of SEQ ID NO: 139.
[0057] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Ras
Related Protein (RAB1) polypeptide. In some embodiments, the RAB1
polypeptide comprises or consists of SEQ ID NO: 140.
[0058] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a DNA
Replication Helicase/Nuclease 2 (DNA2) polypeptide. In some
embodiments, the DNA2 polypeptide comprises or consists of SEQ ID
NO: 141.
[0059] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a FLJ35220
polypeptide. In some embodiments, the FLJ35220 polypeptide
comprises or consists of SEQ ID NO: 142.
[0060] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a FLJ13173
polypeptide. In some embodiments, the FLJ13173 polypeptide
comprises or consists of SEQ ID NO: 143.
[0061] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of Teneurin
Transmembrane Protein (TENM) polypeptide. In some embodiments, the
second RNA binding protein comprises or consists of Teneurin
Transmembrane Protein 1 (TENM1) polypeptide. In some embodiments,
the TENM1 polypeptide comprises or consists of SEQ ID NO: 144. In
some embodiments, the second RNA binding protein comprises or
consists of Teneurin Transmembrane Protein 2 (TENM2) polypeptide.
In some embodiments, the TENM2 polypeptide comprises or consists of
SEQ ID NO: 145.
[0062] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Ribonuclease Kappa (RNAseK) polypeptide. In some embodiments, the
RNAseK polypeptide comprises or consists of SEQ ID NO: 204.
[0063] In some embodiments, the fusion proteins of the disclosure
are used in methods for treating a subject in need thereof, the
methods comprising contacting a target RNA with a fusion protein or
the sequence encoding the fusion protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0065] FIGS. 1A-B is a schematic diagram of an exemplary embodiment
of a composition of the disclosure. (FIG. 1A) An RNA-targeting Cas9
system fused to an endonuclease targets and cleaves a
disease-causing RNA. (FIG. 1B) Depicts an application of (A) in the
context of myotonic dystrophy type 1, wherein an RNA-targeting Cas9
system fused to an endonuclease targets and cleaves a repetitive
RNA composed of repeating CUG units. In the absence of the
RNA-targeting Cas9 system, the repetitive RNA composed of repeating
CUG units binds to a splicing factor MBNL and causes pathology via
dysfunctional RNA splicing. Cleavage of this repetitive RNA
ameliorates disease.
[0066] FIG. 2 is a schematic diagram depicting an exemplary modular
therapeutic platform for treating genetic disease by targeting RNA
molecules.
[0067] FIGS. 3A-B is a pair of schematic diagrams depicting (FIG.
3A) a "high expression" control system (also referred to as "pos
control") comprising a two plasmid system comprising a
cytomegalovirus promoter driving expression of the RNA
endonuclease/Cas9 fusion and (FIG. 3B) a "low expression" control
system (also referred to as "P13") comprising a single plasmid
system comprising a lower-expression promoter (pEFS) driving
expression of the RNA endonuclease/Cas9 fusion.
[0068] FIG. 4A is a pair of schematic diagrams depicting an
exemplary RNA Endonuclease-C. jejuni Cas9 fusion protein (left) and
a vector comprising an exemplary RNA Endonuclease-S. pyogenes Cas9
fusion protein (right)
[0069] FIG. 4B is a graph depicting the ability of a variety of
fusion proteins comprising either C. jejuni Cas9 or S. pyogenes
Cas9, as shown in FIG. 4A, to cleave repetitive RNA molecules.
[0070] FIG. 5A is a pair of schematic diagrams depicting an
exemplary RNA Endonuclease-C. jejuni Cas9 fusion protein (left) and
a vector comprising an exemplary RNA Endonuclease-S. pyogenes Cas9
fusion protein (right)
[0071] FIG. 5B is a graph depicting the ability of a variety of
fusion proteins comprising either C. jejuni Cas9 or S. pyogenes
Cas9, as shown in FIG. 5A, to cleave mRNA molecules encoding a
luciferase protein.
[0072] FIG. 6 is a table providing a key to the endonucleases shown
in FIGS. 4B, 5B, and 9.
[0073] FIG. 7A is a schematic diagram depicting an exemplary RNA
Endonuclease-C. jejuni Cas9 fusion protein.
[0074] FIG. 7B is a graph depicting changes in expression levels of
Zika NS5 in the presence of both E43 and E67 CjeCas9-endonuclease
fusions with sgRNAs containing the various NS5-targeting spacer
sequences as indicated in Table 2. Zika NS5 expression is displayed
as fold change relative to the endonuclease loaded with an sgRNA
containing a control (Lambda) spacer sequence.
[0075] FIG. 8A is a fluorescence microscopy image of cells
transfected with CjeCas9-endonuclease fusions loaded with an sgRNA
containing a Zika NS5-targeting spacer sequence.
[0076] FIG. 8B is a graph depicting changes of expression of Zika
NS5 in the presence of CjeCas9-endonuclease fusions loaded with the
appropriate Zika NS5-targeting sgRNA as compared to a
CjeCas9-endonuclease fusions loaded with a non-Zika NS5 targeting
sgRNA.
[0077] FIG. 9 is a graph depicting the cleavage efficiencies of a
variety of exemplary fusion proteins (SpyCas9 fused to the
annotated endonuclease).
DETAILED DESCRIPTION
[0078] The disclosure provides an RNA-guided fusion protein that
selectively binds and, optionally, cleaves RNA molecules. The
disclosure provides vectors, compositions and cells comprising the
RNA-guided fusion protein. The disclosure provides methods of using
the RNA-guided fusion protein, vectors, compositions and cells of
the disclosure to treat a disease or disorder.
Guide RNA
[0079] The terms guide RNA (gRNA) and single guide RNA (sgRNA) are
used interchangeably throughout the disclosure.
[0080] Guide RNAs (gRNAs) of the disclosure may comprise of a
spacer sequence and a scaffolding sequence. In some embodiments, a
guide RNA is a single guide RNA (sgRNA) comprising a contiguous
spacer sequence and scaffolding sequence. In some embodiments, the
spacer sequence and the scaffolding sequence are not contiguous. In
some embodiments, a scaffold sequence comprises a "direct repeat"
(DR) sequence. DR sequences refer to the repetitive sequences in
the CRISPR locus (naturally-occurring in a bacterial genome or
plasmid) that are interspersed with the spacer sequences. It is
well known that one would be able to infer the DR sequence of a
corresponding Cas protein if the sequence of the associated CRISPR
locus is known. In some embodiments, a sequence encoding a guide
RNA or single guide RNA of the disclosure comprises or consists of
a spacer sequence and a scaffolding sequence, that are separated by
a linker sequence. In some embodiments, the linker sequence may
comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
30, 35, 40, 45, 50 or any number of nucleotides in between. In some
embodiments, the linker sequence may comprise at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of
nucleotides in between.
[0081] Guide RNAs (gRNAs) of the disclosure may comprise
non-naturally occurring nucleotides. In some embodiments, a guide
RNA of the disclosure or a sequence encoding the guide RNA
comprises or consists of modified or synthetic RNA nucleotides.
Exemplary modified RNA nucleotides include, but are not limited to,
pseudouridine (.PSI.), dihydrouridine (D), inosine (I), and
7-methylguanosine (m7G), hypoxanthine, xanthine, xanthosine,
7-methylguanine, 5, 6-Dihydrouracil, 5-methylcytosine,
5-methylcytidine, 5-hydropxymethylcytosine, isoguanine, and
isocytosine.
[0082] Guide RNAs (gRNAs) of the disclosure may bind modified RNA
within a target sequence. Within a target sequence, guide RNAs
(gRNAs) of the disclosure may bind modified RNA. Exemplary
epigenetically or post-transcriptionally modified RNA include, but
are not limited to, 2'-O-Methylation (2'-OMe) (2'-O-methylation
occurs on the oxygen of the free 2'-OH of the ribose moiety),
N6-methyladenosine (m6A), and 5-methylcytosine (m5C).
[0083] In some embodiments of the compositions of the disclosure, a
guide RNA of the disclosure comprises at least one sequence
encoding a non-coding C/D box small nucleolar RNA (snoRNA)
sequence. In some embodiments, the snoRNA sequence comprises at
least one sequence that is complementary to the target RNA, wherein
the target sequence of the RNA molecule comprises at least one
2'-OMe. In some embodiments, the snoRNA sequence comprises at least
one sequence that is complementary to the target RNA, wherein the
at least one sequence that is complementary to the target RNA
comprises a box C motif (RUGAUGA) and a box D motif (CUGA).
[0084] Spacer sequences of the disclosure bind to the target
sequence of an RNA molecule. Spacer sequences of the disclosure may
comprise a CRISPR RNA (crRNA). Spacer sequences of the disclosure
comprise or consist of a sequence having sufficient complementarity
to a target sequence of an RNA molecule to bind selectively to the
target sequence. Upon binding to a target sequence of an RNA
molecule, the spacer sequence may guide one or more of a
scaffolding sequence and a fusion protein to the RNA molecule. In
some embodiments, a sequence having sufficient complementarity to a
target sequence of an RNA molecule to bind selectively to the
target sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in
between to the target sequence. In some embodiments, a sequence
having sufficient complementarity to a target sequence of an RNA
molecule to bind selectively to the target sequence has 100%
identity the target sequence.
[0085] Scaffolding sequences of the disclosure bind the first
RNA-binding polypeptide of the disclosure. Scaffolding sequences of
the disclosure may comprise a trans acting RNA (tracrRNA).
Scaffolding sequences of the disclosure comprise or consist of a
sequence having sufficient complementarity to a target sequence of
an RNA molecule to bind selectively to the target sequence. Upon
binding to a target sequence of an RNA molecule, the scaffolding
sequence may guide a fusion protein to the RNA molecule. In some
embodiments, a sequence having sufficient complementarity to a
target sequence of an RNA molecule to bind selectively to the
target sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in
between to the target sequence. In some embodiments, a sequence
having sufficient complementarity to a target sequence of an RNA
molecule to bind selectively to the target sequence has 100%
identity the target sequence. Alternatively, or in addition, in
some embodiments, scaffolding sequences of the disclosure comprise
or consist of a sequence that binds to a first RNA binding protein
or a second RNA binding protein of a fusion protein of the
disclosure. In some embodiments, scaffolding sequences of the
disclosure comprise a secondary structure or a tertiary structure.
Exemplary secondary structures include, but are not limited to, a
helix, a stem loop, a bulge, a tetraloop and a pseudoknot.
Exemplary tertiary structures include, but are not limited to, an
A-form of a helix, a B-form of a helix, and a Z-form of a helix.
Exemplary tertiary structures include, but are not limited to, a
twisted or helicized stem loop. Exemplary tertiary structures
include, but are not limited to, a twisted or helicized pseudoknot.
In some embodiments, scaffolding sequences of the disclosure
comprise at least one secondary structure or at least one tertiary
structure. In some embodiments, scaffolding sequences of the
disclosure comprise one or more secondary structure(s) or one or
more tertiary structure(s).
[0086] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof selectively binds to a tetraloop
motif in an RNA molecule of the disclosure. In some embodiments, a
target sequence of an RNA molecule comprises a tetraloop motif. In
some embodiments, the tetraloop motif is a "GRNA" motif comprising
or consisting of one or more of the sequences of GAAA, GUGA, GCAA
or GAGA.
[0087] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof that binds to a target sequence of
an RNA molecule hybridizes to the target sequence of the RNA
molecule. In some embodiments, a guide RNA or a portion thereof
that binds to a first RNA binding protein or to a second RNA
binding protein covalently binds to the first RNA binding protein
or to the second RNA binding protein. In some embodiments, a guide
RNA or a portion thereof that binds to a first RNA binding protein
or to a second RNA binding protein non-covalently binds to the
first RNA binding protein or to the second RNA binding protein.
[0088] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof comprises or consists of between 10
and 100 nucleotides, inclusive of the endpoints. In some
embodiments, a spacer sequence of the disclosure comprises or
consists of between 10 and 30 nucleotides, inclusive of the
endpoints. In some embodiments, a spacer sequence of the disclosure
comprises or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the
spacer sequence of the disclosure comprises or consists of 20
nucleotides. In some embodiments, the spacer sequence of the
disclosure comprises or consists of 21 nucleotides. In some
embodiments, a scaffold sequence of the disclosure comprises or
consists of between 10 and 100 nucleotides, inclusive of the
endpoints. In some embodiments, a scaffold sequence of the
disclosure comprises or consists of 30, 35, 40, 45, 50, 55, 60, 65,
70, 76, 80, 87, 90, 95, 100 or any number of nucleotides in
between. In some embodiments, the scaffold sequence of the
disclosure comprises or consists of between 85 and 95 nucleotides,
inclusive of the endpoints. In some embodiments, the scaffold
sequence of the disclosure comprises or consists of 85 nucleotides.
In some embodiments, the scaffold sequence of the disclosure
comprises or consists of 90 nucleotides. In some embodiments, the
scaffold sequence of the disclosure comprises or consists of 93
nucleotides.
[0089] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof does not comprise a nuclear
localization sequence (NLS).
[0090] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof does not comprise a sequence
complementary to a protospacer adjacent motif (PAM).
[0091] Therapeutic or pharmaceutical compositions of the disclosure
do not comprise a PAMmer oligonucleotide. In other embodiments,
optionally, non-therapeutic or non-pharmaceutical compositions may
comprise a PAMmer oligonucleotide. The term "PAMmer" refers to an
oligonucleotide comprising a PAM sequence that is capable of
interacting with a guide nucleotide sequence-programmable RNA
binding protein. Non-limiting examples of PAMmers are described in
O'Connell et al. Nature 516, pages 263-266 (2014), incorporated
herein by reference. A PAM sequence refers to a protospacer
adjacent motif comprising about 2 to about 10 nucleotides. PAM
sequences are specific to the guide nucleotide
sequence-programmable RNA binding protein with which they interact
and are known in the art. For example, Streptococcus pyogenes PAM
has the sequence 5'-NGG-3', where "N" is any nucleobase followed by
two guanine ("G") nucleobases. Cas9 of Francisella novicida
recognizes the canonical PAM sequence 5'-NGG-3', but has been
engineered to recognize the PAM 5'-YG-3' (where "Y" is a
pyrimidine), thus adding to the range of possible Cas9 targets. The
Cpf1 nuclease of Francisella novicida recognizes the PAM 5'-TTTN-3'
or 5'-YTN-3'.
[0092] In some embodiments of the compositions of the disclosure, a
guide RNA or a portion thereof comprises a sequence complementary
to a protospacer flanking sequence (PFS). In some embodiments,
including those wherein a guide RNA or a portion thereof comprises
a sequence complementary to a PFS, the first RNA binding protein
may comprise a sequence isolated or derived from a Cas13 protein.
In some embodiments, including those wherein a guide RNA or a
portion thereof comprises a sequence complementary to a PFS, the
first RNA binding protein may comprise a sequence encoding a Cas13
protein or an RNA-binding portion thereof. In some embodiments, the
guide RNA or a portion thereof does not comprise a sequence
complementary to a PFS.
[0093] In some embodiments of the compositions of the disclosure,
guide RNA sequence of the disclosure comprises a promoter sequence
to drive expression of the guide RNA. In some embodiments, a vector
comprising a guide RNA sequence of the disclosure comprises a
promoter sequence to drive expression of the guide RNA. In some
embodiments, the promoter to drive expression of the guide RNA is a
constitutive promoter. In some embodiments, the promoter sequence
is an inducible promoter. In some embodiments, the promoter is a
sequence is a tissue-specific and/or cell-type specific promoter.
In some embodiments, the promoter is a hybrid or a recombinant
promoter. In some embodiments, the promoter is a promoter capable
of expressing the guide RNA in a mammalian cell. In some
embodiments, the promoter is a promoter capable of expressing the
guide RNA in a human cell. In some embodiments, the promoter is a
promoter capable of expressing the guide RNA and restricting the
guide RNA to the nucleus of the cell. In some embodiments, the
promoter is a human RNA polymerase promoter or a sequence isolated
or derived from a sequence encoding a human RNA polymerase
promoter. In some embodiments, the promoter is a U6 promoter or a
sequence isolated or derived from a sequence encoding a U6
promoter. In some embodiments, the promoter is a human tRNA
promoter or a sequence isolated or derived from a sequence encoding
a human tRNA promoter. In some embodiments, the promoter is a human
valine tRNA promoter or a sequence isolated or derived from a
sequence encoding a human valine tRNA promoter.
[0094] In some embodiments of the compositions of the disclosure, a
promoter to drive expression of the guide RNA further comprises a
regulatory element. In some embodiments, a vector comprising a
promoter sequence to drive expression of the guide RNA further
comprises a regulatory element. In some embodiments, a regulatory
element enhances expression of the guide RNA. Exemplary regulatory
elements include, but are not limited to, an enhancer element, an
intron, an exon, or a combination thereof.
[0095] In some embodiments of the compositions of the disclosure, a
vector of the disclosure comprises one or more of a sequence
encoding a guide RNA, a promoter sequence to drive expression of
the guide RNA and a sequence encoding a regulatory element. In some
embodiments of the compositions of the disclosure, the vector
further comprises a sequence encoding a fusion protein of the
disclosure.
Fusion Proteins
[0096] Fusion proteins of the disclosure comprise a first RNA
binding protein and a second RNA binding protein. In some
embodiments, along a sequence encoding the fusion protein, the
sequence encoding the first RNA binding protein is positioned 5' of
the sequence encoding the second RNA binding protein. In some
embodiments, along a sequence encoding the fusion protein, the
sequence encoding the first RNA binding protein is positioned 3' of
the sequence encoding the second RNA binding protein.
[0097] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a protein capable of binding an
RNA molecule. In some embodiments, the sequence encoding the first
RNA binding protein comprises a sequence isolated or derived from a
protein capable of selectively binding an RNA molecule and not
binding a DNA molecule, a mammalian DNA molecule or any DNA
molecule. In some embodiments, the sequence encoding the first RNA
binding protein comprises a sequence isolated or derived from a
protein capable of binding an RNA molecule and inducing a break in
the RNA molecule. In some embodiments, the sequence encoding the
first RNA binding protein comprises a sequence isolated or derived
from a protein capable of binding an RNA molecule, inducing a break
in the RNA molecule, and not binding a DNA molecule, a mammalian
DNA molecule or any DNA molecule. In some embodiments, the sequence
encoding the first RNA binding protein comprises a sequence
isolated or derived from a protein capable of binding an RNA
molecule, inducing a break in the RNA molecule, and neither binding
nor inducing a break in a DNA molecule, a mammalian DNA molecule or
any DNA molecule.
[0098] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a protein with no DNA nuclease
activity.
[0099] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a protein having DNA nuclease
activity, wherein the DNA nuclease activity does not induce a break
in a DNA molecule, a mammalian DNA molecule or any DNA molecule
when a composition of the disclosure is contacted to an RNA
molecule or introduced into a cell or into a subject of the
disclosure.
[0100] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a protein having DNA nuclease
activity, wherein the DNA nuclease activity is inactivated and
wherein the DNA nuclease activity does not induce a break in a DNA
molecule, a mammalian DNA molecule or any DNA molecule when a
composition of the disclosure is contacted to an RNA molecule or
introduced into a cell or into a subject of the disclosure. In some
embodiments, the sequence encoding the first RNA binding protein
comprises a mutation that inactivates or decreases the DNA nuclease
activity to a level at which the DNA nuclease activity does not
induce a break in a DNA molecule, a mammalian DNA molecule or any
DNA molecule when a composition of the disclosure is contacted to
an RNA molecule or introduced into a cell or into a subject of the
disclosure. In some embodiments, the sequence encoding the first
RNA binding protein comprises a mutation that inactivates or
decreases the DNA nuclease activity and the mutation comprises one
or more of a substitution, inversion, transposition, insertion,
deletion, or any combination thereof to a nucleic acid sequence or
amino acid sequence encoding the first RNA binding protein or a
nuclease domain thereof.
[0101] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein of an
RNA-guided fusion protein disclosed herein comprises a sequence
isolated or derived from a CRISPR Cas protein. In some embodiments,
the CRISPR Cas protein comprises a Type II CRISPR Cas protein. In
some embodiments, the Type II CRISPR Cas protein comprises a Cas9
protein. Exemplary Cas9 proteins of the disclosure may be isolated
or derived from any species, including, but not limited to, a
bacteria or an archaea. Exemplary Cas9 proteins of the disclosure
may be isolated or derived from any species, including, but not
limited to, Streptococcus pyogenes, Haloferax mediteranii,
Mycobacterium tuberculosis, Francisella tularensis subsp. novicida,
Pasteurella multocida, Neisseria meningitidis, Campylobacter
jejune, Streptococcus thermophilus, Campylobacter lari CF89-12,
Mycoplasma gallisepticum str. F, Nitratifractor salsuginis str. DSM
16511, Parvibaculum lavamentivorans, Roseburia intestinalis,
Neisseria cinerea, a Gluconacetobacter diazotrophicus, an
Azospirillum B510, a Sphaerochaeta globus str. Buddy,
Flavobacterium columnare, Fluviicola taffensis, Bacteroides
coprophilus, Mycoplasma mobile, Lactobacillus farciminis,
Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus
pseudintermedius, Filifactor alocis, Treponema denticola,
Legionella pneumophila str. Paris, Sutterella wadsworthensis,
Corynebacter diphtherias, Streptococcus aureus, and Francisella
novicida.
[0102] Exemplary wild type S. pyogenes Cas9 proteins of the
disclosure may comprise or consist of the amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 147) 1 MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK
KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC
YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE
KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181
VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN
241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF
LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR
QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL
VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI
EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS
FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI
601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ
LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD
SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV
MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP
VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH 841 IVPQSFLKDD
SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS
961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY
GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR
PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI
LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV
KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR
KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261
QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA
1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD.
[0103] Nuclease inactivated S. pyogenes Cas9 proteins may comprise
a substitution of an Alanine (A) for an Aspartic Acid (D) at
position 10 and an alanine (A) for a Histidine (H) at position 840.
Exemplary nuclease inactivated S. pyogenes Cas9 proteins of the
disclosure may comprise or consist of the amino acid sequence (D10A
and H840A bolded and underlined):
TABLE-US-00005 (SEQ ID NO: 148) 1 MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK
KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC
YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE
KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181
VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN
241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF
LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR
QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL
VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI
EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS
FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI
601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ
LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD
SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV
MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP
VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA 841 IVPQSFLKDD
SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS
961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY
GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR
PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI
LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV
KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR
KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261
QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA
1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD.
[0104] Nuclease inactivated S. pyogenes Cas9 proteins may comprise
deletion of a RuvC nuclease domain or a portion thereof, an HNH
domain, a DNAse active site, a .beta..beta..alpha.-metal fold or a
portion thereof comprising a DNAse active site or any combination
thereof.
[0105] Other exemplary Cas9 proteins or portions thereof may
comprise or consist of the following amino acid sequences.
[0106] In some embodiments the Cas9 protein can be S. pyogenes Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 149)
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD
LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP
NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI
FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVIEGMRKPAFLSGEQKKAIVD
LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ
LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV
MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
TLANGEIRKRPLIETNGETGEIVVVDKGRDFATVRKVLSMPQVNIVKKTE
VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP
KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP
EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD
KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH
QSITGLYETRIDLSQLGGD
[0107] In some embodiments the Cas9 protein can be S. aureus Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 150)
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK
RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL
SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV
AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT
YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA
YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA
KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ
IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV
KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ
TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP
FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS
YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR
YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH
HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY
KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL
IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE
KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS
RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA
KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT
YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII KKG
[0108] In some embodiments the Cas9 protein can be S. thermophiles
CRISPR1 Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00008 (SEQ ID NO: 151)
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNR
QGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDEL
SNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKT
PGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ
QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDN
IFGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQ
KNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTF
EAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS
FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTIL
TRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEY
GDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAE
LPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI
LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFV
RESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQE
HFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQ
LNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK
SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIK
DIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQIND
KGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITP
KDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFDKGTGTYKIS
QEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMP
KQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVR
TDVLGNQHIIKNEGDKPKLDF
[0109] In some embodiments the Cas9 protein can be N. meningitidis
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00009 (SEQ ID NO: 152)
MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE
VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN
GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET
ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS
HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA
VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT
ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM
KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG
DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR
IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS
KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF
NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ
RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG
QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM
NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA
DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA
KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA
KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVVVVRNHNGIADNATMVR
VDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKF
SLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR
[0110] In some embodiments the Cas9 protein can be Parvibaculum.
lavamentivorans Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00010 (SEQ ID NO: 153)
MERIFGFDIGTTSIGFSVIDYSSTQSAGNIQRLGVRIFPEARDPDGTPLN
QQRRQKRMMRRQLRRRRIRRKALNETLHEAGFLPAYGSADWPVVMADEPY
ELRRRGLEEGLSAYEFGRAIYHLAQHRHFKGRELEESDTPDPDVDDEKEA
ANERAATLKALKNEQTTLGAWLARRPPSDRKRGIHAHRNVVAEEFERLWE
VQSKFHPALKSEEMRARISDTIFAQRPVFWRKNTLGECRFMPGEPLCPKG
SWLSQQRRMLEKLNNLAIAGGNARPLDAEERDAILSKLQQQASMSWPGVR
SALKALYKQRGEPGAEKSLKFNLELGGESKLLGNALEAKLADMFGPDWPA
HPRKQEIRHAVHERLWAADYGETPDKKRVIILSEKDRKAHREAAANSFVA
DFGITGEQAAQLQALKLPTGWEPYSIPALNLFLAELEKGERFGALVNGPD
WEGWRRTNFPHRNQPTGEILDKLPSPASKEERERISQLRNPTVVRTQNEL
RKVVNNLIGLYGKPDRIRIEVGRDVGKSKREREEIQSGIRRNEKQRKKAT
EDLIKNGIANPSRDDVEKWILWKEGQERCPYTGDQIGFNALFREGRYEVE
HIWPRSRSFDNSPRNKTLCRKDVNIEKGNRMPFEAFGHDEDRWSAIQIRL
QGMVSAKGGTGMSPGKVKRFLAKTMPEDFAARQLNDTRYAAKQILAQLKR
LWPDMGPEAPVKVEAVTGQVTAQLRKLWTLNNILADDGEKTRADHRHHAI
DALTVACTHPGMTNKLSRYWQLRDDPRAEKPALTPPWDTIRADAEKAVSE
IVVSHRVRKKVSGPLHKETTYGDTGTDIKTKSGTYRQFVTRKKIESLSKG
ELDEIRDPRIKEIVAAHVAGRGGDPKKAFPPYPCVSPGGPEIRKVRLTSK
QQLNLMAQTGNGYADLGSNHHIAIYRLPDGKADFEIVSLFDASRRLAQRN
PIVQRTRADGASFVMSLAAGEAIMIPEGSKKGIWIVQGVVVASGQVVLER
DTDADHSTTTRPMPNPILKDDAKKVSIDPIGRVRPSND
[0111] In some embodiments the Cas9 protein can be Corynebacter
diphtheria Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00011 (SEQ ID NO: 154)
MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDSGLDPDEIKSA
VTRLASSGIARRTRRLYRRKRRRLQQLDKFIQRQGWPVIELEDYSDPLYP
WKVRAELAASYIADEKERGEKLSVALRHIARHRGWRNPYAKVSSLYLPDG
PSDAFKAIREEIKRASGQPVPETATVGQMVTLCELGTLKLRGEGGVLSAR
LQQSDYAREIQEICRMQEIGQELYRKIIDVVFAAESPKGSASSRVGKDPL
QPGKNRALKASDAFQRYRIAALIGNLRVRVDGEKRILSVEEKNLVFDHLV
NLTPKKEPEWVTIAEILGIDRGQLIGTATMTDDGERAGARPPTHDTNRSI
VNSRIAPLVDWWKTASALEQHAMVKALSNAEVDDFDSPEGAKVQAFFADL
DDDVHAKLDSLHLPVGRAAYSEDTLVRLTRRMLSDGVDLYTARLQEFGIE
PSWTPPTPRIGEPVGNPAVDRVLKTVSRWLESATKTWGAPERVIIEHVRE
GFVTEKRAREMDGDMRRRAARNAKLFQEMQEKLNVQGKPSRADLWRYQSV
QRQNCQCAYCGSPITFSNSEMDHIVPRAGQGSTNTRENLVAVCHRCNQSK
GNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRSTDFKKFTKAVVER
FQRATMDEEIDARSMESVAWMANELRSRVAQHFASHGTTVRVYRGSLTAE
ARRASGISGKLKFFDGVGKSRLDRRHHAIDAAVIAFTSDYVAETLAVRSN
LKQSQAHRQEAPQWREFTGKDAEHRAAWRVWCQKMEKLSALLTEDLRDDR
VVVMSNVRLRLGNGSAHKETIGKLSKVKLSSQLSVSDIDKASSEALWCAL
TREPGFDPKEGLPANPERHIRVNGTHVYAGDNIGLFPVSAGSIALRGGYA
ELGSSFHHARVYKITSGKKPAFAMLRVYTIDLLPYRNQDLFSVELKPQTM
SMRQAEKKLRDALATGNAEYLGWLVVDDELVVDTSKIATDQVKAVEAELG
TIRRWRVDGFFSPSKLRLRPLQMSKEGIKKESAPELSKIIDRPGWLPAVN
KLFSDGNVTVVRRDSLGRVRLESTAHLPVTWKVQ
[0112] In some embodiments the Cas9 protein can be Streptococcus
pasteurianus Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00012 (SEQ ID NO: 155)
MTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNAERRGF
RGSRRLNRRKKHRVKRVRDLFEKYGIVTDFRNLNLNPYELRVKGLTEQLK
NEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRLLKNKTPG
QIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQA
DYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENI
FGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVSTETGKLSTEQK
ESLVEFAKNTATLGPAKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFE
PYRKLKFNLESINIDDLSREVIDKLADILTLNIIREGIEDAIKRNLPNQF
TEEQISEIIKVRKSQSTAFNKGWHSFSAKLMNELIPELYATSDEQMTILT
RLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKY
GDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDK
LPDEVFHGNKQLETKIRLWYQQGERCLYSGKPISIQELVHNSNNFEIDHI
LPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYV
LKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQS
ALRELGKDTKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQ
LKLWEKQDNPMFVDYGKNQVVDKQTGEILSVSDDEYKELVFQPPYQGFVN
TISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLG
KIKDIYSQNGFDTFIKKYNKDKTQFLMYQKDSLTWENVIEVILRDYPTTK
KSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCI
DITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGN
YHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDIASGEQEIYRFLS
RTMPNVNHYVELKPYDKEKFDNVQELVEALGEADKVGRCIKGLNKPNISI
YKVRTDVLGNKYFVKKKGDKPKLDFKNNKK
[0113] In some embodiments the Cas9 protein can be Neisseria
cinerea Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00013 (SEQ ID NO: 156)
MAAFKPNPMNYILGLDIGIASVGWAIVEIDEEENPIRLIDLGVRVFERAE
VPKTGDSLAAARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN
GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET
ADKELGALLKGVADNTHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS
HTFNRKDLQAELNLLFEKQKEFGNPHVSDGLKEGIETLLMTQRPALSGDA
VQKMLGHCTFEPTEPKAAKNTYTAERFVWLTKLNNLRILEQGSERPLTDT
ERATLMDEPYRKSKLTYAQARKLLDLDDTAFFKGLRYGKDNAEASTLMEM
KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGNRYDEACTEIYG
DHYGKKNIEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR
IHIETAREVGKSFKDRKEIEKRQEENRKDREKSAAKFREYFPNFVGEPKS
KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF
NNKVLALGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ
RILLQKFDEDGFKERNLNDTRYINRFLCQFVADHMLLTGKGKRRVFASNG
QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTIAMQQKITRFVRYKEM
NAFDGKTIDKETGEVLHQKAHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA
DTPEKLRTLLAEKLSSRPEAVHKYVTPLFISRAPNRKMSGQGHMETVKSA
KRLDEGISVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA
KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVHNHNGIADNATIVRV
DVFEKGGKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWTVMDDSFEFKFV
LYANDLIKLTAKKNEFLGYFVSLNRATGAIDIRTHDTDSTKGKNGIFQSV
GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR
[0114] In some embodiments the Cas9 protein can be Campylobacter
lari Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00014 (SEQ ID NO: 157)
MRILGFDIGINSIGWAFVENDELKDCGVRIFTKAENPKNKESLALPRRNA
RSSRRRLKRRKARLIAIKRILAKELKLNYKDYVAADGELPKAYEGSLASV
YELRYKALTQNLETKDLARVILHIAKHRGYMNKNEKKSNDAKKGKILSAL
KNNALKLENYQSVGEYFYKEFFQKYKKNTKNFIKIRNTKDNYNNCVLSSD
LEKELKLILEKQKEFGYNYSEDFINEILKVAFFQRPLKDFSHLVGACTFF
EEEKRACKNSYSAWEFVALTKIINEIKSLEKISGEIVPTQTINEVLNLIL
DKGSITYKKFRSCINLHESISFKSLKYDKENAENAKLIDFRKLVEFKKAL
GVHSLSRQELDQISTHITLIKDNVKLKTVLEKYNLSNEQINNLLEIEFND
YINLSFKALGMILPLMREGKRYDEACEIANLKPKTVDEKKDFLPAFCDSI
FAHELSNPVVNRAISEYRKVLNALLKKYGKVHKIHLELARDVGLSKKARE
KIEKEQKENQAVNAWALKECENIGLKASAKNILKLKLWKEQKEICIYSGN
KISIEHLKDEKALEVDHIYPYSRSFDDSFINKVLVFTKENQEKLNKTPFE
AFGKNIEKWSKIQTLAQNLPYKKKNKILDENFKDKQQEDFISRNLNDTRY
IATLIAKYTKEYLNFLLLSENENANLKSGEKGSKIHVQTISGMLTSVLRH
TWGFDKKDRNNHLHHALDAIIVAYSTNSIIKAFSDFRKNQELLKARFYAK
ELTSDNYKHQVKFFEPFKSFREKILSKIDEIFVSKPPRKRARRALHKDTF
HSENKIIDKCSYNSKEGLQIALSCGRVRKIGTKYVENDTIVRVDIFKKQN
KFYAIPIYAMDFALGILPNKIVITGKDKNNNPKQWQTIDESYEFCFSLYK
NDLILLQKKNMQEPEFAYYNDFSISTSSICVEKHDNKFENLTSNQKLLFS
NAKEGSVKVESLGIQNLKVFEKYIITPLGDKIKADFQPRENISLKTSKKY GLR
[0115] In some embodiments the Cas9 protein can be T7 denticola
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00015 (SEQ ID NO: 158)
MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDLWGMRCFETAETA
EVRRLHRGARRRIERRKKRIKLLQELFSQEIAKTDEGFFQRMKESPFYAE
DKTILQENTLFNDKDFADKTYHKAYPTINHLIKAWIENKVKPDPRLLYLA
CHNIIKKRGHFLFEGDFDSENQFDTSIQALFEYLREDMEVDIDADSQKVK
EILKDSSLKNSEKQSRLNKILGLKPSDKQKKAITNLISGNKINFADLYDN
PDLKDAEKNSISFSKDDFDALSDDLASILGDSFELLLKAKAVYNCSVLSK
VIGDEQYLSFAKVKIYEKHKTDLTKLKNVIKKHFPKDYKKVFGYNKNEKN
NNNYSGYVGVCKTKSKKLIINNSVNQEDFYKFLKTILSAKSEIKEVNDIL
TEIETGTFLPKQISKSNAEIPYQLRKMELEKILSNAEKHFSFLKQKDEKG
LSHSEKIIMLLTFKIPYYIGPINDNHKKFFPDRCWVVKKEKSPSGKTTPW
NFFDHIDKEKTAEAFITSRTNFCTYLVGESVLPKSSLLYSEYTVLNEINN
LQIIIDGKNICDIKLKQKIYEDLFKKYKKITQKQISTFIKHEGICNKTDE
VIILGIDKECTSSLKSYIELKNIFGKQVDEISTKNMLEEIIRWATIYDEG
EGKTILKTKIKAEYGKYCSDEQIKKILNLKFSGWGRLSRKFLETVTSEMP
GFSEPVNIITAMRETQNNLMELLSSEFTFTENIKKINSGFEDAEKQFSYD
GLVKPLFLSPSVKKMLWQTLKLVKEISHITQAPPKKIFIEMAKGAELEPA
RTKTRLKILQDLYNNCKNDADAFSSEIKDLSGKIENEDNLRLRSDKLYLY
YTQLGKCMYCGKPIEIGHVFDTSNYDIDHIYPQSKIKDDSISNRVLVCSS
CNKNKEDKYPLKSEIQSKQRGFWNFLQRNNFISLEKLNRLTRATPISDDE
TAKFIARQLVETRQATKVAAKVLEKMFPETKIVYSKAETVSMFRNKFDIV
KCREINDFHHAHDAYLNIVVGNVYNTKFTNNPWNFIKEKRDNPKIADTYN
YYKVFDYDVKRNNITAWEKGKTIITVKDMLKRNTPIYTRQAACKKGELFN
QTIMKKGLGQHPLKKEGPFSNISKYGGYNKVSAAYYTLIEYEEKGNKIRS
LETIPLYLVKDIQKDQDVLKSYLTDLLGKKEFKILVPKIKINSLLKINGF
PCHITGKTNDSFLLRPAVQFCCSNNEVLYFKKIIRFSEIRSQREKIGKTI
SPYEDLSFRSYIKENLWKKTKNDEIGEKEFYDLLQKKNLEIYDMLLTKHK
DTIYKKRPNSATIDILVKGKEKFKSLIIENQFEVILEILKLFSATRNVSD
LQHIGGSKYSGVAKIGNKISSLDNCILIYQSITGIFEKRIDLLKV
[0116] In some embodiments the Cas9 protein can be S. mutans Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00016 (SEQ ID NO: 159)
MKKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGA
LLFDSGNTAEDRRLKRTARRRYTRRRNRILYLQEIFSEEMGKVDDSFFHR
LEDSFLVIEDKRGERHPIFGNLEEEVKYHENFPTIYHLRQYLADNPEKVD
LRLVYLALAHIIKFRGHFLIEGKFDTRNNDVQRLFQEFLAVYDNTFENSS
LQEQNVQVEEILTDKISKSAKKDRVLKLFPNEKSNGRFAEFLKLIVGNQA
DFKKHFELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAKKLYDSI
LLSGILTVTDVGTKAPLSASMIQRYNEHQMDLAQLKQFIRQKLSDKYNEV
FSDVSKDGYAGYIDGKTNQEAFYKYLKGLLNKIEGSGYFLDKIEREDFLR
KQRTFDNGSIPHQIHLQEMRAIIRRQAEFYPFLADNQDRIEKLLTFRIPY
YVGPLARGKSDFAWLSRKSADKITPWNFDEIVDKESSAEAFINRMTNYDL
YLPNQKVLPKHSLLYEKFTVYNELTKVKYKTEQGKTAFFDANMKQEIFDG
VFKVYRKVTKDKLMDFLEKEFDEFRIVDLTGLDKENKVFNASYGTYHDLC
KILDKDFLDNSKNEKILEDIVLTLTLFEDREMIRKRLENYSDLLTKEQVK
KLERRHYTGWGRLSAELIHGIRNKESRKTILDYLIDDGNSNRNFMQLIND
DALSFKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDELVK
IMGHQPENIVVEMARENQFTNQGRRNSQQRLKGLTDSIKEFGSQILKEHP
VENSQLQNDRLFLYYLQNGRDMYTGEELDIDYLSQYDIDHIIPQAFIKDN
SIDNRVLTSSKENRGKSDDVPSKDVVRKMKSYWSKLLSAKLITQRKFDNL
TKAERGGLTDDDKAGFIKRQLVETRQITKHVARILDERFNTETDENNKKI
RQVKIVTLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVIGKALLGV
YPQLEPEFVYGDYPHFHGHKENKATAKKFFYSNIMNFFKKDDVRTDKNGE
IIWKKDEHISNIKKVLSYPQVNIVKKVEEQTGGFSKESILPKGNSDKLIP
RKTKKFYWDTKKYGGFDSPIVAYSILVIADIEKGKSKKLKTVKALVGVTI
MEKMTFERDPVAFLERKGYRNVQEENIIKLPKYSLFKLENGRKRLLASAR
ELQKGNEIVLPNHLGTLLYHAKNIHKVDEPKHLDYVDKHKDEFKELLDVV
SNFSKKYTLAEGNLEKIKELYAQNNGEDLKELASSFINLLTFTAIGAPAT
FKFFDKNIDRKRYTSTTEILNATLIHQSITGLYETRIDLNKLGGD
[0117] In some embodiments the Cas9 protein can be S. thermophilus
CRISPR 3 Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00017 (SEQ ID NO: 160)
MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGV
LLFDSGITAEGRRLKRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQR
LDDSFLVPDDKRDSKYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKAD
LRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAIFESDL
SLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQA
DFRKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAI
LLSGFLTVTDNETEAPLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEV
FKDDTKNGYAGYIDGKTNQEDFYVYLKKLLAEFEGADYFLEKIDREDFLR
KQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPY
YVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRMTSFDL
YLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVR
LYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNII
NDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKL
SRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDA
LSFKKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVK
VMGGRKPESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKELGSKILKEN
IPAKLSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIP
QAFLKDNSIDNKVLVSSASNRGKSDDVPSLEVVKKRKTFWYQLLKSKLIS
QRKFDNLTKAERGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKK
DENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVV
ASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEKVYFYSNIMNIFKKSI
SLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEE
QNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISN
SFTVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGYKD
IELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVK
LLYHAKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKL
LNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKI
PRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG
[0118] In some embodiments the Cas9 protein can be C. jejuni Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00018 (SEQ ID NO: 161)
MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRL
ARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLIS
PYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIK
QNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFL
KDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFT
DEKRAPKNSPLAFWVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKN
GTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDD
LNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKAL
KLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPV
VLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNEN
YKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQD
EKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKW
QKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYT
KDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDR
NNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKN
KRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQS
YGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYT
MDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDM
QEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKS
IGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK
[0119] In some embodiments the Cas9 protein can be P. multocida
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00019 (SEQ ID NO: 162)
MQTTNLSYILGLDLGIASVGWAVVEINENEDPIGLIDVGVRIFERAEVPK
TGESLALSRRLARSTRRLIRRRAHRLLLAKRFLKREGILSTIDLEKGLPN
QAWELRVAGLERRLSAIEWGAVLLHLIKHRGYLSKRKNESQTNNKELGAL
LSGVAQNHQLLQSDDYRTPAELALKKFAKEEGHIRNQRGAYTHTFNRLDL
LAELNLLFAQQHQFGNPHCKEHIQQYMTELLMWQKPALSGEAILKMLGKC
THEKNEFKAAKHTYSAERFVWLTKLNNLRILEDGAERALNEEERQLLINH
PYEKSKLTYAQVRKLLGLSEQAIFKHLRYSKENAESATFMELKAWHAIRK
ALENQGLKDTWQDLAKKPDLLDEIGTAFSLYKTDEDIQQYLTNKVPNSVI
NALLVSLNFDKFIELSLKSLRKILPLMEQGKRYDQACREIYGHHYGEANQ
KTSQLLPAIPAQEIRNPVVLRTLSQARKVINAIIRQYGSPARVHIETGRE
LGKSFKERREIQKQQEDNRTKRESAVQKFKELFSDFSSEPKSKDILKFRL
YEQQHGKCLYSGKEINIHRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLA
SENQNKGNQTPYEWLQGKINSERWKNFVALVLGSQCSAAKKQRLLTQVID
DNKFIDRNLNDTRYIARFLSNYIQENLLLVGKNKKNVFTPNGQITALLRS
RWGLIKARENNNRHHALDAIVVACATPSMQQKITRFIRFKEVHPYKIENR
YEMVDQESGEIISPHFPEPWAYFRQEVNIRVFDNHPDTVLKEMLPDRPQA
NHQFVQPLFVSRAPTRKMSGQGHMETIKSAKRLAEGISVLRIPLTQLKPN
LLENMVNKEREPALYAGLKARLAEFNQDPAKAFATPFYKQGGQQVKAIRV
EQVQKSGVLVRENNGVADNASIVRTDVFIKNNKFFLVPIYTWQVAKGILP
NKAIVAHKNEDEWEEMDEGAKFKFSLFPNDLVELKTKKEYFFGYYIGLDR
ATGNISLKEHDGEISKGKDGVYRVGVKLALSFEKYQVDELGKNRQICRPQ QRQPVR
[0120] In some embodiments the Cas9 protein can be F. novicida Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00020 (SEQ ID NO: 163)
MNFKILPIAIDLGVKNTGVFSAFYQKGTSLERLDNKNGKVYELSKDSYTL
LMNNRTARRHQRRGIDRKQLVKRLFKLIWTEQLNLEWDKDTQQAISFLFN
RRGFSFITDGYSPEYLNIVPEQVKAILMDIFDDYNGEDDLDSYLKLATEQ
ESKISEIYNKLMQKILEFKLMKLCTDIKDDKVSTKTLKEITSYEFELLAD
YLANYSESLKTQKFSYTDKQGNLKELSYYHHDKYNIQEFLKRHATINDRI
LDTLLTDDLDIWNFNFEKFDFDKNEEKLQNQEDKDHIQAHLHHFVFAVNK
IKSEMASGGRHRSQYFQEITNVLDENNHQEGYLKNFCENLHNKKYSNLSV
KNLVNLIGNLSNLELKPLRKYFNDKIHAKADHWDEQKFIETYCHWILGEW
RVGVKDQDKKDGAKYSYKDLCNELKQKVTKAGLVDFLLELDPCRTIPPYL
DNNNRKPPKCQSLILNPKFLDNQYPNWQQYLQELKKLQSIQNYLDSFETD
LKVLKSSKDQPYFVEYKSSNQQIASGQRDYKDLDARILQFIFDRVKASDE
LLLNEIYFQAKKLKQKASSELEKLESSKKLDEVIANSQLSQILKSQHTNG
IFEQGTFLHLVCKYYKQRQRARDSRLYIMPEYRYDKKLHKYNNTGRFDDD
NQLLTYCNHKPRQKRYQLLNDLAGVLQVSPNFLKDKIGSDDDLFISKWLV
EHIRGFKKACEDSLKIQKDNRGLLNHKINIARNTKGKCEKEIFNLICKIE
GSEDKKGNYKHGLAYELGVLLFGEPNEASKPEFDRKIKKFNSIYSFAQIQ
QIAFAERKGNANTCAVCSADNAHRMQQIKIIEPVEDNKDKIILSAKAQRL
PAIPTRIVDGAVKKMATILAKNIVDDNWQNIKQVLSAKHQLHIPIIIESN
AFEFEPALADVKGKSLKDRRKKALERISPENIFKDKNNRIKEFAKGISAY
SGANLTDGDFDGAKEELDHIIPRSHKKYGTLNDEANLICVTRGDNKNKGN
RIFCLRDLADNYKLKQFETTDDLEIEKKIADTIWDANKKDFKFGNYRSFI
NLTPQEQKAFRHALFLADENPIKQAVIRAINNRNRTFVNGTQRYFAEVLA
NNIYLRAKKENLNTDKISFDYFGIPTIGNGRGIAEIRQLYEKVDSDIQAY
AKGDKPQASYSHLIDAMLAFCIAADEHRNDGSIGLEIDKNYSLYPLDKNT
GEVFTKDIFSQIKITDNEFSDKKLVRKKAIEGFNTHRQMTRDGIYAENYL
PILIHKELNEVRKGYTWKNSEEIKIFKGKKYDIQQLNNLVYCLKFVDKPI
SIDIQISTLEELRNILTTNNIAATAEYYYINLKTQKLHEYYIENYNTALG
YKKYSKEMEFLRSLAYRSERVKIKSIDDVKQVLDKDSNFIIGKITLPFKK
EWQRLYREWQNTTIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEG
KFLVKRKTWDNNFIYQILNDSDSRADGTKPFIPAFDISKNEIVEAIIDSF
TSKNIFWLPKNIELQKVDNKNIFAIDTSKWFEVETPSDLRDIGIATIQYK
IDNNSRPKVRVKLDYVIDDDSKINYFMNHSLLKSRYPDKVLEILKQSTII
EFESSGFNKTIKEMLGMKLAGIYNETSNN
[0121] In some embodiments the Cas9 protein can be Lactobacillus
buchneri Cas9 and may comprise or consist of the amino acid
sequence:
TABLE-US-00021 (SEQ ID NO: 164)
MKVNNYHIGLDIGTSSIGWVAIGKDGKPLRVKGKTAIGARLFQEGNPAAD
RRMFRTTRRRLSRRKWRLKLLEEIFDPYITPVDSTFFARLKQSNLSPKDS
RKEFKGSMLFPDLTDMQYHKNYPTIYHLRHALMTQDKKFDIRMVYLAIHH
IVKYRGNFLNSTPVDSFKASKVDFVDQFKKLNELYAAINPEESFKINLAN
SEDIGHQFLDPSIRKFDKKKQIPKIVPVMMNDKVTDRLNGKIASEIIHAI
LGYKAKLDVVLQCTPVDSKPWALKFDDEDIDAKLEKILPEMDENQQSIVA
ILQNLYSQVTLNQIVPNGMSLSESMIEKYNDHHDHLKLYKKLIDQLADPK
KKAVLKKAYSQYVGDDGKVIEQAEFWSSVKKNLDDSELSKQIMDLIDAEK
FMPKQRTSQNGVIPHQLHQRELDEIIEHQSKYYPWLVEINPNKHDLHLAK
YKIEQLVAFRVPYYVGPMITPKDQAESAETVFSWMERKGTETGQITPWNF
DEKVDRKASANRFIKRMTTKDTYLIGEDVLPDESLLYEKFKVLNELNMVR
VNGKLLKVADKQAIFQDLFENYKHVSVKKLQNYIKAKTGLPSDPEISGLS
DPEHFNNSLGTYNDFKKLFGSKVDEPDLQDDFEKIVEWSTVFEDKKILRE
KLNEITWLSDQQKDVLESSRYQGWGRLSKKLLTGIVNDQGERIIDKLWNT
NKNFMQIQSDDDFAKRIHEANADQMQAVDVEDVLADAYTSPQNKKAIRQV
VKVVDDIQKAMGGVAPKYISIEFTRSEDRNPRRTISRQRQLENTLKDTAK
SLAKSINPELLSELDNAAKSKKGLTDRLYLYFTQLGKDIYTGEPINIDEL
NKYDIDHILPQAFIKDNSLDNRVLVLTAVNNGKSDNVPLRMFGAKMGHFW
KQLAEAGLISKRKLKNLQTDPDTISKYAMHGFIRRQLVETSQVIKLVANI
LGDKYRNDDTKIIEITARMNHQMRDEFGFIKNREINDYHHAFDAYLTAFL
GRYLYHRYIKLRPYFVYGDFKKFREDKVTMRNFNFLHDLTDDTQEKIADA
ETGEVIWDRENSIQQLKDVYHYKFMLISHEVYTLRGAMFNQTVYPASDAG
KRKLIPVKADRPVNVYGGYSGSADAYMAIVRIHNKKGDKYRVVGVPMRAL
DRLDAAKNVSDADFDRALKDVLAPQLTKTKKSRKTGEITQVIEDFEIVLG
KVMYRQLMIDGDKKFMLGSSTYQYNAKQLVLSDQSVKTLASKGRLDPLQE
SMDYNNVYlEILDKVNQYFSLYDMNKFRHKLNLGFSKFISFPNHNVLDGN
TKVSSGKREILQEILNGLHANPTFGNLKDVGITTPFGQLQQPNGILLSDE
TKIRYQSPTGLFERTVSLKDL
[0122] In some embodiments the Cas9 protein can be Listeria innocua
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00022 (SEQ ID NO: 165)
MKKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKIAGDSEKKQIKKNFWGV
RLFDEGQTAADRRMARTARRRIERRRNRISYLQGIFAEEMSKTDANFFCR
LSDSFYVDNEKRNSRHPFFATIEEEVEYHKNYPTIYHLREELVNSSEKAD
LRLVYLALAHIIKYRGNFLIEGALDTQNTSVDGIYKQFIQTYNQVFASGI
EDGSLKKLEDNKDVAKILVEKVTRKEKLERILKLYPGEKSAGMFAQFISL
IVGSKGNFQKPFDLIEKSDIECAKDSYEEDLESLLALIGDEYAELFVAAK
NAYSAVVLSSIITVAETETNAKLSASMIERFDTHEEDLGELKAFIKLHLP
KHYEEIFSNTEKHGYAGYIDGKTKQADFYKYMKMTLENIEGADYFIAKIE
KENFLRKQRTFDNGAIPHQLHLEELEAILHQQAKYYPFLKENYDKIKSLV
TFRIPYFVGPLANGQSEFAWLTRKADGEIRPWNIEEKVDFGKSAVDFIEK
MTNKDTYLPKENVLPKHSLCYQKYLVYNELTKVRYINDQGKTSYFSGQEK
EQIFNDLFKQKRKVKKKDLELFLRNMSHVESPTIEGLEDSFNSSYSTYHD
LLKVGIKQEILDNPVNTEMLENIVKILTVFEDKRMIKEQLQQFSDVLDGV
VLKKLERRHYTGWGRLSAKLLMGIRDKQSHLTILDYLMNDDGLNRNLMQL
INDSNLSFKSIIEKEQVTTADKDIQSIVADLAGSPAIKKGILQSLKIVDE
LVSVMGYPPQTIVVEMARENQTTGKGKNNSRPRYKSLEKAIKEFGSQILK
EHPTDNQELRNNRLYLYYLQNGKDMYTGQDLDIHNLSNYDIDHIVPQSFI
TDNSIDNLVLTSSAGNREKGDDVPPLEIVRKRKVFWEKLYQGNLMSKRKF
DYLTKAERGGLTEADKARFIHRQLVETRQITKNVANILHQRFNYEKDDHG
NTMKQVRIVTLKSALVSQFRKQFQLYKVRDVNDYHHAHDAYLNGVVANTL
LKVYPQLEPEFVYGDYHQFDWFKANKATAKKQFYTNIMLFFAQKDRIIDE
NGEILWDKKYLDTVKKVMSYRQMNIVKKTEIQKGEFSKATIKPKGNSSKL
IPRKTNWDPMKYGGLDSPNMAYAVVIEYAKGKNKLVFEKKIIRVTIMERK
AFEKDEKAFLEEQGYRQPKVLAKLPKYTLYECEEGRRRMLASANEAQKGN
QQVLPNHLVTLLHHAANCEVSDGKSLDYIESNREMFAELLAHVSEFAKRY
TLAEANLNKINQLFEQNKEGDIKAIAQSFVDLMAFNAMGAPASFKFFETT
IERKRYNNLKELLNSTIIYQSITGLYESRKRLDD
[0123] In some embodiments the Cas9 protein can be L. pneumophilia
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00023 (SEQ ID NO: 166)
MESSQILSPIGIDLGGKFTGVCLSHLEAFAELPNHANTKYSVILIDHNNF
QLSQAQRRATRHRVRNKKRNQFVKRVALQLFQHILSRDLNAKEETALCHY
LNNRGYTYVDTDLDEYIKDETTINLLKELLPSESEHNFIDWFLQKMQSSE
FRKILVSKVEEKKDDKELKNAVKNIKNFITGFEKNSVEGHRHRKVYFENI
KSDITKDNQLDSIKKKIPSVCLSNLLGHLSNLQWKNLHRYLAKNPKQFDE
QTFGNEFLRMLKNFRHLKGSQESLAVRNLIQQLEQSQDYISILEKTPPEI
TIPPYEARTNTGMEKDQSLLLNPEKLNNLYPNWRNLIPGIIDAHPFLEKD
LEHTKLRDRKRIISPSKQDEKRDSYILQRYLDLNKKIDKFKIKKQLSFLG
QGKQLPANLIETQKEMETHFNSSLVSVLIQIASAYNKEREDAAQGIWFDN
AFSLCELSNINPPRKQKILPLLVGAILSEDFINNKDKWAKFKIFWNTHKI
GRTSLKSKCKEIEEARKNSGNAFKIDYEEALNHPEHSNNKALIKIIQTIP
DIIQAIQSHLGHNDSQALIYHNPFSLSQLYTILETKRDGFHKNCVAVTCE
NYWRSQKTEIDPEISYASRLPADSVRPFDGVLARMMQRLAYEIAMAKWEQ
IKHIPDNSSLLIPIYLEQNRFEFEESFKKIKGSSSDKTLEQAIEKQNIQW
EEKFQRIINASMNICPYKGASIGGQGEIDHIYPRSLSKKHFGVIFNSEVN
LIYCSSQGNREKKEEHYLLEHLSPLYLKHQFGTDNVSDIKNFISQNVANI
KKYISFHLLTPEQQKAARHALFLDYDDEAFKTITKFLMSQQKARVNGTQK
FLGKQIMEFLSTLADSKQLQLEFSIKQITAEEVHDHRELLSKQEPKLVKS
RQQSFPSHAIDATLTMSIGLKEFPQFSQELDNSWFINHLMPDEVHLNPVR
SKEKYNKPNISSTPLFKDSLYAERFIPVWVKGETFAIGFSEKDLFEIKPS
NKEKLFTLLKTYSTKNPGESLQELQAKSKAKWLYFPINKTLALEFLHHYF
HKEIVTPDDTTVCHFINSLRYYTKKESITVKILKEPMPVLSVKFESSKKN
VLGSFKHTIALPATKDWERLFNHPNFLALKANPAPNPKEFNEFIRKYFLS
DNNPNSDIPNNGHNIKPQKHKAVRKVFSLPVIPGNAGTMMRIRRKDNKGQ
PLYQLQTIDDTPSMGIQINEDRLVKQEVLMDAYKTRNLSTIDGINNSEGQ
AYATFDNWLTLPVSTFKPEIIKLEMKPHSKTRRYIRITQSLADFIKTIDE
ALMIKPSDSIDDPLNMPNEIVCKNKLFGNELKPRDGKMKIVSTGKIVTYE
FESDSTPQWIQTLYVTQLKKQP
[0124] In some embodiments the Cas9 protein can be N. lactamica
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00024 (SEQ ID NO: 167)
MAAFKPNPMNYILGLDIGIASVGWAMVEVDEEENPIRLIDLGVRVFERAE
VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQDADFDEN
GLVKSLPNTPWQLRAAALDRKLTCLEWSAVLLHLVKHRGYLSQRKNEGET
ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS
HTFSRKDLQAELNLLFEKQKEFGNPHVSDGLKEDIETLLMAQRPALSGDA
VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT
ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM
KAYHAISRALEKEGLKDKKSPLNLSTELQDEIGTAFSLFKTDKDITGRLK
DRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG
DHYCKKNAEEKIYLPPIPADEIRNPVVLRALSQARKVINCVVRRYGSPAR
IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS
KDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSF
NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ
RILLQKFDEEGFKERNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNG
QITNLLRGFWGLRKVRIENDRHHALDAVVVACSTVAMQQKITRFVRYKEM
NAFDGKTIDKETGEVLHQKAHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA
DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA
KRLDEGISVLRVPLTQLKLKGLEKMVNREREPKLYDALKAQLETHKDDPA
KAFAEPFYKYDKAGSRTQQVKAVRIEQVQKTGVWVRNHNGIADNATMVRV
DVFEKGGKYYLVPIYSWQVAKGILPDRAVVAFKDEEDWTVMDDSFEFRFV
LYANDLIKLTAKKNEFLGYFVSLNRATGAIDIRTHDTDSTKGKNGIFQSV
GVKTALSFQKNQIDELGKEIRPCRLKKRPPVR
[0125] In some embodiments the Cas9 protein can be N. meningitides
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00025 (SEQ ID NO: 168)
MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE
VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN
GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET
ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS
HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA
VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT
ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM
KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG
DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR
IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS
KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF
NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ
RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG
QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM
NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA
DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA
KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA
KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV
DVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS
LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGI
GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR
[0126] In some embodiments the Cas9 protein can be B. longum Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00026 (SEQ ID NO: 169)
MLSRQLLGASHLARPVSYSYNVQDNDVHCSYGERCFMRGKRYRIGIDVGL
NSVGLAAVEVSDENSPVRLLNAQSVIHDGGVDPQKNKEAITRKNMSGVAR
RTRRMRRRKRERLHKLDMLLGKFGYPVIEPESLDKPFEEWHVRAELATRY
IEDDELRRESISIALRHMARHRGWRNPYRQVDSLISDNPYSKQYGELKEK
AKAYNDDATAAEEESTPAQLVVAMLDAGYAEAPRLRWRTGSKKPDAEGYL
PVRLMQEDNANELKQIFRVQRVPADEWKPLFRSVFYAVSPKGSAEQRVGQ
DPLAPEQARALKASLAFQEYRIANVITNLRIKDASAELRKLTVDEKQSIY
DQLVSPSSEDITWSDLCDFLGFKRSQLKGVGSLTEDGEERISSRPPRLTS
VQRIYESDNKIRKPLVAWWKSASDNEHEAMIRLLSNTVDIDKVREDVAYA
SAIEFIDGLDDDALTKLDSVDLPSGRAAYSVETLQKLTRQMLTTDDDLHE
ARKTLFNVTDSWRPPADPIGEPLGNPSVDRVLKNVNRYLMNCQQRWGNPV
SVNIEHVRSSFSSVAFARKDKREYEKNNEKRSIFRSSLSEQLRADEQMEK
VRESDLRRLEAIQRQNGQCLYCGRTITFRTCEMDHIVPRKGVGSTNTRTN
FAAVCAECNRMKSNTPFAIWARSEDAQTRGVSLAEAKKRVTMFTFNPKSY
APREVKAFKQAVIARLQQTEDDAAIDNRSIESVAWMADELHRRIDWYFNA
KQYVNSASIDDAEAETMKTTVSVFQGRVTASARRAAGIEGKIHFIGQQSK
TRLDRRHHAVDASVIAMMNTAAAQTLMERESLRESQRLIGLMPGERSWKE
YPYEGTSRYESFHLWLDNMDVLLELLNDALDNDRIAVMQSQRYVLGNSIA
HDATIHPLEKVPLGSAMSADLIRRASTPALWCALTRLPDYDEKEGLPEDS
HREIRVHDTRYSADDEMGFFASQAAQIAVQEGSADIGSAIHHARVYRCWK
TNAKGVRKYFYGMIRVFQTDLLRACHDDLFTVPLPPQSISMRYGEPRVVQ
ALQSGNAQYLGSLVVGDEIEMDFSSLDVDGQIGEYLQFFSQFSGGNLAWK
HWVVDGFFNQTQLRIRPRYLAAEGLAKAFSDDVVPDGVQKIVTKQGWLPP
VNTASKTAVRIVRRNAFGEPRLSSAHHMPCSWQWRHE
[0127] In some embodiments the Cas9 protein can be A. muciniphila
Cas9 and may comprise or consist of the amino acid sequence:
TABLE-US-00027 (SEQ ID NO: 170)
MSRSLTFSFDIGYASIGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFK
RREYRRLRRNIRSRRVRIERIGRLLVQAQIITPEMKETSGHPAPFYLASE
ALKGHRTLAPIELWHVLRWYAHNRGYDNNASWSNSLSEDGGNGEDTERVK
HAQDLMDKHGTATMAETICRELKLEEGKADAPMEVSTPAYKNLNTAFPRL
IVEKEVRRILELSAPLIPGLTAEIIELIAQHHPLTTEQRGVLLQHGIKLA
RRYRGSLLFGQLIPRFDNRIISRCPVTWAQVYEAELKKGNSEQSARERAE
KLSKVPTANCPEFYEYRMARILCNIRADGEPLSAEIRRELMNQARQEGKL
TKASLEKAISSRLGKETETNVSNYFTLHPDSEEALYLNPAVEVLQRSGIG
QILSPSVYRIAANRLRRGKSVTPNYLLNLLKSRGESGEALEKKIEKESKK
KEADYADTPLKPKYATGRAPYARTVLKKVVEEILDGEDPTRPARGEAHPD
GELKAHDGCLYCLLDTDSSVNQHQKERRLDTMTNNHLVRHRMLILDRLLK
DLIQDFADGQKDRISRVCVEVGKELTTFSAMDSKKIQRELTLRQKSHTDA
VNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGATYGDHELENLELEH
IVPHSFRQSNALSSLVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLH
ICSLNNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQNHEA
MKEIGMTEGMMTQSSHLMKLACKSIKTSLPDAHIDMIPGAVTAEVRKAWD
VFGVFKELCPEAADPDSGKILKENLRSLTHLHHALDACVLGLIPYIIPAH
HNGLLRRVLAMRRIPEKLIPQVRPVANQRHYVLNDDGRMMLRDLSASLKE
NIREQLMEQRVIQHVPADMGGALLKETMQRVLSVDGSGEDAMVSLSKKKD
GKKEKNQVKASKLVGVFPEGPSKLKALKAAIEIDGNYGVALDPKPVVIRH
IKVFKRIMALKEQNGGKPVRILKKGMLIHLTSSKDPKHAGVWRIESIQDS
KGGVKLDLQRAHCAVPKNKTHECNWREVDLISLLKKYQMKRYPTSYTGTP R
[0128] In some embodiments the Cas9 protein can be O. laneus Cas9
and may comprise or consist of the amino acid sequence:
TABLE-US-00028 (SEQ ID NO: 171)
METTLGIDLGTNSIGLALVDQEEHQILYSGVRIFPEGINKDTIGLGEKEE
SRNATRRAKRQMRRQYFRKKLRKAKLLELLIAYDMCPLKPEDVRRWKNWD
KQQKSTVRQFPDTPAFREWLKQNPYELRKQAVTEDVTRPELGRILYQMIQ
RRGFLSSRKGKEEGKIFTGKDRMVGIDETRKNLQKQTLGAYLYDIAPKNG
EKYRFRTERVRARYTLRDMYIREFEIIWQRQAGHLGLAHEQATRKKNIFL
EGSATNVRNSKLITHLQAKYGRGHVLIEDTRITVTFQLPLKEVLGGKIEI
EEEQLKFKSNESVLFWQRPLRSQKSLLSKCVFEGRNFYDPVHQKWIIAGP
TPAPLSHPEFEEFRAYQFINNIIYGKNEHLTAIQREAVFELMCTESKDFN
FEKIPKHLKLFEKFNFDDTTKVPACTTISQLRKLFPHPVWEEKREEIWHC
FYFYDDNTLLFEKLQKDYALQTNDLEKIKKIRLSESYGNVSLKAIRRINP
YLKKGYAYSTAVLLGGIRNSFGKRFEYFKEYEPEIEKAVCRILKEKNAEG
EVIRKIKDYLVHNRFGFAKNDRAFQKLYHHSQAITTQAQKERLPETGNLR
NPIVQQGLNELRRTVNKLLATCREKYGPSFKFDHIHVEMGRELRSSKTER
EKQSRQIRENEKKNEAAKVKLAEYGLKAYRDNIQKYLLYKEIEEKGGTVC
CPYTGKTLNISHTLGSDNSVQIEHIIPYSISLDDSLANKTLCDATFNREK
GELTPYDFYQKDPSPEKWGASSWEEIEDRAFRLLPYAKAQRFIRRKPQES
NEFISRQLNDTRYISKKAVEYLSAICSDVKAFPGQLTAELRHLWGLNNIL
QSAPDITFPLPVSATENHREYYVITNEQNEVIRLFPKQGETPRIEKGELL
LTGEVERKVFRCKGMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAPKPISA
DGQIVLKGRIEKGVFVCNQLKQKLKTGLPDGSYWISLPVISQTFKEGESV
NNSKLTSQQVQLFGRVREGIFRCHNYQCPASGADGNFWCTLDTDTAQPAF
TPIKNAPPGVGGGQIILTGDVDDKGIFHADDDLHYELPASLPKGKYYGIF
TVESCDPTLIPIELSAPKTSKGENLIEGNIWVDEHTGEVRFDPKKNREDQ
RHHAIDAIVIALSSQSLFQRLSTYNARRENKKRGLDSTEHFPSPWPGFAQ
DVRQSVVPLLVSYKQNPKTLCKISKTLYKDGKKIHSCGNAVRGQLHKETV
YGQRTAPGATEKSYHIRKDIRELKTSKHIGKVVDITIRQMLLKHLQENYH
IDITQEFNIPSNAFFKEGVYRIFLPNKHGEPVPIKKIRMKEELGNAERLK
DNINQYVNPRNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPR
EGRNIVSILQINDTFLIGLKEEEPEVYRNDLSTLSKHLYRVQKLSGMYYT
FRHHLASTLNNEREEFRIQSLEAWKRANPVKVQIDEIGRITFLNGPLC.
[0129] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a CRISPR Cas protein or portion
thereof. In some embodiments, the CRISPR Cas protein comprises a
Type V CRISPR Cas protein. In some embodiments, the Type V CRISPR
Cas protein comprises a Cpf1 protein. Exemplary Cpf1 proteins of
the disclosure may be isolated or derived from any species,
including, but not limited to, a bacteria or an archaea. Exemplary
Cpf1 proteins of the disclosure may be isolated or derived from any
species, including, but not limited to, Francisella tularensis
subsp. novicida, Acidaminococcus sp. BV3L6 and Lachnospiraceae
bacterium sp. ND2006. Exemplary Cpf1 proteins of the disclosure may
be nuclease inactivated.
[0130] Exemplary wild type Francisella tularensis subsp. Novicida
Cpf1 (FnCpf1) proteins of the disclosure may comprise or consist of
the amino acid sequence:
TABLE-US-00029 (SEQ ID NO: 172) 1 MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN
IKARGLILDD EKRAKDYKKA KQIIDKYHQF 61 FIEEILSSVC ISEDLLQNYS
DVYFKLKKSD DDNLQKDFKS AKDTIKKQIS EYIKDSEKFK 121 NLFNQNLIDA
KKGQESDLIL WLKQSKDNGI ELFKANSDIT DIDEALEIIK SFKGWTTYFK 181
GFHENRKNVY SSNDIPTSII YRIVDDNLPK FLENKAKYES LKDKAPEAIN YEQIKKDLAE
241 ELTFDIDYKT SEVNQRVFSL DEVFEIANFN NYLNQSGITK FNTIIGGKFV
NGENTKRKGI 301 NEYINLYSQQ INDKTLKKYK MSVLFKQILS DTESKSFVID
KLEDDSDVVT TMQSFYEQIA 361 AFKTVEEKSI KETLSLLFDD LKAQKLDLSK
IYFKNDKSLT DLSQQVFDDY SVIGTAVLEY 421 ITQQIAPKNL DNPSKKEQEL
IAKKTEKAKY LSLETIKLAL EEFNKHRDID KQCRFEEILA 481 NFAAIPMIFD
EIAQNKDNLA QISIKYQNQG KKDLLQASAE DDVKAIKDLL DQTNNLLHKL 541
KIFHISQSED KANILDKDEH FYLVFEECYF ELANIVPLYN KIRNYITQKP YSDEKFKLNF
601 ENSTLANGWD KNKEPDNTAI LFIKDDKYYL GVMNKKNNKI FDDKAIKENK
GEGYKKIVYK 661 LLPGANKMLP KVFFSAKSIK FYNPSEDILR IRNHSTHTKN
GSPQKGYEKF EFNIEDCRKF 721 IDFYKQSISK HPEWKDFGFR FSDTQRYNSI
DEFYREVENQ GYKLTFENIS ESYIDSVVNQ 781 GKLYLFQIYN KDFSAYSKGR
PNLHTLYWKA LFDERNLQDV VYKLNGEAEL FYRKQSIPKK 841 ITHPAKEAIA
NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF HCPITINFKS SGANKFNDEI 901
NLLLKEKAND VHILSIDRGE RHLAYYTLVD GKGNIIKQDT FNIIGNDRMK TNYHDKLAAI
961 EKDRDSARKD WKKINNIKEM KEGYLSQVVH EIAKLVIEYN AIVVFEDLNF
GFKRGRFKVE 1021 KQVYQKLEKM LIEKLNYLVF KDNEFDKTGG VLRAYQLTAP
FETFKKMGKQ TGIIYYVPAG 1081 FTSKICPVTG FVNQLYPKYE SVSKSQEFFS
KFDKICYNLD KGYFEFSFDY KNFGDKAAKG 1141 KWTIASFGSR LINFRNSDKN
HNWDTREVYP TKELEKLLKD YSIEYGHGEC IKAAICGESD 1201 KKFFAKLTSV
LNTILQMRNS KTGTELDYLI SPVADVNGNF FDSRQAPKNM PQDADANGAY 1261
HIGLKGLMLL GRIKNNQEGK KLNLVIKNEE YFEFVQNRNN.
[0131] Exemplary wild type Lachnospiraceae bacterium sp. ND2006
Cpf1 (LbCpf1) proteins of the disclosure may comprise or consist of
the amino acid sequence:
TABLE-US-00030 (SEQ ID NO: 173) 1 AASKLEKFTN CYSLSKTLRF KAIPVGKTQE
NIDNKRLLVE DEKRAEDYKG VKKLLDRYYL 61 SFINDVLHSI KLKNLNNYIS
LFRKKTRTEK ENKELENLEI NLRKEIAKAF KGAAGYKSLF 121 KKDIIETILP
EAADDKDEIA LVNSFNGFTT AFTGFFDNRE NMFSEEAKST SIAFRCINEN 181
LTRYISNMDI FEKVDAIFDK HEVQEIKEKI LNSDYDVEDF FEGEFFNFVL TQEGIDVYNA
241 IIGGFVTESG EKIKGLNEYI NLYNAKTKQA LPKFKPLYKQ VLSDRESLSF
YGEGYTSDEE 301 VLEVFRNTLN KNSEIFSSIK KLEKLFKNFD EYSSAGIFVK
NGPAISTISK DIFGEWNLIR 361 DKWNAEYDDI HLKKKAVVTE KYEDDRRKSF
KKIGSFSLEQ LQEYADADLS VVEKLKEIII 421 QKVDEIYKVY GSSEKLFDAD
FVLEKSLKKN DAVVAIMKDL LDSVKSFENY IKAFFGEGKE 481 TNRDESFYGD
FVLAYDILLK VDHIYDAIRN YVTQKPYSKD KFKLYFQNPQ FMGGWDKDKE 541
TDYRATILRY GSKYYLAIMD KKYAKCLQKI DKDDVNGNYE KINYKLLPGP NKMLPKVFFS
601 KKWMAYYNPS EDIQKIYKNG TFKKGDMFNL NDCHKLIDFF KDSISRYPKW
SNAYDFNFSE 661 TEKYKDIAGF YREVEEQGYK VSFESASKKE VDKLVEEGKL
YMFQIYNKDF SDKSHGTPNL 721 HTMYFKLLFD ENNHGQIRLS GGAELFMRRA
SLKKEELVVH PANSPIANKN PDNPKKTTTL 781 SYDVYKDKRF SEDQYELHIP
IAINKCPKNI FKINTEVRVL LKHDDNPYVI GIDRGERNLL 841 YIVVVDGKGN
IVEQYSLNEI INNFNGIRIK TDYHSLLDKK EKERFEARQN WTSIENIKEL 901
KAGYISQVVH KICELVEKYD AVIALEDLNS GFKNSRVKVE KQVYQKFEKM LIDKLNYMVD
961 KKSNPCATGG ALKGYQITNK FESFKSMSTQ NGFIFYIPAW LTSKIDPSTG
FVNLLKTKYT 1021 SIADSKKFIS SFDRIMYVPE EDLFEFALDY KNFSRTDADY
IKKWKLYSYG NRIRIFAAAK 1081 KNNVFAWEEV CLTSAYKELF NKYGINYQQG
DIRALLCEQS DKAFYSSFMA LMSLMLQMRN 1141 SITGRTDVDF LISPVKNSDG
IFYDSRNYEA QENAILPKNA DANGAYNIAR KVLWAIGQFK 1201 KAEDEKLDKV
KIAISNKEWL EYAQTSVK.
[0132] Exemplary wild type Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1)
proteins of the disclosure may comprise or consist of the amino
acid sequence:
TABLE-US-00031 (SEQ ID NO: 174) 1 MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH
IQEQGFIEED KARNDHYKEL KPIIDRIYKT 61 YADQCLQLVQ LDWENLSAAI
DSYRKEKTEE TRNALIEEQA TYRNAIHDYF IGRTDNLTDA 121 INKRHAEIYK
GLFKAELFNG KVLKQLGTVT TTEHENALLR SFDKFTTYFS GFYENRKNVF 181
SAEDISTAIP HRIVQDNFPK FKENCHIFTR LITAVPSLRE HFENVKKAIG IFVSTSIEEV
241 FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV LNLAIQKNDE
TAHIIASLPH 301 RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL
RNENVLETAE ALFNELNSID 361 LTHIFISHKK LETISSALCD HWDTLRNALY
ERRISELTGK ITKSAKEKVQ RSLKHEDINL 421 QEIISAAGKE LSEAFKQKTS
EILSHAHAAL DQPLPTTLKK QEEKEILKSQ LDSLLGLYHL 481 LDWFAVDESN
EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK FKLNFQMPTL 541
ASGWDVNKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPD
601 AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLEITK EIYDLNNPEK
EPKKFQTAYA 661 KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP
SSQYKDLGEY YAELNPLLYH 721 ISFQRIAEKE IMDAVETGKL YLFQIYNKDF
AKGHHGKPNL HTLYWTGLFS PENLAKTSIK 781 LNGQAELFYR PKSRMKRMAH
RLGEKMLNKK LKDQKTPIPD TLYQELYDYV NHRLSHDLSD 841 EARALLPNVI
TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ AANSPSKFNQ RVNAYLKEHP 901
ETPIIGIDRG ERNLIYITVI DSTGKILEQR SLNTIQQFDY QKKLDNREKE RVAARQAWSV
961 VGTIKDLKQG YLSQVIHEIV DLMIHYQAVV VLENLNFGFK SKRTGIAEKA
VYQQFEKMLI 1021 DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG
FLFYVPAPYT SKIDPLTGFV 1081 DPFVWKTIKN HESRKHFLEG FDFLHYDVKT
GDFILHFKMN RNLSFQRGLP GFMPAWDIVF 1141 EKNETQFDAK GTPFIAGKRI
VPVIENHRFT GRYRDLYPAN ELIALLEEKG IVFRDGSNIL 1201 PKLLENDDSH
AIDTMVALIR SVLQMRNSNA ATGEDYINSP VRDLNGVCFD SRFQNPEWPM 1261
DADANGAYHI ALKGQLLLNH LKESKDLKLQ NGISNQDWLA YIQELRN.
[0133] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a CRISPR Cas protein. In some
embodiments, the CRISPR Cas protein comprises a Type VI CRISPR Cas
protein or portion thereof. In some embodiments, the Type VI CRISPR
Cas protein comprises a Cas13 protein or portion thereof. Exemplary
Cas13 proteins of the disclosure may be isolated or derived from
any species, including, but not limited to, a bacteria or an
archaea. Exemplary Cas13 proteins of the disclosure may be isolated
or derived from any species, including, but not limited to,
Leptotrichia wadei, Listeria seeligeri serovar 1/2b (strain ATCC
35967/DSM 20751/CIP 100100/SLCC 3954), Lachnospiraceae bacterium,
Clostridium aminophilum DSM 10710, Carnobacterium gallinarum DSM
4847, Paludibacter propionicigenes WB4, Listeria weihenstephanensis
FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL
M6-0635 (Listeria newyorkensis), Leptotrichia wadei F0279,
Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121,
Rhodobacter capsulatus DE442 and Corynebacterium ulcerans.
Exemplary Cas13 proteins of the disclosure may be DNA nuclease
inactivated. Exemplary Cas13 proteins of the disclosure include,
but are not limited to, Cas13a, Cas13b, Cas13c, Cas13d and
orthologs thereof. Exemplary Cas13b proteins of the disclosure
include, but are not limited to, subtypes 1 and 2 referred to
herein as Csx27 and Csx28, respectively.
[0134] Exemplary Cas13a proteins include, but are not limited
to:
TABLE-US-00032 Cas13a Cas13a number abbreviation Organism name
Accession number Direct Repeat sequence Cas13a1 LshCas13a
Leptotrichia WP_018451595.1 CCACCCCAATATCGAAGGGGACTAA shahii AAC
(SEQ ID NO: 175) Cas13a2 LwaCas13a Leptotrichia WP_021746774.1
GATTTAGACTACCCCAAAAACGAAG wadei GGGACTAAAAC (SEQ ID NO: 176)
Cas13a3 LseCas13a Listeria seeligeri WP_012985477.1
GTAAGAGACTACCTCTATATGAAAG AGGACTAAAAC (SEQ ID NO: 177) Cas13a4
LbmCas13a Lachnospiraceae WP_044921188.1 GTATTGAGAAAAGCCAGATATAGTT
bacterium GGCAATAGAC (SEQ ID NO: 178) MA2020 Cas13a5 LbnCas13a
Lachnospiraceae WP_022785443.1 GTTGATGAGAAGAGCCCAAGATAG bacterium
AGGGCAATAAC (SEQ ID NO: NK4A179 179) Cas13a6 CamCas13a
[Clostridium] WP_031473346.1 GTCTATTGCCCTCTATATCGGGCTGT aminophilum
TCTCCAAAC (SEQ ID NO: 180) DSM 10710 Cas13a7 CgaCas13a
Carnobacterium WP_034560163.1 ATTAAAGACTACCTCTAAATGTAAG gallinarum
DSM AGGACTATAAC (SEQ ID NO: 4847 181) Cas13a8 Cga2Cas13a
Carnobacterium WP_034563842.1 AATATAAACTACCTCTAAATGTAAG gallinarum
DSM AGGACTATAAC (SEQ ID NO: 4847 182) Cas13a9 Pprcas13a
Paludibacter WP_013443710.1 CTTGTGGATTATCCCAAAATTGAAG
propionicigenes GGAACTACAAC (SEQ ID NO: WB4 183) Cas13a10 LweCas13a
Listeria WP_036059185.1 GATTTAGAGTACCTCAAAATAGAAG
weihenstephanensis AGGTCTAAAAC (SEQ ID NO: FSL R9-0317 184)
Cas13a11 LbfCas13a Listeriaceae WP_036091002.1
GATTTAGAGTACCTCAAAACAAAAG bacterium FSL AGGACTAAAAC (SEQ ID NO:
M6-0635 185) (Listeria newyorkensis) Cas13a12 Lwa2cas13a
Leptotrichia WP_021746774.1 GATATAGATAACCCCAAAAACGAA wadei F0279
GGGATCTAAAAC (SEQ ID NO: 186) Cas13a13 RcsCas13a Rhodobacter
WP_013067728.1 GCCTCACATCACCGCCAAGACGACG capsulatus SB GCGGACTGAAC
(SEQ ID NO: 187) 1003 Cas13a14 RcrCas13a Rhodobacter WP_023911507.1
GCCTCACATCACCGCCAAGACGACG capsulatus R121 GCGGACTGAAC (SEQ ID NO:
188) Cas13a15 RcdCas13a Rhodobacter WP_023911507.1
GCCTCACATCACCGCCAAGACGACG capsulatus GCGGACTGAAC (SEQ ID NO: DE442
189)
[0135] Exemplary wild type Cas13a proteins of the disclosure may
comprise or consist of the amino acid sequence:
TABLE-US-00033 (SEQ ID NO: 190) 1 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN
YDGNKYILNI NENNNKEKID NNKFIRKYIN 61 YKKNDNILKE FTRKFHAGNI
LFKLKGKEGI IRIENNDDFL ETEEVVLYIE AYGKSEKLKA 121 LGITKKKIID
EAIRQGITKD DKKIEIKRQE NEEEIEIDIR DEYTNKTLND CSIILRIIEN 181
DELETKKSIY EIFKNINMSL YKIIEKIIEN ETEKVFENRY YEEHLREKLL KDDKIDVILT
241 NFMEIREKIK SNLEILGFVK FYLNVGGDKK KSKNKKMLVE KILNINVDLT
VEDIADFVIK 301 ELEFWNITKR IEKVKKVNNE FLEKRRNRTY IKSYVLLDKH
EKFKIERENK KDKIVKFFVE 361 NIKNNSIKEK IEKILAEFKI DELIKKLEKE
LKKGNCDTEI FGIFKKHYKV NFDSKKFSKK 421 SDEEKELYKI IYRYLKGRIE
KILVNEQKVR LKKMEKIEIE KILNESILSE KILKRVKQYT 481 LEHIMYLGKL
RHNDIDMTTV NTDDFSRLHA KEELDLELIT FFASTNMELN KIFSRENINN 541
DENIDFFGGD REKNYVLDKK ILNSKIKIIR DLDFIDNKNN ITNNFIRKFT KIGTNERNRI
601 LHAISKERDL QGTQDDYNKV INIIQNLKIS DEEVSKALNL DVVFKDKKNI
ITKINDIKIS 661 EENNNDIKYL PSFSKVLPEI LNLYRNNPKN EPFDTIETEK
IVLNALIYVN KELYKKLILE 721 DDLEENESKN IFLQELKKTL GNIDEIDENI
IENYYKNAQI SASKGNNKAI KKYQKKVIEC 781 YIGYLRKNYE ELFDFSDFKM
NIQEIKKQIK DINDNKTYER ITVKTSDKTI VINDDFEYII 841 SIFALLNSNA
VINKIRNRFF ATSVWLNTSE YQNIIDILDE IMQLNTLRNE CITENWNLNL 901
EEFIQKMKEI EKDFDDFKIQ TKKEIFNNYY EDIKNNILTE FKDDINGCDV LEKKLEKIVI
961 FDDETKFEID KKSNILQDEQ RKLSNINKKD LKKKVDQYIK DKDQEIKSKI
LCRIIFNSDF 1021 LKKYKKEIDN LIEDMESENE NKFQEIYYPK ERKNELYIYK
KNLFLNIGNP NFDKIYGLIS 1081 NDIKMADAKF LFNIDGKNIR KNKISEIDAI
LKNLNDKLNG YSKEYKEKYI KKLKENDDFF 1141 AKNIQNKNYK SFEKDYNRVS
EYKKIRDLVE FNYLNKIESY LIDINWKLAI QMARFERDMH 1201 YIVNGLRELG
IIKLSGYNTG ISRAYPKRNG SDGFYTTTAY YKFFDEESYK KFEKICYGFG 1261
IDLSENSEIN KPENESIRNY ISHFYIVRNP FADYSIAEQI DRVSNLLSYS TRYNNSTYAS
1321 VFEVFKKDVN LDYDELKKKF KLIGNNDILE RLMKPKKVSV LELESYNSDY
IKNLIIELLT 1381 KIENTNDTL.
[0136] Exemplary Cas13b proteins include, but are not limited
to:
TABLE-US-00034 Cas13b Species Cas13b Accession Size (aa)
Paludibacter propionicigenes WB4 WP_013446107.1 1155 Prevotella sp.
P5-60 WP_044074780.1 1091 Prevotella sp. P4-76 WP_044072147.1 1091
Prevotella sp. P5-125 WP_044065294.1 1091 Prevotella sp. P5-119
WP_042518169.1 1091 Capnocytophaga canimorsus Cc5 WP_013997271.1
1200 Phaeodactylibacter xiamenensis WP_044218239.1 1132
Porphyromonas gingivalis W83 WP_005873511.1 1136 Porphyromonas
gingivalis F0570 WP_021665475.1 1136 Porphyromonas gingivalis ATCC
33277 WP_012458151.1 1136 Porphyromonas gingivalis F0185 ERJ81987.1
1136 Porphyromonas gingivalis F0185 WP_021677657.1 1136
Porphyromonas gingivalis SJD2 WP_023846767.1 1136 Porphyromonas
gingivalis F0568 ERJ65637.1 1136 Porphyromonas gingivalis W4087
ERJ87335.1 1136 Porphyromonas gingivalis W4087 WP_021680012.1 1136
Porphyromonas gingivalis F0568 WP_021663197.1 1136 Porphyromonas
gingivalis WP_061156637.1 1136 Porphyromonas gulae WP_039445055.1
1136 Bacteroides pyogenes F0041 ERI81700.1 1116 Bacteroides
pyogenes JCM 10003 WP_034542281.1 1116 Alistipes sp. ZOR0009
WP_047447901.1 954 Flavobacterium branchiophilum FL-15
WP_014084666.1 1151 Prevotella sp. MA2016 WP_036929175.1 1323
Myroides odoratimimus CCUG 10230 EH006562.1 1160 Myroides
odoratimimus CCUG 3837 EKB06014.1 1158 Myroides odoratimimus CCUG
3837 WP_006265509.1 1158 Myroides odoratimimus CCUG 12901
WP_006261414.1 1158 Myroides odoratimimus CCUG 12901 EHO08761.1
1158 Myroides odoratimimus (NZ_CP013690.1) WP_058700060.1 1160
Bergeyella zoohelcum ATCC 43767 EKB54193.1 1225 Capnocytophaga
cynodegmi WP_041989581.1 1219 Bergeyella zoohelcum ATCC 43767
WP_002664492.1 1225 Flavobacterium sp. 316 WP_045968377.1 1156
Psychroflexus torquis ATCC 700755 WP_015024765.1 1146
Flavobacterium columnare ATCC 49512 WP_014165541.1 1180
Flavobacterium columnare WP_060381855.1 1214 Flavobacterium
columnare WP_063744070.1 1214 Flavobacterium columnare
WP_065213424.1 1215 Chryseobacterium sp. YR477 WP_047431796.1 1146
Riemerella anatipestifer ATCC 11845 = WP_004919755.1 1096 DSM 15868
Riemerella anatipestifer RA-CH-2 WP_015345620.1 949 Riemerella
anatipestifer WP_049354263.1 949 Riemerella anatipestifer
WP_061710138.1 951 Riemerella anatipestifer WP_064970887.1 1096
Prevotella saccharolytica F0055 EKY00089.1 1151 Prevotella
saccharolytica JCM 17484 WP_051522484.1 1152 Prevotella buccae ATCC
33574 EFU31981.1 1128 Prevotella buccae ATCC 33574 WP_004343973.1
1128 Prevotella buccae D17 WP_004343581.1 1128 Prevotella sp. MSX73
WP_007412163.1 1128 Prevotella pallens ATCC 700821 EGQ18444.1 1126
Prevotella pallens ATCC 700821 WP_006044833.1 1126 Prevotella
intermedia ATCC 25611 = WP_036860899.1 1127 DSM 20706 Prevotella
intermedia WP_061868553.1 1121 Prevotella intermedia 17 AFJ07523.1
1135 Prevotella intermedia WP_050955369.1 1133 Prevotella
intermedia BAU18623.1 1134 Prevotella intermedia ZT KJJ86756.1 1126
Prevotella aurantiaca JCM 15754 WP_025000926.1 1125 Prevotella
pleuritidis F0068 WP_021584635.1 1140 Prevotella pleuritidis JCM
14110 WP_036931485.1 1117 Prevotella falsenii DSM 22864 = JCM
WP_036884929.1 1134 15124 Porphyromonas gulae WP_039418912.1 1176
Porphyromonas sp. COT-052 OH4946 WP_039428968.1 1176 Porphyromonas
gulae WP_039442171.1 1175 Porphyromonas gulae WP_039431778.1 1176
Porphyromonas gulae WP_046201018.1 1176 Porphyromonas gulae
WP_039434803.1 1176 Porphyromonas gulae WP_039419792.1 1120
Porphyromonas gulae WP_039426176.1 1120 Porphyromonas gulae
WP_039437199.1 1120 Porphyromonas gingivalis TDC60 WP_013816155.1
1120 Porphyromonas gingivalis ATCC 33277 WP_012458414.1 1120
Porphyromonas gingivalis A7A1-28 WP_058019250.1 1176 Porphyromonas
gingivalis JCVI SC001 E0A10535.1 1176 Porphyromonas gingivalis W50
WP_005874195.1 1176 Porphyromonas gingivalis WP_052912312.1 1176
Porphyromonas gingivalis AJW4 WP_053444417.1 1120 Porphyromonas
gingivalis WP_039417390.1 1120 Porphyromonas gingivalis
WP_061156470.1 1120
[0137] Exemplary wild type Bergeyella zoohelcum ATCC 43767 Cas13b
(BzCas13b) proteins of the disclosure may comprise or consist of
the amino acid sequence:
TABLE-US-00035 (SEQ ID NO: 191) 1 menktslgnn iyynpfkpqd ksyfagyfna
amentdsvfr elgkrlkgke ytsenffdai 61 fkenislvey eryvkllsdy
fpmarlldkk evpikerken fkknfkgiik avrdlrnfyt 121 hkehgeveit
deifgvldem lkstvltvkk kkvktdktke ilkksiekql dilcqkkley 181
lrdtarkiee krrnqrerge kelvapfkys dkrddliaai yndafdvyid kkkdslkess
241 kakyntksdp qqeegdlkip iskngvvfll slfltkqeih afkskiagfk
atvideatvs 301 eatvshgkns icfmatheif shlaykklkr kvrtaeinyg
eaenaeqlsv yaketlmmqm 361 ldelskvpdv vyqnlsedvq ktfiedwney
lkenngdvgt meeeqvihpv irkryedkfn 421 yfairfldef aqfptlrfqv
hlgnylhdsr pkenlisdrr ikekitvfgr lselehkkal 481 fikntetned
rehyweifpn pnydfpkeni svndkdfpia gsildrekqp vagkigikvk 541
llnqqyvsev dkavkahqlk qrkaskpsiq niieeivpin esnpkeaivf ggqptaylsm
601 ndihsilyef fdkwekkkek lekkgekelr keigkelekk ivgkiqaqiq
qiidkdtnak 661 ilkpyqdgns taidkeklik dlkqeqnilq klkdeqtvre
keyndfiayq dknreinkvr 721 drnhkqylkd nlkrkypeap arkevlyyre
kgkvavwlan dikrfmptdf knewkgeqhs 781 llqkslayye qckeelknll
pekvfqhlpf klggyfqqky lyqfytcyld krleyisglv 841 qqaenfksen
kvfkkvenec fkflkkqnyt hkeldarvqs ilgypifler gfmdekptii 901
kgktfkgnea lfadwfryyk eyqnfqtfyd tenyplvele kkqadrkrkt kiyqqkkndv
961 ftllmakhif ksvfkqdsid qfsledlyqs reerlgnqer arqtgerntn
yiwnktvdlk 1021 lcdgkitven vklknvgdfi kyeydqrvqa flkyeeniew
qaflikeske eenypyvver 1081 eieqyekvrr eellkevhli eeyilekvkd
keilkkgdnq nfkyyilngl lkqlknedve 1141 sykvfnlnte pedvninqlk
qeatdleqka fvltyirnkf ahnqlpkkef wdycqekygk 1201 ektyaey faevfkkeke
alik.
[0138] In some embodiments of the compositions of the disclosure,
the sequence encoding the first RNA binding protein comprises a
sequence isolated or derived from a CasRX/Cas13d protein.
CasRX/Cas13d is an effector of the type VI-D CRISPR-Cas systems. In
some embodiments, the CasRX/Cas13d protein is an RNA-guided RNA
endonuclease enzyme that can cut or bind RNA. In some embodiments,
the CasRX/Cas13d protein can include one or more higher eukaryotes
and prokaryotes nucleotide-binding (HEPN) domains. In some
embodiments, the CasRX/Cas13d protein can include either a
wild-type or mutated HEPN domain. In some embodiments, the
CasRX/Cas13d protein includes a mutated HEPN domain that cannot cut
RNA but can process guide RNA. In some embodiments, the
CasRX/Cas13d protein does not require a protospacer flanking
sequence. Also see WO Publication No. WO2019/040664 &
US2019/0062724, which is incorporated herein by reference in its
entirety, for further examples and sequences of CasRX/Cas13d
protein, without limitation, specific reference is made to
[0139] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig6049000251:
TABLE-US-00036 (SEQ ID NO: 54) LYLTSFGKGN AAVIEQKIEP ENGYRVTGMQ
ITPSITVNKA TDESVRFRVK RKIAQKDEFI 60 ADNPMHEGRH RIEPSAGSDM
LGLKTKLEKY YFGKEFDDNL HIQIIYNILD IEKILAVYST 120 NITA. 124
[0140] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig546000275:
TABLE-US-00037 (SEQ ID NO: 57) MDSYRPKLYK LIDFCIFKHY HEYTEISEKN
VDTLRAAVSE EQKESFYADE AKRLWGIFDK 60 QFLGFCKKIN VWVNGSHEKE
ILGYIDKDAY RKKSDVSYFS KFLYAMSFFL DGKEINDLLT 120 TLINKFDNIA
SFISTAKELD AEIDRILEKK LDPVTGKPLK GKNSFRNFIA NNVIENKRFI 180
YVIKFCNPKN VLKLVKNTKV TEFVLKRMPE SQIDRYYSSC IDTEKNPSVD KKISDLAEMI
240 KKIAFDDFRN VRQKTRTREE SLEKERFKAV IGLYLTVVYL LIKNLVNVNS
RYVMAFHCLE 300 RDAKLYGINI GKNYIELTED LCRENENSRS AYLARNKRLR
DCVKQNIDNA KNMKSKEK. 358
[0141] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig4114000374:
TABLE-US-00038 (SEQ ID NO: 61) DTKINPQTWL YQLENTPDLD NEYRDTLDHF
FDERFNEINE HFVTQNATNL CIMKEVFPDE 60 DFKSIADLYY DFIVVKSYKN
IGFSIKKLRE KMLELPEAKR VTSTEMDSVR SKLYKLIDFC 120 IFKHYHEKPE
TVEMIVSMLR AYTSEDMKE. 149
[0142] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig721000619:
TABLE-US-00039 (SEQ ID NO: 67) KEGSTMAKNE KKKSTAKALG LKSSFVVNND
IYMTSFGKGN KAVLEKKITE NTIENKSDTT 60 YFDVINRDPK GFTLEGRRIA
DMTAFSNDPK YHVNVVNGKF LEDQLGARSE LEKKVFGRTF 120 DDNVHIQLIH
NILDIEKIMA QYVSDIVYLL HNTIKRDMND DIMGYISIRN SFDDFCHPER 180
IPDRKAKDNL QKQHDIFFDE ILKCGRLAYF GNAFFEDGSD NKEIAKLKRY KEIYHIIALM
240 GSLRQSYFHG ENSDKNFQGP TWAYTLESNL TGKYKEFKDT LDKTFDERYE
MISKDFGSTN 300 MVNLQILEEL LKMLYGNVSP. 320
[0143] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig2002000411:
TABLE-US-00040 (SEQ ID NO: 69) EKQNKAKYQA IISLYLMVMY QIVKNMIYVN
SRYVIAFHCL ERDSNQLLGR FNSRDASMYN 60 KLTQKFITDK YLNDGAQGCS
KKVGNYLSHN ITCCSDELRK EYRNQVDHFA VVRMIGKYAA 120 DIGKFSTWFE
LYHYVMQRII FDKRNPLSET ERTYKQLIAK HHTYCKDLVK ALNTPFGYNL 180
ARYKNLSIGE LFDRNNYNAK TKET. 204
[0144] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contigl3552000311:
TABLE-US-00041 (SEQ ID NO: 71) LIDFLIYDLY YNRKPARIEE IVDKLRESVN
DEEKESIYSA ETKYVYEALG KVLVRSLKKY 60 LNGATIRDLK NRYDAKTANR
IWDISEHSKS GHVNCFCKLI YMMTLMLDGK EINDLLTTLV 120 NKFDNIASFI
DVMDELGLEH SFTDNYKMFA DSKAICLDLQ FINSFARMSK IDDEKSKRQL 180
FRDALVVLDI GDKNEDWIEK YLTSDIFKRD ENGNKIDGEK RDFRNFIANN VIKSARFKYL
240 VKYSSADGMI KLKKNEKLIS FVLEQLPETQ IDRYYESCGL DCAVADRKVR
IEKLTGLIRD 300 MRFDNFRGVN YSNDACKKDK QAKAKYQAII SLYLMVLYQI
VKNMIYVNSR YVIAFHCLER 360 DLLFFNIELD NSYQYSNCNE LTEKFIKDKY
MKEGALGFNM KAGRYLTKNI GNCSNELRKI 420 YRNQVDHFAV VRKIGNYAAD
IASVGSWFE. 449
[0145] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig10037000527:
TABLE-US-00042 (SEQ ID NO: 72) YMDQNFANSD AWAIHVYRNK IQHLDAVRHA
DMYIGDIREF HSWFELYHYI IQRRIIDQYA 60 YESTPGSSRD GSAIIDEERL
NPATRRYFRL ITTYKT. 96
[0146] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig238000329:
TABLE-US-00043 (SEQ ID NO: 73) RYDKDRSKIY TMMDFVIYRY YIDNNNDSID
FINKLRSSID EKSKEKLYNE EANRLWNKLK 60 EYMLYIKEFN GKLASRTPDR
DGNISEFVES LPKIHRLLPR GQKISNFSKL MYLLTMFLDG 120 KEINDLLTTL
INKFENIQGF LDIMPEINVN AKFEPEYVFF NKSHEIAGEL KLIKGFAQMG 180
EPAATLKLEM TADAIKILGT EKEDAELIKL AESLFKDENG KLLGNKQHGM RNFIGNNVIK
240 SKRFHYLIRY GDPAHLHKIA TNKNVVRFVL GRIADMQKKQ GQKGKNQIDR
YYEVCVGNKD 300 IKKTIEEKID ALTDIIVNMN YDQFEKKKAV IENQNRGKTF
EEKNKYKRDN AEREKFKKII 360 SLYLTVIYHI LKNIVNVNSR YILGFHCLER
DKQLYIEKYN KDKLDGFVAL TKFCLGDEER 420 YEDLKAKAQA SIQALETANP
KLYAKYMNYS DEEKKEEFKK QLNRERVKNA RNAYLKNIKN 480 YIMIRLQLRD
QTDSSGYLCG EFRDKVAHLE VARHAHEYI. 519
[0147] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut meta enome conti 2643000492:
TABLE-US-00044 (SEQ ID NO: 84) NGEIVSLAEK EAFSAKIADK NIGCKIENKQ
FRHPKGYDVI ADNPIYKGSP RQDMLGLKET 60 LEKRYFSPSD SIDNVRVQVA
HNILDIEKIL AEYITNAVYS FDNIAGFGKD IIGDDFSPVY 120 TYDKFEKSDR
YEYFKNLLNN SRLGYYGQAF FECDDSKENK KKKDAIKCYN IIALLSGLRH 180 W.
181
[0148] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig874000057:
TABLE-US-00045 (SEQ ID NO: 85) MSKNKESYAK GMGLKSALVS GSKVYMTSFE
GGNDAKLEKV VENSEIVSLA EKESFSAEIF 60 KKNIGCKIEN KKFKHPKRYD
VIADNPLYKG SVRQDMLGLK ETLEKRYFNS ADGTDNVCIQ 120 VIHNILDIEK
ILAEYITNAV YSFDNIAGFG EDIIGMGGFK PIYTYKQFKE PDKYNKKFDD 180
ILNNSRLGYY GKAFFEKNDL KHNPNKKKRD KNPYILKYDN ECYYIIALLS GLRHWNIHSH
240 AKDDLVSYRW LYNLDSILNR EYISTLNYLY DDIADELTES FSKNSSANVN
YIAETLNIDP 300 SEFAQQYFRF SIMKEQKNMG FNVSKLREIM LDRKELSDIR
DNHRVFDSIR SKLYTMMDFV 360 IYRYYIEEAA KTEAENRNLP ENEKKISEKD
FFVINLRGSF DENQKEKLYI EEAKRLWEKL 420 KDIMLKIKEF RGEKVKEYKK. 440
[0149] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig4781000489:
TABLE-US-00046 (SEQ ID NO: 86) LDKQLDYEYI RTLNYMFNDI ADELTRTFSK
NSAANVNYIA ETLNIDPNKF AEQYFRFSIM 60 KEQKNLGFNL TKLRESMLDR
RELSDIRDNH NVFDSIRPKL YTMMDFVIYK HYIDEAKKTE 120 AENKSLPDDR KNLSEKD.
137
[0150] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig12144000352:
TABLE-US-00047 (SEQ ID NO: 87) RMGEPVANTK RVMMIDAVKI LGTDLSDDEL
KEMADSFFKD SDGNLLKKGK HGMRNFITNN 60 VIKNKRFHYL IRYGDPAHLH EIAKNEA.
87
[0151] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig5590000448:
TABLE-US-00048 (SEQ ID NO: 88) VHNNEEKDLI KYTWLYNLDK YLDAEYITTL
NYMYNDIGDE LTDSFSKNSA ANINYIAETL 60 GIDPKTFAEQ YFRFSIMKEQ
KNLGFNLTKL REVMLDRKDM SEIRENHNDF DSIRAKVYTM 120 MDFVIYRYYI
EEAAKVNAAN KSLPDNEKSL SEKDIFVISL RGSFNEDQKD RLYYDEAQRL 180
WSKVGKLMLK IKKFRGKDTR KYKNMGTPRI RRLIPEGRDI STFSKLMYAL TMFLDGKEIN
240 DLLTTLINKF DNIQSFLKVM PLIGVNAKFA EEYSFFNNSE KIADELRLIK
SFARMGEPVA 300 DARRAMYIDA IRILGTDLSD DELKALADSF SLDENGNKLG
KGKHGMRNFI INNVITNKRF 360 HYLIRYGNPV HLHEIAKNEA VVKFVLGRIA
DIQKKQGQNG KNQIDRYYET CIGK. 414
[0152] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig525000349:
TABLE-US-00049 (SEQ ID NO: 89) MSKKENRKSY VKGLGLKSTL VSDSKVYLTT
FADGSNAKLE KCVENNKIIC ISNDKEAFAA 60 SIANKNVGYK IKNDEKFRHP
KGYDIISNNP LLHNNSVQQD MLGLKNVLEK RYFGKSSGGD 120 NNLCIQIIHN
IIDIEKILSE YIPNVVYAFN NIAGFKDEHN NIIDIIGTQT YNSSYTYADF 180
SKDKSDKKYI EFQKLLKNKR LGYWGKAFFT GQGNNAKVRQ ENQCFHIIAL LISLRNWATH
240 SNELDKHTKR TWLYKLDDTN ILNAEYVKTL NYLYDTIADE LTKSFSKNGA
VNVNYLAKKY 300 NIKDDLPGFS EQYFRFSIMK EQKNLGFNIS KLRENMLDFK DMSVI.
345
[0153] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig7229000302:
TABLE-US-00050 (SEQ ID NO: 90) KKISSLTKFC LGESDEKKLK ALAKKSLEEL
KTTNSKLYEN YIKYSDERKA EEAKRQINRE 60 RAKTAMNAHL RNTKWNDIMY
GQLKDLADSK SRICSEFRNK AAHLEVARYA HMYINDISEV 120 KSYFRLYHYI
MQRRIIDVIE NNPKAKYEGK VKVYFEDVKK NKKYNKNLLK LMCVPFGYCI 180
PRFKNLSIEQ MFDMNETDNS DKKKEK. 206
[0154] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig3227000343:
TABLE-US-00051 (SEQ ID NO: 91) IGDISEVNSY FQLYHYIMQR ILIDKIGSKT
TGKAKEYFDS VIVNKKYDDR LLKLLCSPLG 60 YCLTRYKDLS IEALFDMNEA
AKYDKLNKER KNKKK. 95
[0155] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Gut_metagenome_contig7030000469:
TABLE-US-00052 (SEQ ID NO: 92) SIRSKLYTMM DFVIYRYYIE ESAKAAAENK
PSESDSFVIR LRGSFNENQK EELYIEEAER 60 LWKKFGEIML KIKEFRGEKV
KEYKKEVPRI ERILPHGKDI SAFSKLMYML SMFLD. 115
[0156] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d gut_metagenome_P17E0k2120140920,
c87000043:
TABLE-US-00053 (SEQ ID NO: 93) MYFSKMIYML TYFLDGKEIN DLLTTLISKF
DNIKEFLKIM KSSAVDVECE LTAGYKLFND 60 SQRITNELFI VKNIASMRKP
AASAKLTMFR DALTILGIDD KITDDRISEI LKLKEKGKGI 120 HGLRNFITNN
VIESSRFVYL IKYANAQKIR EVAKNEKVVM FVLGGIPDTQ IERYYKSCVE 180
FPDMNSSLEA KRSELARMIK NISFDDFKNV KQQAKGRENV AKERAKAVIG LYLT.
234
[0157] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OBVH01003037.1, human gut metagenome sequence (also
found in WGS contigs emb|OBXZ01000094.11 and
emb|OBJF01000033.11):
TABLE-US-00054 (SEQ ID NO: 94) MAKKKRITAK ERKQNHRELL MKKADSNAEK
EKAKKPVVEN KPDTAISKDN TPKPNKEIKK 60 SKAKLAGVKW VIKANDDVAY
ISSFGKGNNS VLEKRIMGDV SSNVNKDSHM YVNPKYTKKN 120 YEIKNGFSSG
SSLVTYPNKP DKNSGMDALC LKPYFEKDFF GHIFTDNMHI QAIYNIFDIE 180
KILAKHITNI IYTVNSFDRN YNQSGNDTIG FGLNYRVPYS EYGGGKDSNG EPKNQSKWEK
240 RDNFIKFYNE SKPHLGYYEN IFYDHGEPIS EEKFYNYLNI LNFIRNNTFH
YKDDDIELYS 300 ENYSEEFVFI NCLNKFVKNK FKNVNKNFIS NEKNNLYIIL
NAYGKDTENV EVVKKYSKEL 360 YKLSVLKTNK NLGVNVKKLR ESAIEYGYCP
LPYDKEKEVA KLSSVKHKLY KTYDFVITHY 420 LNSNDKLLLE IVETLRLSKN
DDEKENVYKK YAEKLFKADD VINPIKAISK LFARKGNKLF 480 KEKIIIKKEY
IEDVSIDKNI YDFTKVIFFM TCFLDGKEIN DLLTNIISKL QVIEDHNNVI 540
KFISNNKDAV YKDYSDKYAI FRNAGKIATE LEAIKSIARM ENKIENAPQE PLLKDALLSL
600 GVSDDTKVLE NTYNKYFDSK EKTDKQSQKV STFLMNNVIN NNRFKYVIKY
INPADINGLA 660 KNRYLVKFVL SKIPEEQIDS YYKLFSNEEE PGCEEKIKLL
TKKISKLNFQ TLFENNKIPN 720 VEKEKKKAII TLYFTIVYIL VKNLVNINGL
YTLALYFVER DGYFYKDICG KKDKKKSYND 780 VDYLLLPEIF SGSKYREETK
NLKLPKEKDR DIMKKYLPND KDREKYNKFF TAYRNNIVHL 840 NIIAKLSELT
KNIDKDINSY FDIYHYCTQR VMFNYCKEKN DVVLAKMKDL AHIKSDCNEF 900
SSKHTYPFSS AVLRFMNLPF AYNVPRFKNL SYKKFFDKQ. 939
[0158] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig tpg|DJXD01000002.11 (uncultivated Ruminococcus assembly,
UBA7013, from sheep gut metagenome):
TABLE-US-00055 (SEQ ID NO: 95) MKKQKSKKTV SKTSGLKEAL SVQGTVIMTS
FGKGNMANLS YKIPSSQKPQ NLNSSAGLKN 60 VEVSGKKIKF QGRHPKIATT
DNPLFKPQPG MDLLCLKDKL EMHYFGKTFD DNIHIQLIYQ 120 ILDIEKILAV
HVNNIVFTLD NVLHPQKEEL TEDFIGAGGW RINLDYQTLR GQTNKYDRFK 180
NYIKRKELLY FGEAFYHENE RRYEEDIFAI LTLLSALRQF CFHSDLSSDE SDHVNSFWLY
240 QLEDQLSDEF KETLSILWEE VTERIDSEFL KTNTVNLHIL CHVFPKESKE
TIVRAYYEFL 300 IKKSFKNMGF SIKKLREIML EQSDLKSFKE DKYNSVRAKL
YKLFDFIITY YYDHHAFEKE 360 ALVSSLRSSL TEENKEEIYI KTARTLASAL
GADFKKAAAD VNAKNIRDYQ KKANDYRISF 420 EDIKIGNTGI GYFSELIYML
TLLLDGKEIN DLLTTLINKF DNIISFIDIL KKLNLEFKFK 480 PEYADFFNMT
NCRYTLEELR VINSIARMQK PSADARKIMY RDALRILGMD NRPDEEIDRE 540
LERTMPVGAD GKFIKGKQGF RNFIASNVIE SSRFHYLVRY NNPHKTRTLV KNPNVVKFVL
600 EGIPETQIKR YFDVCKGQEI PPTSDKSAQI DVLARIISSV DYKIFEDVPQ
SAKINKDDPS 660 RNFSDALKKQ RYQAIVSLYL TVMYLITKNL VYVNSRYVIA
FHCLERDAFL HGVTLPKMNK 720 KIVYSQLTTH LLTDKNYTTY GHLKNQKGHR
KWYVLVKNNL QNSDITAVSS FRNIVAHISV 780 VRNSNEYISG IGELHSYFEL
YHYLVQSMIA KNNWYDTSHQ PKTAEYLNNL KKHHTYCKDF 840 VKAYCIPFGY
VVPRYKNLTI NELFDRNNPN PEPKEEV. 877
[0159] An exemplary direct repeat sequence of CasRX/Cas13d
Metagenomic hit (no protein accession): contig tpg|DJXD01000002.11
(uncultivated Ruminococcus assembly, UBA7013, from sheep gut
metagenome) (SEQ ID NO: 95) comprises or consists of the nucleic
acid sequence:
CasRX/Cas13d DR:
TABLE-US-00056 [0160] (SEQ ID NO: 96) caactacaac cccgtaaaaa
tacggggttc tgaaac. 36
[0161] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig OGZC01000639.1 (human gut metagenome assembly):
TABLE-US-00057 (SEQ ID NO: 97) MKKKNIRATR EALKAQKIKK SQENEALKKQ
KLAEEAAQKR REELEKKNLA QWEETSAEGR 60 RSRVKAVGVK SVFVVGDDLY
LATFGNGNET VLEKKITPDG KITTFPEEET FTAKLKFAQT 120 EPTVATSIGI
SNGRIVLPEI SVDNPLHTTM QKNTIKRSAG EDILQLKDVL ENRYFDRSFN 180
DDLHIRLIYN ILDIEKILAE YTTNAVFAID NVSGCSDDFL SNFSTRNQWD EFQNPEQHRE
240 HFGNKDNVIC SVKKQQDLFF NFFKNNRIGY FGKAFFHAES ERKIVKKTEK
EVYHILTLIG 300 SLRQWITHST EGGISRLWLY QLEDALSREY QETMNNCYNS
TIYGLQKDFE KTNAPNLNFL 360 AEILGKNASE LAEPYFRFII TKEYKNLGFS
IKTLREMLLD QPDLQEIREN HNVYDSIRSK 420 LYKMIDFVLV YAYSNERKSK
ADALASNLRS AITEDAKKRI YQNEADQLWT SYQELFKRIR 480 GFKGAQVKEY
SSKNMPIPIQ KQIQNILKPA EQVTYFTKLM YLLTMFLDGK EINDLLTTLI 540
NKFDNISSLL KTMEQLELQT TFKEDYTFFQ QSSRLCKEIT QLKSFARMGN PISNLKEVMM
600 VDAIQILGTE KSEQELQSMA CFFFRDKNGK KLNTGEHGMR NFIGNNVISN
TRFQYLIRYG 660 NPQKLHTLSQ NETVVRFVLS RIAKNQRVQG MNGKNQIDRY
YETCGGTNSW SVSEEEKINF 720 LCKILTNMSY DQFQDVKQSG AEITAEEKRK
KERYKAIISL YLTVLYQLIK NLVNINARYI 780 IAFHCLERDA ILYSSKFNTS
INLKKRYTAL TEMILGYETD EKARRKDTRT VYEKAEAAKN 840 RHLKNVKWNC
KTRENLENAD KNAIVAFRNI VAHLWIIRDA DRFITGMGAM KRYFDCYHYL 900
LQRELGYILE KSNQGSEYTK KSLEKVQQYH SYCKDFLHML CLPFAYCIPR YKNLSIAELF
960 DRHEPEAEPK EEASSVNNSQ FITT. 984
[0162] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OHBM01000764.1 (human gut metagenome assembly):
TABLE-US-00058 (SEQ ID NO: 98) XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX
XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX 60 XXXXXXXXXX XXXXXXXXXX
XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX 120 XXXXXXXXXX
XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX 180
XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXHPLQKRYR YLTSTNLKSF
240 ETYKNNLVNK KKFDLDRVKK IPQLAYFGSA FYNTPEDTSA KITKTKIKSN
EEIYYTFMLL 300 STARNFSAHY LDRNRAKSSD AEDFDGTSVI MYNLDNEELY
KKLYNKKVHM ALTGMKKVLD 360 ANFNKKVEHL NNSFIKNSAK DFVILCEVLG
IKSRDEKTKF VKDYYDFVVR KNYKHLGFSV 420 KELRELLFAN HDSNKYIKEF
DKISNKKFDS VRSRLNRLAD YIIYDYYNKN NAKVSDLVKY 480 LRAAADDEQK
KKIYLNESIN LVKSGILERI KKILPKLNGK IIGNMQPDST ITASMLHNTG 540
KDWHPISENA HYFTKWIYTL TLFMDGKEIN DLVTTLINKF DNIASFIEVL KSQSVCTHFS
600 EERKMFIDSA EICSELSAMN SFARMEAPGA SSKRAMFVEA ARILGDNRSK
EELEEYFDTL 660 FDKSASKKEK GFRNFIRNNV VDSNRFKYLT RYTDTSSVKA
FSNNKALVKF AIKDIPQEQI 720 LRYYNSCFGA SERYYNDGMS DKLVEAIGKI
NLMQFNGVIQ QADRNMLPEE KKKANAQKEK 780 YKSIIRLYLT VCYLFFKNLV
YVNSRYYSAF YNLEKDRSLF EINGELKPTG KFDEGHYTGL 840 VKLFIDNGWI
NPRASAYLTV NLANSDETAI RTFRNTAEHL EALRNADKYL NDLKQFDSYF 900
EIYHYITQRN IKEKCEMLKE QTVKYNNDLL KYHGYSKDFV KALCVPFGYN LPRFKNLSID
960 ALFDKNDKRE KLKKGFED. 978
[0163] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OHCP01000044.1 (human gut metagenome assembly):
TABLE-US-00059 (SEQ ID NO: 99) MAKKITAKQK REEKERLNKQ KWAKNDSVII
VPETKEEIKT GEIQDNNRKR SRQKSQAKAM 60 GLKAVLSFDN KIAIASFVSS
KNAKSSHIER ITDKEGTTIS VNSKMFESSV NKRDINIEKR 120 ITIEEPQQDG
TIKKEEKGVK STTCNPYFKV GGKDYIGIKE IAEEHFFGRA FPNENLRVQI 180
AYNIFDVQKI LGTFVNNIIY SFYNLSRDEV QSDNDVIGML YSISDYDRQK ETETFLQAKS
240 LLKQTEAYYA YFDDVFKKNK KPDKNKEGDN SKQYQENLRH NFNILRVLSF
LRQICMHAEV 300 HVSDDEGCTR TQNYTDSLEA LFNISKAFGK KMPELKTLID
NIYSKGINAI NDEFVKNGKN 360 NLYILSKVYP NEKREVLLRE YYNFVVCKEG
SNIGISTRKL KETMIAQNMP SLKEENTYRN 420 KLYTVMNFIL VRELKNCATI
REQMIKELRA NMDEEEGRDR IYSKYAKEIY LYVKDKLKLM 480 LNVFKEEAEG
IIIPGKEDPV KFSHGKLDKK EIESFCLTTK NTEDITKVIY FLCKFLDGKE 540
INELCCAMMN KLDGISDLIE TAKQCGEDVE FVDQFKCLSK CATMSNQIRI VKNISRMKKE
600 MTIDNDTIFL DALELLGRKI EKYQKDKNGD YVKDEKGKKV YTKDYNNFQD
MFFEGKNHRV 660 RNFVSNNVIK SKWFSYVVRY NKPAECQALM RNSKLVKFAL
DELPDSQIEK YYISVFGEKS 720 SSSNEEMRRE LLKKLCDFSV RGFLDEIVLL
SEDEMKQKDK FSEKEKKKSL IRLYLTIVYL 780 ITKSMVKINT RFSIACATYE
RDYILLCQSE KAERAWEKGA TAFALTRKFL NHDKPTFEQY 840 YTREREISAM
PQEKRKELRK ENDQLLKKTH YSKHAYCYIV DNVNNLTGAV ANDNGRGLPC 900
LSEKNDNANL FLEMRNKIVH LNVVHDMVKY INEIKNITSY YAFFCYVLQR MIIGNNSNEQ
960 NKFKAKYSKT LQEFGTYSKD LMWVLNLPFA YNLPRYKNLS NEQLFYDEEE
RMEKIVGRKN 1020 DSR. 1023
[0164] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OGDF01008514.11 (human gut metagenome assembly):
TABLE-US-00060 (SEQ ID NO: 100) MTETKPKRED IAKTPAAKSR SKAAGLKSTF
AVNGSVLLTS FGRGNDAVPE KLITEKAVSE 60 INTVKPRFSV EKPATSYSSS
FGIKSHISAT ADNPLAGRAP VGEDAIHAKE VLEQRVFGKT 120 FSDDNIHIQL
IYNILDIRKI LSTYANNVVF TINSMRRLDE YDREQDYLGY LYTGNSYERL 180
LDIADKYAVD GEDWRNTAAG ISNDFEKKQF QTINGFWDLL DMIEPYMCYF SEAFFCETTV
240 KDPDSGRIVP CLEQRSDGDI YNILRILSIV RQTCMHDNAS MRTVMFTLGQ
NSVRDRKNGF 300 DELAELLDYL YDEKIDIVNR DFLRNQKNNI ELLSRIYGSS
ADSPERDRLV QNFYDFRVLS 360 QDKNLGFSIK KLREKLLDSP ALSVVRSKKY
DTMRSKIYSL IDFMIYRKFS ENHVAVDDFV 420 EELRSLLTED EKESAYSRWA
ETLINDGFAQ EILVKLLPQT DPAVIGKIKG KKLLNDSIAG 480 IKLKKDASFF
TKIINVLCMF QDGKEINELV SSLVNKFANI QSFVDVMRSQ GIDSGFTADY 540
AMFAESGRIS RELHILKGIA RMQHSIAGLG DVKIYGSDDK FHGVSRRVYT DAAYILGFGE
600 RSEDNDGYVD DYVSSKLLGG ADKNLRNFIT NNVIKNRRFL YTVRYMNPKR
AKKLVQNDAL 660 VVLALSGIPE TQIDRYYKSC IEKRSFNPDL NEKIAALSEM
ITTLKIDDFE DVKQNPEKNA 720 NYEAKKNQRI SKERYKACIG LYLTVLYLIC
KNLVKINARY SIAIGCLERD TQLHGVDFKG 780 AAYMTRDVFI AKGWINPKKP
TVKSIKEQYA FLTPYIFTTY RNMIAHLAAV TNAYKYIPQM 840 DRFKSWFHLY
HTVIQHSLIQ QYEYDRDYGR KGAPVVSERV LQLLEQCREH SNYSRDLLHI 900
LNLPFGYNLP RYLNLSSEKY FDANAI. 926
[0165] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OGPN01002610.1 (human gut metagenome assembly):
TABLE-US-00061 (SEQ ID NO: 101) MAKKITAKQK REEKERLNKQ KWAKQDTPVV
PKSKTEEKPV AASDDKLLKT TQVKKVQTKS 60 KAKAMGLKTV LSFDDKIAIA
SFVNDKKTKL PHIERITDKS GTTIHENARM FDSSVDEQNV 120 NIEKRMTIEE
KQNDGTFKKD EKDVKATICN PYFKTCGKDY IGIKDVAEKY FFGKTFPNEN 180
LRVQIAYNVF DIQKILGTYV NNIIYSFYNL RRDGKSDVDI IGSLYAFADF DNQLKDKPAF
240 REAKDLLKNT EAYFSYFGDV FKKSKKGKKD ENNEDYEKNL RHNFNVLRVL
SFLRQICTHA 300 YVKCTGGAKN NGDSTKVEAE SLDALFNITE YFAKTAPELS
KTINEIYKEG IDRINNDFVT 360 NGKNNLYILS KVYPDMQRNE LVKKYYQFVV
CKEGNNVGIN TRKLKESIIS QHPWITTPQD 420 NNKANDYESC RHKLYTIMCF
ILVAELDAHE SIRDNMVAEL RANMDGDDGR DAIYEKYAKD 480 IYHIVKDKLL
AMQKVFDEEL VPVKVEGKND PQQFTHGKLG KKEIESFCLS DKNTSDIAKV 540
VYFLCNFLDG KEINELCCAM MNKFDGIGDL IDTAKQCGEE VKFIEEFACL SNCRKITNDI
600 RVAKSISKMK NKVNIDNDII YLDAIELLGR KIEKYQKDEN GKILLGTDGK
RLYTQEYKYF 660 NDMFFNAGNH KVRNFIANNV MQSKWFFYVV RYNKPAECQI
IMRNKTLVKF TLDDLPDMQI 720 QRYYSSVFGD NNMPAVDEMR KRLLDKINQF
SVRGFLDELD EIVLMSDEES KRNKSSEKEQ 780 KKSLIRLYLT IAYLITKSMV
KINTRFSIAC AMYERDYALL CQSEMKGGPW DGGAQALAVT 840 RKFLNHDREV
FDRYCAREAE IARLPSEERK PLRKANDKLL KQTHYTNHSY TYIVNNLNSF 900
TDIDYCAKDV GLPAPNDKND NASILGEMRN DIAHLNIVHD MVKYIEELKD ISSYYAFYCY
960 VLQRRLVGKD PNCQNKFKAK YAKELNDYGT YNKNLMWMLN LPFAYNLPRY
KNLSSEFLFY 1020 DMEYNKKDDE. 1030
[0166] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
from contig emb|OBLI01020244 and emb OBLIO1038679 (from pig gut
metagenome):
TABLE-US-00062 (SEQ ID NO: 102) MAKKITAKQR REERERQNKQ KWAKKQADAT
AVFECEADIK PADSKDEDCT NIYIKREKKK 60 TQAKAMGLKT VLGFDNKIAI
ASFMSSKDSK SSHIERITDP NGKTIREDVR MFDSNVDECS 120 INLEKRMTVE
ERQKDGTIKK DEKDVKSTIC NPYSNECGKD YIGIKSVAEE LFFGRTFPND 180
NLRVQIAYNI FDIQKILGTY INNIIYSFYN LSRDESQSDN DVIGTLYMLK DFDGQKETDT
240 FRQARALLER TEAYYSYFDN VFKKIDKNKK KSDDCKRERN EILRYNFNVL
RVLSFLRQIC 300 AHAQVKISNE HDREKGGGLV DSLDALFNIS RFFDAVAPEL
NEVINSVYSK GIDDINDNFV 360 KNGKNNFYIL SKIYPEVARE DLLREYYYFV
VSKEGNNIGI STKKLKEAII VQDMSYIKSE 420 DYDTYRNKLY TVLCFILVKE
LNERTTIREQ MVADLRANMN GDIGREDIYS KYAKIIYAQV 480 KPRFDTMKSA
FEEEAKDVIV PDKKKPVKFS HGKLDKNEIE RFCITSANTD SVAKIIYFLC 540
KFLDGKEINE LCCAMMNKLD GINDLIETAE QCGAKVEFVD KFSVLSNCET ISDQIRIVKS
600 ISKMKKEIAI DNDTIFLDAL ELLGRKIDKY KKDATGKYLK DENGKYLYSK
EYDDFQYMFF 660 KDSHRVRNFI SNSVIKSKWF SYIVRYNQPS ECRAIMKNKT
LVKFALDELP DLQIQRYFVA 720 LYGDEDLPSY GEMRKILLKK LHDFSIKGFL
DEIVLLSDLD MESQDKYCEK EQKKSLFRLY 780 LTIAYLITKS MVKINTRFSI
ACATYERDYA LLCASNKQER AWSSGATALA LTRRFLNQDK 840 LIFEKHYARE
GEISKLPKEE RKAMRKVNDQ LLKRTHFSKH SYCYIVDNVN RLTGGECRTD 900
KRVLPVLNEK NDNAGILLDF RKTIAHLNVV HKMVDYVDEI KGITSYYAFF CYVLQRMLVG
960 NNLNEKNAIK EKYSATVKSF GTYSKDFMWL INLPFAYNLP RYKNLSNEQL
FYDEEERNET 1020 EEQIDRL. 1027
[0167] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig OIZX01000427.1:
TABLE-US-00063 (SEQ ID NO: 103) MAKKKKTARQ LREEMQQQRK QAIQKQQEQR
QEKAAAARET AAPEQPAAAP VPKRQRKSLA 60 KAAGLKSNFI LDPQRRTTVM
TAFGQGSTAI LEKQIVDRAI SDLQPVQQFQ VEPASAAKYR 120 LKNSRVRFPN
VTADDPLYRR KDGGFVPGMD ALRRKNVLEQ RFFGKSFADN IHIQMIYSIL 180
DIHKILAAAS GHIVHLLNIV NGSKDRDFIG MLAAHVLYNE LNEEAKRSIA DFCKSPRLIY
240 YSAAFYETLD NGKSERRSNE DIFNILALMT CLRNFSSHHS IAIKVKDYSA
AGLYNLRRLG 300 PDMKKMLDTF YTEAFIQLNQ SFQDHNTTNL TCLFDILNIS
DSARQKQLAE EFYRYVVFKE 360 QKNLGFSVRK LREEMLLLPD AAVIADKRYD
TCRSKLYNLM DFLILRVYRT GRADRCDKLP 420 EALRAALTDE EKAVVYHKEA
LSLWNEMRTL ILDGLLPQMT PENLSRLSGQ KRKGELSLDD 480 AMLKECLYEP
GPVPEDAAPE EANAEYFCRM IYLATLFMDG KEINTLLTTL ISKFENIAAF 540
LQTMEQLNIE AELGPEYAMF TRSRAVAEQL RVINSFALMK KPQVNAKQQL YRAAVTLLGT
600 EDPDGVTDEM LCIDPVTGKM LPPNQRHHGD TGLRNFIANN VVESRRFQYL
IRYSDPAQLH 660 QLASNKKLVR FVLSSIPDTQ INRYYETCGQ TRLAGRAAKV
EFLTDMIAAI RFDQFRDVNQ 720 KERGANTQKE RYKAMLGLYQ TVLYLAVKNL
VNINARYVMA FHCVERDMFL YDGELTDPKG 780 ESVSAFLAVN GKKGVQPQYL
LLTQLFIRRD YLKRSACEQI QHNMENISDR LLREYRNAVA 840 HLNVIAHLAD
YSADMREITS YYGLYHYLMQ RHLFKRHAWQ IRQPERPTEE EQKLIEQEQK 900
QLAWEKALFD KTLQYHSYNK DLVKALNAPF GYNLARYKNL SIEPLFSKEA APAAEIKATH
960 A. 961
[0168] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig OCTW011587266.1:
TABLE-US-00064 (SEQ ID NO: 104) MKQNDRENNN KIKKSAAKAV GVKSLARLSD
GSTVVSSFGK GAAAELESLI TGGEIRKLSD 60 KAILEITDDT QNKNAYNVKS
SRIPNLTART DKLSDKSGMD DLGFKRELEL EVFGQCFDDS 120 IHIQIAHAVF
DIQKSLAAVI PNVLYTLNNL DRSYSTDNTS DKKDIIGNTL NYQHSYESFN 180
VEKRGEFTEY YNAAKDRFSY FPDILCVLEK VNGKDRYQPK SEKDAFNVLS SVNMLRNSLF
240 HFAPKSNDGK ARIAVFKNQF DSDFSHITST VNKIYSAKIA GVNENFLNNE
GNNLYIILKA 300 TNWDIKKIVP QLYRFSVLKS DKNMGFNMRK LREFAVESKN
IDLSRLNDKF LTNNRKKLYK 360 VIDFIIYYHL NKVLKDSFVD DFVAALRASQ
SEEEKEKLYA QYSERLFADE GLKSAIKKAV 420 DMISDTKSNI FKMKTPLDKA
LIENIKVNSD ASDFCKLIYV FTRFLDGKEI NILLNSLIKK 480 FQDIHSFNTT
VKKLSENNLI INADYVDDYS LFEQSGTVAR ELMLIKSISK MDFGLDNINL 540
SFMYDDALRT LGVSDENLPE VKREYFGKTK NLSAYIRNNV LENRRFKYVI KYIHPSDVQK
600 IACNKAIAGF VLNRMPDTQI KRYYDSLINK GATDIQAQAK ALLDCITGIS
FDAIKDDKHL 660 HKSKEKSPQR SADRERKKAM LTLYYTIVYI FVKQMLHINS
LYTIGFFYLE RDQRFIYSRA 720 KKENKNPSKN SYLNDFRSVT AYFIPSEIMK
RIEKNENKGF LEDFEALWNS CGKTSRLRKE 780 DVLLYARYIS PDHALKNYKM
ILNSYRNKIA HINVIMSAGK YTGGIKRMDS YFSVFQHLVQ 840 CDILSNPNNK
GKCFESESLK PLLLDMKFDG TDEKLYSKRL TRALNIPFGY NVPRYKNLTF 900
EKIYLKSSIN E. 911
[0169] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OGNF01009141.1:
TABLE-US-00065 (SEQ ID NO: 105) MADIDKKKSS AKAAGLKSTF VLENNKLLMT
SFGNGNKAVI EKIIDEKVDS INEPEVFSVT 60 PCDKKFELQP AKRGLAADSL
VDNPLKSKKT AGDDAIHSRK FLERQFFDGN TFNDNIHIQL 120 IYNILDIEKI
LSVHVNDIVY SVNNILSRGE GMEYNDYIGT LNLKSFETYK NNLVNKKKFD 180
LDRVKKIPQL AYFGSAFYNT PEDTSAKITK TKIKSNEEIY YTFMLLSTAR NFSAHYLDRN
240 RAKSSDAEDF DGTSVIMYNL DNEELYKKLY NKKVHMALTG MKKVLDANFN
KKVEHLNNSF 300 IKNSAKDFVI LCEVLGIKSR DEKTKFVKDY YDFVVRKNYK
HLGFSVKELR ELLFANHDSN 360 KYIKEFDKIS NKKFDSVRSR LNRLADYIIY
DYYNKNNAKV SDLVKYLRAA ADDEQKKKIY 420 LNESINLVKS GILERIKKIL
PKLNGKIIGN MQPDSTITAS MLHNTGKDWH PISENAHYFT 480 KWIYTLTLFM
DGKEINDLVT TLINKFDNIA SFIEVLKSQS VCTHFSEERK MFIDSAEICS 540
ELSAMNSFAR MEAPGASSKR AMFVEAARIL GDNRSKEELE EYFDTLFDKS ASKKEKGFRN
600 FIRNNVVDSN RFKYLTRYTD TSSVKAFSNN KALVKFAIKD IPQEQILRYY
NSCFGASERY 660 YNDGMSDKLV EAIGKINLMQ FNGVIQQADR NMLPEEKKKA
NAQKEKYKSI IRLYLTVCYL 720 FFKNLVYVNS RYYSAFYNLE KDRSLFEING
ELKPTGKFDE GHYTGLVKLF IDNGWINPRA 780 SAYLTVNLAN SDETAIRTFR
NTAEHLEALR NADKYLNDLK QFDSYFEIYH YITQRNIKEK 840 CEMLKEQTVK
YNNDLLKYHG YSKDFVKALC VPFGYNLPRF KNLSIDALFD KNDKREKLKK 900 GFED.
904
[0170] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig emb|OIEN01002196.1:
TABLE-US-00066 (SEQ ID NO: 106) MERQKRKMKS KSKMAGVKSV FVIGDELLMT
SFGDGDDAVL EKDIDENGVV NDCRNPAAYD 60 AVYGTDSIRV KKTNNNIRAK
VNNPLAKSNI RSEESALFRT RVNEYKREQK DKYETLFFGK 120 TFDDNIHIQL
ISKILDIEKT FSVVIGNIVY AINNLSLEQS IDRPIDIFGD KNTQGISLRE 180
DNDYLKTMLP RCEYLFHNIL NSDSDNNSKM NYNKVNKGKE EKDNRNNENI EKLKKALEVI
240 KIIRVDSFHG VDGIKGDQKF PRSKYNLAVN YNEEIQKTIS EPFNRKVEEV
QQDFYRNSCV 300 NIDFLKEIMY GSNYTDRGSD SLECSYFNFA ILKQNKNMGF
SITSIRECLL DLYELNFESM 360 QNLRPRANSF CDFLIYDYYC KNESERANLV
DCLRSAASEE EKKNIYFQTA ERVKEKFRNA 420 FNRISRFDAS YIKNSREKNL
SGGSSLPKYS FIEGFTKRSK KINDNDEKNA DLFCNMLYYL 480 AQFLDGKEIN
IFLTSIHNIF QNIDSFLKVM KEKGMECKFQ KDFKMFSHAG HVAKKIEIVI 540
SLAKMKKTLD FYNAQALKDA VTILGVSKKH QYLDMNSYLD FYMFDNRSGA TGKNAGKDHN
600 LRNFLVSNVI RSRKFNYLSR YSNLAEVKKL AQNPSLVQFV LSRIEPSLIC
RYYESSQGIS 660 SEGITIDEQI KKLTGIIVDM NIDSFENINN GEIGMRYSKA
TPQSIERRNQ MRVCVGLYLN 720 VLYQIEKNLM NVNARYVLAF AFAERDALML
NFTLEECKKN KKRSSGGFSF IEMTQFFIDK 780 KLFKVATEAI KKNVLKYNGN
PESLNHIPGE YICKNMEGYH ENTVRNFRNM VAHLTAVARV 840 PLYISEVTQI
DSYYALYHYC MQMNILQGIE QSGKILDNIK LKNALENARV HRTYSKDAVK 900
YLCLPFAYNI SRYKALTIKD LFDWTEYSCK KDE. 933
[0171] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Metagenomic hit (no protein accession):
contig e-k87_11092736:
TABLE-US-00067 (SEQ ID NO: 107) MKRQKTFAKR IGIKSTVAYG QGKYAITTFG
KGSKAEIAVR SADPPEETLP TESDATLSIH 60 AKFAKAGRDG REFKCGDVDE
TRIHTSRSEY ESLISNPAES PREDYLGLKG TLERKFFGDE 120 YPKDNLRIQI
IYSILDIQKI LGLYVEDILH FVDGLQDEPE DLVGLGLGDE KMQKLLSKAL 180
PYMGFFGSTD VFKVTKKREE RAAADEHNAK VFRALGAIRQ KLAHFKWKES LAIFGANANM
240 PIRFFQGATG GRQLWNDVIA PLWKKRIERV RKSFLSNSAK NLWVLYQVFK
DDTDEKKKAR 300 ARQYYHFSVL KEGKNLGFNL TKTREYFLDK FFPIFHSSAP
DVKRKVDTFR SKFYAILDFI 360 IYEASVSVAN SGQMGKVAPW KGAIDNALVK
LREAPDEEAK EKIYNVLAAS IRNDSLFLRL 420 KSACDKFGAE QNRPVFPNEL
RNNRDIRNVR SEWLEATQDV DAAAFVQLIA FLCNFLEGKE 480 INELVTALIK
KFEGIQALID LLRNLEGVDS IRFENEFALF NDDKGNMAGR IARQLRLLAS 540
VGKMKPDMTD AKRVLYKSAL EILGAPPDEV SDEWLAENIL LDKSNNDYQK AKKTVNPFRN
600 YIAKNVITSR SFYYLVRYAK PTAVRKLMSN PKIVRYVLKR LPEKQVASYY
SAIWTQSESN 660 SNEMVKLIEM IDRLTTEIAG FSFAVLKDKK DSIVSASRES
RAVNLEVERL KKLTTLYMSI 720 AYIAVKSLVK VNARYFIAYS ALERDLYFFN
EKYGEEFRLH FIPYELNGKT CQFEYLAILK 780 YYLARDEETL KRKCEICEEI
KVGCEKHKKN ANPPYEYDQE WIDKKKALNS ERKACERRLH 840 FSTHWAQYAT
KRDENMAKHP QKWYDILASH YDELLALQAT GWLATQARND AEHLNPVNEF 900
DVYIEDLRRY PEGTPKNKDY HIGSYFEIYH YIRQRAYLEE VLAKRKEYRD SGSFTDEQLD
960 KLQKILDDIR ARGSYDKNLL KLEYLPFAYN LPRYKNLTTE ALFDDDSVSG
KKRVAEWRER 1020 EKTREAEREQ RRQR. 1034
[0172] An exemplary direct repeat sequence of CasRX/Cas13d
Metagenomic hit (no protein accession): contig e-k87_11092736 (SEQ
ID NO: 107) comprises or consists of the nucleic acid sequence:
TABLE-US-00068 CasRX/Cas13d Direct repeat 1: (SEQ ID NO: 108)
gtgagaagtc tccttatggg gagatgctac.
[0173] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Ga0129306_1000735:
TABLE-US-00069 (SEQ ID NO: 109) MQKQREQQTV TDESERKKKP LKSGAKAAGL
KSVFVLSEGK ELLTSFGRGN EAVPEKRVTG 60 GTIANARTDN KEAFSAALQN
KRFEVFGRTA GSSDDPLAVS RAPGQDLIGA KTALEERYFG 120 RAFADNIHMQ
VIYAIQDINK ILAVHANNIV YTLNNLDREA DPETDDFIGS GYLTLKNTFE 180
TYCDPAALNE REREKVTVSK QHFDAFMQNP RLAYYGNAFF RKLSKAERLA RGREIFDKES
240 PERRQEILGS RGKNKSVDDE IRALAPEWVK REERDVYSEL VLMSELRQSC
FHGQQKNSAR 300 IFRLDNDLGP GVDGARELLD RLYAEKINDL RSFDKTSASS
NFRLLFNAYH ADNEKKKELA 360 QEFYRFSVLK VSKNTGFSIR TLREKIIEDH
AAQYRDKIYD SMRKKLFSTF DFFLWRFYEE 420 REDEAEELRA CLRAARSDEE
KEQIYAEAAA SCWPSVKPFV ESVAATLCDV VKGRTKLNKL 480 KLSADESTLV
RNAIDGVRIS PRASYFTKLI YLMTLFLDGK EINDLLTTLI HAFENIDSFL 540
SVLGSERLER TFDANYRIFA DSGVIAQELR AVNSFARMTT EPFNSKLVMF EDAAQLFGMS
600 GGLVEHAEEL REYLDNKMLD KTKLRLLPDG KVDTGFRNFI ISNVTESRRF
RYLVRYCEPR 660 AVRDYMSCRP LIRLTLRDMP DTILRRYYEQ SVGAATVDRE
RILDTLADKL LSLRFTDFEN 720 VNQRANAERN REKQKMMGII SLYLNVAYQI
VKNLVYVNAR YTMAYHCAER DTELLLNAAG 780 EGNLLRRDRS WPARLHLPRR
ALARRRDRVE VMERDVARGP EAYNRDEWLG LVRTLRREKR 840 VCDNLHNNYA
YLCGADAEPG DASLSLLFVY RNKAAHLSVL NKGGRLSGDL KEAKSWFYVY 900
HFLMQRVLEE EFRNTQALPE RLRELLMMAE RYRGCSKDLI KVLNLTFAYN LPRYKNLSID
960 GRFDKNHPDP SDE. 973
[0174] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Ga0129317_1008067:
TABLE-US-00070 (SEQ ID NO: 110) MKKQKKSLVK AAGLKSAFVV GDSVYLTSFG
KGNAARLDTK INPDNSTERY VSDSEKHTLK 60 INSITDTELR LSGPFPKQAE
AKNPTHKKDN EQKNTRQDML GLKSTLEKFY FGSTFDDNIH 120 IQIIHNIQDI
AKILAAHSNN AGYALDNMLA YQGVEFSDMI GYMGTSRTFD NYDPNHKNNK 180
DFFRFLKLPR LGYFGSAFYS QKGKDFEKRS DEEVYNICAL MGQIRQCCFH GKQEKYQLKW
240 LYNFHNFKSN KPFLDTLDKH FDEMIDRINK NFIKNNTPDL IILSGLYPDM
AKKELVRLFY 300 DFTTVKEYKN MGFSVKKLRE KMLESEEASD FRDKDYDSVR
RKLYKLMDFC IYYLYYSDSE 360 RNENLVSRLR ESLTDENKDI IYSKEAKIVW
NELRKKFSTI LDNVKGSNIK KLENVKEKFI 420 SEDEFDDIKL DIDISYFSKL
MYVMCYFLDG KEINDLLTTL VSKFDNIGSI IEAATQIGIN 480 IEFIDDFKFF
DRSKDISVEL NIIRNFARMQ APVPNAKRAM QEDAIRILGG SEEDIFSILD 540
DMTGYDKSGK KLAQSKKGFR NFIINNVVES SRFKYIVRYS NPQKIRKLAN NSVVVGFVLG
600 KLPDAQIESY FNSCLPNRVY STPDKARESL RDMLHNISFN DFADVKQDDR
RATPEEKVEK 660 ERYKAIIGLY LTVMYHLVKN LVYVNSRYVM AFHCLERDAM
HYDVSLDNYR DLIRHLISEG 720 DSSCNHFISH NRRMRDCIEE NVKNSEQLIF
GKEDAVIRFR NNVAHLSAIR NANEYIGDIR 780 EITSYFALYH YLMQRKLIDD
CKVNDTAHKY FEQLTKYKTY VMDMVKALCS PFGYNLPRFK 840 NLSIEGKFDM HESK.
854
[0175] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d Ga0224415_10048792:
TABLE-US-00071 (SEQ ID NO: 111) MSKKENRKSY VKGLGLKSTL VSDSKVYLTT
FADGSNAKLE KCVENNKIIC ISNDKEAFAA 60 SIANKNVGYK IKNDEKFRHP
KGYDIISNNP LLHNNSVQQD MLGLKNVLEK RYFGKSSGGD 120 NNLCIQIIHN
IIDIEKILSE YIPNVVYAFN NIAGFKDEHN NIIDIIGTQT YNSSYTYADF 180
SKDKSDKKYI EFQKLLKNKR LGYWGKAFFT GQGNNAKVRQ ENQCFHIIAL LISLRNWATH
240 SNELDKHTKR TWLYKLDDTN ILNAEYVKTL NYLYDTIADE LTKSFSKNGA
VNVNYLAKKY 300 NIKDDLPGFS EQYFRFSIMK EQKNLGFNIS KLRENMLDFK
DMSVIRDDHN RYDKDRSKIY 360 TMMDFVIYRY YIDNNNDSID FINKLRSSID
EKSKEKLYNE EANRLWNKLK EYMLYIKEFN 420 GKLASRTPDR DGNISEFVES
LPKIHRLLPR GQKISNFSKL MYLLTMFLDG KEINDLLTTL 480 INKFENIQGF
LDIMPEINVN AKFEPEYVFF NKSHEIAGEL KLIKGFAQMG EPAATLKLEM 540
TADAIKILGT EKEDAELIKL AESLFKDENG KLLGNKQHGM RNFIGNNVIK SKRFHYLIRY
600 GDPAHLHKIA TNKNVVRFVL GRIADMQKKQ GQKGKNQIDR YYEVCVGNKD
IKKTIEEKID 660 ALTDIIVNMN YDQFEKKKAV IENQNRGKTF EEKNKYKRDN
AEREKFKKII SLYLTVIYHI 720 LKNIVNVNSR YILGFHCLER DKQLYIEKYN
KDKLDGFVAL TKFCLGDEER FEDLKAKAQA 780 SIQALETANP KLYAKYMNYS
DEEKKEEFKK QLNRERVKNA RNAYLKNIKN YIMIRLQLRD 840 QTDSSGYLCG
EFRDKVAHLE VARHAHEYIG NIKEVNSYFQ LYHYIMQCRL YDVLKNNTKA 900
EAMVKGKAKE YFEALEKEGT YNDKLLKIAC VPFGYCIPRY KNLSMEELFD MNEEKKFKKK
960 APENT. 965
[0176] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence CasRX/Cas13d 160582958 gene49834:
TABLE-US-00072 (SEQ ID NO: 112) MKNSVTFKLI QAQENKEAAR KKAKDIAEQA
RIAKRNGVVK KEENRINRIQ IEIQTQKKSN 60 TQNAYHLKSL AKAAGVKSVF
AIGNDLLMTG FGPGNDATIE KRVFQNRAIE TLSSPEQYSA 120 EFQNKQFKIK
GNIKVLNHST QKMEEIQTEL QDNYNRPHFD LLGCKNVLEQ KYFGRTFSDN 180
IHVQIAYNIM DIEKLLTPYI NNIIYTLNEL MRDNSKDDFF GCDSHFSVAY LYDELKAGYS
240 DRLKTKPNLS KNIDRIWNNF CNYMNSDSGN TEARLAYFGE LFYKPKETGD
AKSDYKTHLS 300 NNQKEEWELK SDKEVYNIFA ILCDLRHFCT HGESITPSGK
PFPYNLEKNL FPEAKQVLNS 360 LFEEKAESLG AEAFGKTAGK TDVSILLKVF
EKEQASQKEQ QALLKEYYDF KVQKTYKNMG 420 FSIKKLREAI MEIPDAAKFK
DDLYSSLRHK LYGLFDFILV KHFLDTSDSE NLQNNDIFRQ 480 LRACRCEEEK
DQVYRSIAVK VWEKVKKKEL NMFKQVVVIP SLSKDELKQM EMTKNTELLS 540
SIETISTQAS LFSEMIFMMT YLLDGKEINL LCTSLIEKFE NIASFNEVLK SPQIGYETKY
600 TEGYAFFKNA DKTAKELRQV NNMARMTKPL GGVNTKCVMY NEAAKILGAK
PMSKAELESV 660 FNLDNHDYTY SPSGKKIPNK NFRNFIINNV ITSRRFLYLI
RYGNPEKIRK IAINPSIISF 720 VLKQIPDEQI KRYYPPCIGK RTDDVTLMRD
ELGKMLQSVN FEQFSRVNNK QNAKQNPNGE 780 KARLQACVRL YLTVPYLFIK
NMVNINARYV LAFHCLERDH ALCFNSRKLN DDSYNEMANK 840 FQMVRKAKKE
QYEKEYKCKK QETGTAHTKK IEKLNQQIAY IDKDIKNMHS YTCRNYRNLV 900
AHLNVVSKLQ NYVSELPNDY QITSYFSFYH YCMQLGLMEK VSSKNIPLVE SLKNEANDAQ
960 SYSAKKTLEY FDLIEKNRTY CKDFLKALNA PFSYNLPRFK NLSIEALFDK
NIVYEQADLK 1020 KE. 1022
[0177] An exemplary direct repeat sequence of CasRX/Cas13d proteins
may comprise or consist of the sequence
CasRX/Cas13d 160582958_gene49834 (SEQ ID NO: 112) comprises or
consists of the nucleic acid sequence: CasRX/Cas13d DR:
TABLE-US-00073 (SEQ ID NO: 113) gaactacacc cctctgttct tgtaggggtc
taacac. 36
[0178] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d 250twins_35838_GL0110300:
TABLE-US-00074 (SEQ ID NO: 114) MGNKQRVSAQ KRRENAKLCN QQKARQAESQ
RDKIKNMNVE KMKNINTNDI KHTKTTAKKL 60 GLKSTIIADK KIILTSFINE
QSSKTANIEK VAGFKGDTID TISYTPRMFR SEINPGEIVI 120 SKGDDLSEFA
NPANFPIGRD YVKIRSALEK QYFGKEFPED NLHVQIAYNV ADIKKILSVY 180
INNIIYMFYN LARSEEYDIF YNSQSENSGR DCDVIGSLYY QASYRNQDAN RFEKDGKKKA
240 IDSLLDDTRA YYTYFDGLFS VPKREDDGKI KESEKEKAKD QNFDVLRLLS
VGRQLTFHSD 300 KSNNEAYLFD LSKLTRAAQD ENRRQDIQSL LNILNSTCRS
NLEGVNGDFV KHAKNNLYVL 360 NQLYPSLKAN DLIGEYYNFI VKKENRNIGI
RLITVRELII EHNYTNLKDS KYDTYRNKIY 420 TVLNFILFRE IQENSIAIKN
FREKLRSTEK AEQPALYQAF ANKIYPMVQA KFAKAIDLFE 480 EQYKTKFKSE
FKGGISIENM QQQNILLQTE NIDYFSKYVL FLTKFLDGKE INELLCALIN 540
KFDNIADLLD ISKQIGTPVV FCADYESLND AAKIAENIRL IKNIAHLRPA IQEAQSSKDN
600 ADAAGTPATL LIDAYNMLNT DIQLVYGEAA YEELRKDLFE RKNGTKYNKK
GKKVDVYDHK 660 FRNFLINNVI KSKWFFYIAK YVKPADCAKM MSNKKMIEFA
LRDLPETQIK RYYYTITGNE 720 ALGDAESLKG VIIEQLHAFS IKNTLLSIKN
MGEGEYKIQQ IGSSKEKLKA IVNLYLTVAY 780 LLTKSLVKVN IRFSIAFGCL
ERDLVLQKKS EKKFDAIINE ILLEDDKIRK ECDKERAQAK 840 TLPRELAQER
FAQIKRRESG CYFKSYHVYD YLSKNSNEFK QNHIDFAVTS YRNNVEHLNV 900
VHCMTKYFSE VKDVKSYYGV YCYIMQRMLC DELIIKNQDK PDVRQTFEEY NRLLKDHGTY
960 SKNLMWLLNF PFAYNLARYK NLSNEDLFNA KNNDQKSK. 998
[0179] Exemplary CasRX/Cas13d proteins may comprise or consist of
the sequence: CasRX/Cas13d 250twins_36050_GL0158985:
TABLE-US-00075 (SEQ ID NO: 115) MKKKHQSAAE KRQVKKLKNQ EKAQKYASEP
SPLQSDTAGV ECSQKKTVVS HIASSKTLAK 60 AMGLKSTLVM GDKLVITSFA
ASKAVGGAGY KSANIEKITD LQGRVIEEHE RMFSADVGEK 120 NIELSKNDCH
TNVNNPVVTN IGKDYIGLKS RLEQEFFGKT FENDNLHVQL AYNILDIKKI 180
LGTYVNNIIY IFYNLNRAGT GRDERMYDDL IGTLYAYKPM EAQQTYLLKG DKDMRRFEEV
240 KQLLQNTSAY YVYYGTLFEK VKAKSKKEQR AKEAEIDACT AHNYDVLRLL
SLMRQLCMHS 300 VAGTAFKLAE SALFNIEDVL SADLKEILDE AFSGAVNKLN
DGFVQHSGNN LYVLQQLYPN 360 ETIERIAEKY YRLTVRKEDL NMGVNIKKLR
ELIVGQYFPE VLDKEYDLSK NGDSVVTYRS 420 KIYTVMNYIL LYYLEDHDSS
RESMVEALRQ NREGDEGKEE IYRQFAKKVW NGVSGLFGVC 480 LNLFKTEKRN
KFRSKVALPD VSGAAYMLSS ENIDYFVKML FFVCKFLDGK EINELLCALI 540
NKFDNIADIL DAAAQCGSSV WFVDSYRFFE RSRRISAQIR IVKNIASKDF KKSKKDSDES
600 YPEQLYLDAL ALLGDVISKY KQNRDGSVVI DDQGNAVLTE QYKRFRYEFF
EEIKRDESGG 660 IKYKKSGKPE YNHQRRNFIL NNVLKSKWFF YVVKYNRPSS
CRELMKNKEI LRFVLRDIPD 720 SQVRRYFKAV QGEEAYASAE AMRTRLVDAL
SQFSVTACLD EVGGMTDKEF ASQRAVDSKE 780 KLRAIIRLYL TVAYLITKSM
VKVNTRFSIA FSVLERDYYL LIDGKKKSSD YTGEDMLALT 840 RKFVGEDAGL
YREWKEKNAE AKDKYFDKAE RKKVLRQNDK MIRKMHFTPH SLNYVQKNLE 900
SVQSNGLAAV IKEYRNAVAH LNIINRLDEY IGSARADSYY SLYCYCLQMY LSKNFSVGYL
960 INVQKQLEEH HTYMKDLMWL LNIPFAYNLA RYKNLSNEKL FYDEEAAAEK
ADKAENERGE. 1020
[0180] Yan et al. (2018) Mol Cell. 70(2):327-339 (doi:
10.1016/j.molcel.2018.02.2018) and Konermann et al. (2018) Cell
173(3):665-676 (doi: 10.1016/j.cell/2018.02.033) have described
CasRX/Cas13d proteins and both of which are incorporated by
reference herein in their entireties. Also see WO Publication Nos.
WO2018/183703 (CasM) and WO2019/006471 (Cas13d), which are
incorporated herein by reference in their entirety.
[0181] Exemplary wild type Cas13d proteins of the disclosure may
comprise or consist of the amino acid sequence:
[0182] Cas13d (Ruminococcus flavefaciens XPD3002) Sequence:
TABLE-US-00076 (SEQ ID NO: 45) 1 IEKKKSFAKG MGVKSTLVSG SKVYMTTFAE
GSDARLEKIV EGDSIRSVNE GEAFSAEMAD 61 KNAGYKIGNA KFSHPKGYAV
VANNPLYTGP VQQDMLGLKE TLEKRYFGES ADGNDNICIQ 121 VIHNILDIEK
ILAEYITNAA YAVNNISGLD KDIIGFGKFS TVYTYDEFKD PEHHRAAFNN 181
NDKLINAIKA QYDEFDNFLD NPRLGYFGQA FFSKEGRNYI INYGNECYDI LALLSGLAHW
241 VVANNEEESR ISRTWLYNLD KNLDNEYIST LNYLYDRITN ELTNSFSKNS
AANVNYIAET 301 LGINPAEFAE QYFRFSIMKE QKNLGFNITK LREVMLDRKD
MSEIRKNHKV FDSIRTKVYT 361 MMDFVIYRYY IEEDAKVAAA NKSLPDNEKS
LSEKDIFVIN LRGSFNDDQK DALYYDEANR 421 IWRKLENIMH NIKEFRGNKT
REYKKKDAPR LPRILPAGRD VSAFSKLMYA LTMFLDGKEI 481 NDLLTTLINK
FDNIQSFLKV MPLIGVNAKF VEEYAFFKDS AKIADELRLI KSFARMGEPI 541
ADARRAMYID AIRILGTNLS YDELKALADT FSLDENGNKL KKGKHGMRNF IINNVISNKR
601 FHYLIRYGDP AHLHEIAKNE AVVKFVLGRI ADIQKKQGQN GKNQIDRYYE
TCIGKDKGKS 661 VSEKVDALTK IITGMNYDQF DKKRSVIEDT GRENAEREKF
KKIISLYLTV IYHILKNIVN 721 INARYVIGFH CVERDAQLYK EKGYDINLKK
LEEKGFSSVT KLCAGIDETA PDKRKDVEKE 781 MAERAKESID SLESANPKLY
ANYIKYSDEK KAEEFTRQIN REKAKTALNA YLRNTKWNVI 841 IREDLLRIDN
KTCTLFANKA VALEVARYVH AYINDIAEVN SYFQLYHYIM QRIIMNERYE 901
KSSGKVSEYF DAVNDEKKYN DRLLKLLCVP FGYCIPRFKN LSIEALFDRN EAAKFDKEKK
961 KVSGNS.
[0183] Exemplary wild type Cas13d proteins of the disclosure may
comprise or consist of the amino acid sequence:
[0184] Cas13d (contig e-k87_11092736):
TABLE-US-00077 (SEQ ID NO: 46)
MKRQKTFAKRIGIKSTVAYGQGKYAITTFGKGSKAEIAVRSADPPEETLP
TESDATLSIHAKFAKAGRDGREFKCGDVDETRIHTSRSEYESLISNPAES
PREDYLGLKGTLERKFFGDEYPKDNLRIQIIYSILDIQKILGLYVEDILH
FVDGLQDEPEDLVGLGLGDEKMQKLLSKALPYMGFFGSTDVFKVTKKREE
RAAADEHNAKVFRALGAIRQKLAHFKWKESLAIFGANANMPIRFFQGATG
GRQLWNDVIAPLWKKRIERVRKSFLSNSAKNLWVLYQVFKDDTDEKKKAR
ARQYYHFSVLKEGKNLGFNLTKTREYFLDKFFPIFHSSAPDVKRKVDTFR
SKFYAILDFIIYEASVSVANSGQMGKVAPWKGAIDNALVKLREAPDEEAK
EKIYNVLAASIRNDSLFLRLKSACDKFGAEQNRPVFPNELRNNRDIRNVR
SEWLEATQDVDAAAFVQLIAFLCNFLEGKEINELVTALIKKFEGIQALID
LLRNLEGVDSIRFENEFALFNDDKGNMAGRIARQLRLLASVGKMKPDMTD
AKRVLYKSALEILGAPPDEVSDEWLAENILLDKSNNDYQKAKKTVNPFRN
YIAKNVITSRSFYYLVRYAKPTAVRKLMSNPKIVRYVLKRLPEKQVASYY
SAIWTQSESNSNEMVKLIEMIDRLTTEIAGFSFAVLKDKKDSIVSASRES
RAVNLEVERLKKLTTLYMSIAYIAVKSLVKVNARYFIAYSALERDLYFFN
EKYGEEFRLHFIPYELNGKTCQFEYLAILKYYLARDEETLKRKCEICEEI
KVGCEKHKKNANPPYEYDQEWIDKKKALNSERKACERRLHFSTHWAQYAT
KRDENMAKHPQKWYDILASHYDELLALQATGWLATQARNDAEHLNPVNEF
DVYIEDLRRYPEGTPKNKDYHIGSYFEIYHYIRQRAYLEEVLAKRKEYRD
SGSFTDEQLDKLQKILDDIRARGSYDKNLLKLEYLPFAYNLPRYKNLTTE
ALFDDDSVSGKKRVAEWREREKTREAEREQRRQR.
[0185] An exemplary direct repeat sequence of Cas13d (contig
e-k87_11092736) (SEQ ID NO: 46) comprises or consists of the
nucleic acid sequence:Cas13d (contig e-k87_11092736) Direct Repeat
Sequence): GTGAGAAGTCTCCTTATGGGGAGATGCTAC (SEQ ID NO: 47).
[0186] Exemplary wild type Cas13d proteins of the disclosure may
comprise or consist of the amino acid sequence:
[0187] Cas13d (160582958_gene49834):
TABLE-US-00078 (SEQ ID NO: 48)
MKNSVTFKLIQAQENKEAARKKAKDIAEQARIAKRNGVVKKEENRINRIQ
IEIQTQKKSNTQNAYHLKSLAKAAGVKSVFAIGNDLLMTGFGPGNDATIE
KRVFQNRAIETLSSPEQYSAEFQNKQFKIKGNIKVLNHSTQKMEEIQTEL
QDNYNRPHFDLLGCKNVLEQKYFGRTFSDNIHVQIAYNIMDIEKLLTPYI
NNIIYTLNELMRDNSKDDFFGCDSHFSVAYLYDELKAGYSDRLKTKPNLS
KNIDRIWNNFCNYMNSDSGNTEARLAYFGELFYKPKETGDAKSDYKTHLS
NNQKEEWELKSDKEVYNIFAILCDLRHFCTHGESITPSGKPFPYNLEKNL
FPEAKQVLNSLFEEKAESLGAEAFGKTAGKTDVSILLKVFEKEQASQKEQ
QALLKEYYDFKVQKTYKNMGFSIKKLREAIMEIPDAAKFKDDLYSSLRHK
LYGLFDFILVKHFLDTSDSENLQNNDIFRQLRACRCEEEKDQVYRSIAVK
VWEKVKKKELNMFKQVVVIPSLSKDELKQMEMTKNTELLSSIETISTQAS
LFSEMIFMMTYLLDGKEINLLCTSLIEKFENIASFNEVLKSPQIGYETKY
TEGYAFFKNADKTAKELRQVNNMARMTKPLGGVNTKCVMYNEAAKILGAK
PMSKAELESVFNLDNHDYTYSPSGKKIPNKNFRNFIINNVITSRRFLYLI
RYGNPEKIRKIAINPSIISFVLKQIPDEQIKRYYPPCIGKRTDDVTLMRD
ELGKMLQSVNFEQFSRVNNKQNAKQNPNGEKARLQACVRLYLTVPYLFIK
NMVNINARYVLAFHCLERDHALCFNSRKLNDDSYNEMANKFQMVRKAKKE
QYEKEYKCKKQETGTAHTKKIEKLNQQIAYIDKDIKNMHSYTCRNYRNLV
AHLNVVSKLQNYVSELPNDYQITSYFSFYHYCMQLGLMEKVSSKNIPLVE
SLKNEANDAQSYSAKKTLEYFDLIEKNRTYCKDFLKALNAPFSYNLPRFK
NLSIEALFDKNIVYEQADLKKE.
[0188] An exemplary direct repeat sequence of Cas13d
(160582958_gene49834) (SEQ ID NO: 48) comprises or consists of the
nucleic acid sequence:
[0189] Cas13d (160582958_gene49834) Direct Repeat Sequence:
TABLE-US-00079 (SEQ ID NO: 49)
GAACTACACCCCTCTGTTCTTGTAGGGGTCTAACAC.
[0190] Exemplary wild type Cas13d proteins of the disclosure may
comprise or consist of the amino acid sequence:
[0191] Cas13d (contig tpg|DJXD01000002.1|; uncultivated
Ruminococcus assembly, UBA7013, from sheep gut metagenome):
TABLE-US-00080 (SEQ ID NO: 50)
MKKQKSKKTVSKTSGLKEALSVQGTVIMTSFGKGNMANLSYKIPSSQKPQ
NLNSSAGLKNVEVSGKKIKFQGRHPKIATTDNPLFKPQPGMDLLCLKDKL
EMHYFGKTFDDNIHIQLIYQILDIEKILAVHVNNIVFTLDNVLHPQKEEL
TEDFIGAGGWRINLDYQTLRGQTNKYDRFKNYIKRKELLYFGEAFYHENE
RRYEEDIFAILTLLSALRQFCFHSDLSSDESDHVNSFWLYQLEDQLSDEF
KETLSILWEEVTERIDSEFLKTNTVNLHILCHVFPKESKETIVRAYYEFL
IKKSFKNMGFSIKKLREIMLEQSDLKSFKEDKYNSVRAKLYKLFDFIITY
YYDHHAFEKEALVSSLRSSLTEENKEEIYIKTARTLASALGADFKKAAAD
VNAKNIRDYQKKANDYRISFEDIKIGNTGIGYFSELIYMLTLLLDGKEIN
DLLTTLINKFDNIISFIDILKKLNLEFKFKPEYADFFNMTNCRYTLEELR
VINSIARMQKPSADARKIMYRDALRILGMDNRPDEEIDRELERTMPVGAD
GKFIKGKQGFRNFIASNVIESSRFHYLVRYNNPHKTRTLVKNPNVVKFVL
EGIPETQIKRYFDVCKGQEIPPTSDKSAQIDVLARIISSVDYKIFEDVPQ
SAKINKDDPSRNFSDALKKQRYQAIVSLYLTVMYLITKNLVYVNSRYVIA
FHCLERDAFLHGVTLPKMNKKIVYSQLTTHLLTDKNYTTYGHLKNQKGHR
KWYVLVKNNLQNSDITAVSSFRNIVAHISVVRNSNEYISGIGELHSYFEL
YHYLVQSMIAKNNWYDTSHQPKTAEYLNNLKKHHTYCKDFVKAYCIPFGY
VVPRYKNLTINELFDRNNPNPEPKEEV.
[0192] An exemplary direct repeat sequence of Cas13d (contig
tpg|DJXD01000002.1|; uncultivated Ruminococcus assembly, UBA7013,
from sheep gut metagenome) (SEQ ID NO: 50) comprises or consists of
the nucleic acid sequence:Cas13d (contig tpg|DJXD01000002.1|;
uncultivated Ruminococcus assembly, UBA7013, from sheep gut
metagenome) Direct Repeat Sequence:
CAACTACAACCCCGTAAAAATACGGGGTTCTGAAAC (SEQ ID NO: 51).
gRNA Target Sequences
[0193] In some embodiments of the compositions of the disclosure, a
target sequence of an RNA molecule comprises a sequence motif
corresponding to the first RNA binding protein and/or the second
RNA binding protein.
[0194] In some embodiments of the compositions and methods of the
disclosure, the sequence motif is a signature of a disease or
disorder.
[0195] A sequence motif of the disclosure may be isolated or
derived from a sequence of foreign or exogenous sequence found in a
genomic sequence, and therefore translated into an mRNA molecule of
the disclosure or a sequence of foreign or exogenous sequence found
in an RNA sequence of the disclosure.
[0196] A sequence motif of the disclosure may comprise or consist
of a mutation in an endogenous sequence that causes a disease or
disorder. The mutation may comprise or consist of a sequence
substitution, inversion, deletion, insertion, transposition, or any
combination thereof.
[0197] A sequence motif of the disclosure may comprise or consist
of a repeated sequence. In some embodiments, the repeated sequence
may be associated with a microsatellite instability (MSI). MSI at
one or more loci results from impaired DNA mismatch repair
mechanisms of a cell of the disclosure. A hypervariable sequence of
DNA may be transcribed into an mRNA of the disclosure comprising a
target sequence comprising or consisting of the hypervariable
sequence.
[0198] A sequence motif of the disclosure may comprise or consist
of a biomarker. The biomarker may indicate a risk of developing a
disease or disorder. The biomarker may indicate a healthy gene (low
or no determinable risk of developing a disease or disorder. The
biomarker may indicate an edited gene. Exemplary biomarkers
include, but are not limited to, single nucleotide polymorphisms
(SNPs), sequence variations or mutations, epigenetic marks, splice
acceptor sites, exogenous sequences, heterologous sequences, and
any combination thereof.
[0199] A sequence motif of the disclosure may comprise or consist
of a secondary, tertiary or quaternary structure. The secondary,
tertiary or quaternary structure may be endogenous or naturally
occurring. The secondary, tertiary or quaternary structure may be
induced or non-naturally occurring. The secondary, tertiary or
quaternary structure may be encoded by an endogenous, exogenous, or
heterologous sequence.
[0200] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule comprises or
consists of between 2 and 100 nucleotides or nucleic acid bases,
inclusive of the endpoints. In some embodiments, the target
sequence of an RNA molecule comprises or consists of between 2 and
50 nucleotides or nucleic acid bases, inclusive of the endpoints.
In some embodiments, the target sequence of an RNA molecule
comprises or consists of between 2 and 20 nucleotides or nucleic
acid bases, inclusive of the endpoints.
[0201] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule is continuous. In
some embodiments, the target sequence of an RNA molecule is
discontinuous. For example, the target sequence of an RNA molecule
may comprise or consist of one or more nucleotides or nucleic acid
bases that are not contiguous because one or more intermittent
nucleotides are positioned in between the nucleotides of the target
sequence.
[0202] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule is naturally
occurring. In some embodiments, the target sequence of an RNA
molecule is non-naturally occurring. Exemplary non-naturally
occurring target sequences may comprise or consist of sequence
variations or mutations, chimeric sequences, exogenous sequences,
heterologous sequences, chimeric sequences, recombinant sequences,
sequences comprising a modified or synthetic nucleotide or any
combination thereof.
[0203] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule binds to a guide
RNA of the disclosure.
[0204] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule binds to a first
RNA binding protein of the disclosure.
[0205] In some embodiments of the compositions and methods of the
disclosure, a target sequence of an RNA molecule binds to a second
RNA binding protein of the disclosure.
RNA Molecules
[0206] In some embodiments of the compositions and methods of the
disclosure, an RNA molecule of the disclosure comprises a target
sequence. In some embodiments, the RNA molecule of the disclosure
comprises at least one target sequence. In some embodiments, the
RNA molecule of the disclosure comprises one or more target
sequence(s). In some embodiments, the RNA molecule of the
disclosure comprises two or more target sequences.
[0207] In some embodiments of the compositions and methods of the
disclosure, an RNA molecule of the disclosure is a naturally
occurring RNA molecule. In some embodiments, the RNA molecule of
the disclosure is a non-naturally occurring molecule. Exemplary
non-naturally occurring RNA molecules may comprise or consist of
sequence variations or mutations, chimeric sequences, exogenous
sequences, heterologous sequences, chimeric sequences, recombinant
sequences, sequences comprising a modified or synthetic nucleotide
or any combination thereof.
[0208] In some embodiments of the compositions and methods of the
disclosure, an RNA molecule of the disclosure comprises or consists
of a sequence isolated or derived from a virus.
[0209] In some embodiments of the compositions and methods of the
disclosure, an RNA molecule of the disclosure comprises or consists
of a sequence isolated or derived from a prokaryotic organism. In
some embodiments, an RNA molecule of the disclosure comprises or
consists of a sequence isolated or derived from a species or strain
of archaea or a species or strain of bacteria.
[0210] In some embodiments of the compositions and methods of the
disclosure, the RNA molecule of the disclosure comprises or
consists of a sequence isolated or derived from a eukaryotic
organism. In some embodiments, an RNA molecule of the disclosure
comprises or consists of a sequence isolated or derived from a
species of protozoa, parasite, protist, algae, fungi, yeast,
amoeba, worm, microorganism, invertebrate, vertebrate, insect,
rodent, mouse, rat, mammal, or a primate. In some embodiments, an
RNA molecule of the disclosure comprises or consists of a sequence
isolated or derived from a human.
[0211] In some embodiments of the compositions and methods of the
disclosure, the RNA molecule of the disclosure comprises or
consists of a sequence derived from a coding sequence from a genome
of an organism or a virus. In some embodiments, the RNA molecule of
the disclosure comprises or consists of a primary RNA transcript, a
precursor messenger RNA (pre-mRNA) or messenger RNA (mRNA). In some
embodiments, the RNA molecule of the disclosure comprises or
consists of a gene product that has not been processed (e.g. a
transcript). In some embodiments, the RNA molecule of the
disclosure comprises or consists of a gene product that has been
subject to post-transcriptional processing (e.g. a transcript
comprising a 5'cap and a 3' polyadenylation signal). In some
embodiments, the RNA molecule of the disclosure comprises or
consists of a gene product that has been subject to alternative
splicing (e.g. a splice variant). In some embodiments, the RNA
molecule of the disclosure comprises or consists of a gene product
that has been subject to removal of non-coding and/or intronic
sequences (e.g. a messenger RNA (mRNA)).
[0212] In some embodiments of the compositions and methods of the
disclosure, the RNA molecule of the disclosure comprises or
consists of a sequence derived from a non-coding sequence (e.g. a
non-coding RNA (ncRNA)). In some embodiments, the RNA molecule of
the disclosure comprises or consists of a ribosomal RNA. In some
embodiments, the RNA molecule of the disclosure comprises or
consists of a small ncRNA molecule. Exemplary small RNA molecules
of the disclosure include, but are not limited to, microRNAs
(miRNAs), small interfering (siRNAs), piwi-interacting RNAs
(piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs
(snRNAs), extracellular or exosomal RNAs (exRNAs), and small Cajal
body-specific RNAs (scaRNAs). In some embodiments, the RNA molecule
of the disclosure comprises or consists of a long ncRNA molecule.
Exemplary long RNA molecules of the disclosure include, but are not
limited to, X-inactive specific transcript (Xist) and HOX
transcript antisense RNA (HOTAIR).
[0213] In some embodiments of the compositions and methods of the
disclosure, the RNA molecule of the disclosure contacted by a
composition of the disclosure in an intracellular space. In some
embodiments, the RNA molecule of the disclosure contacted by a
composition of the disclosure in a cytosolic space. In some
embodiments, the RNA molecule of the disclosure contacted by a
composition of the disclosure in a nucleus. In some embodiments,
the RNA molecule of the disclosure contacted by a composition of
the disclosure in a vesicle, membrane-bound compartment of a cell,
or an organelle.
[0214] In some embodiments of the compositions and methods of the
disclosure, the RNA molecule of the disclosure contacted by a
composition of the disclosure in an extracellular space. In some
embodiments, the RNA molecule of the disclosure contacted by a
composition of the disclosure in an exosome. In some embodiments,
the RNA molecule of the disclosure contacted by a composition of
the disclosure in a liposome, a polymersome, a micelle or a
nanoparticle. In some embodiments, the RNA molecule of the
disclosure contacted by a composition of the disclosure in an
extracellular matrix. In some embodiments, the RNA molecule of the
disclosure contacted by a composition of the disclosure in a
droplet. In some embodiments, the RNA molecule of the disclosure
contacted by a composition of the disclosure in a microfluidic
droplet.
[0215] In some embodiments of the compositions and methods of the
disclosure, a RNA molecule of the disclosure comprises or consists
of a single-stranded sequence. In some embodiments, the RNA
molecule of the disclosure comprises or consists of a
double-stranded sequence. In some embodiments, the double-stranded
sequence comprises two RNA molecules. In some embodiments, the
double-stranded sequence comprises one RNA molecule and one DNA
molecule. In some embodiments, including those wherein the
double-stranded sequence comprises one RNA molecule and one DNA
molecule, compositions of the disclosure selectively bind and,
optionally, selectively cut the RNA molecule.
RNA-Binding Endonucleases
[0216] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a nuclease
domain. In some embodiments, the second RNA binding protein binds
RNA in a manner in which it associates with RNA. In some
embodiments, the second RNA binding protein associates with RNA in
a manner in which it cleaves RNA.
[0217] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
RNAse.
[0218] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse1. In some embodiments, the RNAse1
protein comprises or consists of:
TABLE-US-00081 (SEQ ID NO: 20)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGLCKPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVHFDASVEDST.
[0219] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse4. In some embodiments, the RNAse4
protein comprises or consists of:
TABLE-US-00082 (SEQ ID NO: 21)
QDGMYQRFLRQHVHPEETGGSDRYCDLMMQRRKMTLYHCKRFNTFIHEDI
WNIRSICSTTNIQCKNGKMNCHEGVVKVTDCRDTGSSRAPNCRYRAIAST
RRVVIACEGNPQVPVHFDG.
[0220] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse6. In some embodiments, the RNAse6
protein comprises or consists of:
TABLE-US-00083 (SEQ ID NO: 22)
WPKRLTKAHWFEIQHIQPSPLQCNRAMSGINNYTQHCKHQNTFLHDSFQN
VAAVCDLLSIVCKNRRHNCHQSSKPVNMTDCRLTSGKYPQCRYSAAAQYK
FFIVACDPPQKSDPPYKLVPVHLDSIL.
[0221] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse7. In some embodiments, the RNAse7
protein comprises or consists of:
TABLE-US-00084 (SEQ ID NO: 23)
APARAGFCPLLLLLLLGLWVAEIPVSAKPKGMTSSQWFKIQHMQPSPQAC
NSAMKNINKHTKRCKDLNTFLHEPFSSVAATCQTPKIACKNGDKNCHQSH
GPVSLTMCKLTSGKYPNCRYKEKRQNKSYVVACKPPQKKDSQQFHLVPVH LDRVL.
[0222] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse8. In some embodiments, the RNAse8
protein comprises or consists of:
TABLE-US-00085 (SEQ ID NO: 24)
TSSQWFKTQHVQPSPQACNSAMSIINKYTERCKDLNTFLHEPFSSVAITC
QTPNIACKNSCKNCHQSHGPMSLTMGELTSGKYPNCRYKEKHLNTPYIVA
CDPPQQGDPGYPLVPVHLDKVV.
[0223] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse2. In some embodiments, the RNAse2
protein comprises or consists of:
TABLE-US-00086 (SEQ ID NO: 25)
KPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFAN
VVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQ
TPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII.
[0224] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse6PL. In some embodiments, the
RNAse6PL protein comprises or consists of:
TABLE-US-00087 (SEQ ID NO: 26)
DKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEG
CNRSWPFNLEEIKKNWMEITDSSLPSPSMGPAPPRWMRSTPRRSTLAEAW
NSTGSWTSTGGCALPPAALPSGDLCCRPSLTAGSRGVGVDLTALHQLLHV
HYSATGIIPEECSEPTKPFQIILHHDHTEWVQSIGMPIWGTISSSESAIG
KNEESQPACAVLSHDS.
[0225] In some embodiments, the second RNA binding protein
comprises or consists of an RNAseL. In some embodiments, the RNAseL
protein comprises or consists of:
TABLE-US-00088 (SEQ ID NO: 27)
AAVEDNHLLIKAVQNEDVDLVQQLLEGGANVNFQEEEGGWTPLHNAVQMS
REDIVELLLRHGADPVLRKKNGATPFILAAIAGSVKdLLKLFLSKGADVN
ECDFYGFTAFMEAAVYGKVKALKFLYKRGANVNLRRKTKEDQERLRKGGA
TALMDAAEKGHVEVLKILLDEMGADVNACDNMGRNALIHALLSSDDSDVE
AITHLLLDHGADVNVRGERGKTPLILAVEKKHLGLVQRLLEQEHIEINDT
DSDGKTALLLAVELKLKKIAELLCKRGASTDCGDLVMTARRNYDHSLVKV
LLSHGAKEDFHPPAEDWKPQSSHWGAALKDLHRIYRPMIGKLKFFIDEKY
KIADTSEGGIYLGFYEKQEVAVKTFCEGSPRAQREVSCLQSSRENSHLVT
FYGSESHRGHLFVCVTLCEQTLEACLDVHRGEDVENEEDEFARNVLSSIF
KAVQELHLSCGYTHQDLQPQNILIDSKKAAHLADFDKSIKWAGDPQEVKR
DLEDLGRLVLYVVKKGSISFEDLKAQSNEEVVQLSPDEETKDLIHRLFHP
GEHVRDCLSDLLGHPFFWTWESRYRTLRNVGNESDIKTRKSESEILRLLQ
PGPSEHSKSFDKWTTKINECVMKKMNKFYEKRGNFYQNTVGDLLKFIRNL
GEHIDEEKHKKMKLKIGDPSLYFQKTFPDLVIYVYTKLQNTEYRKHFPQT
HSPNKPQCDGAGGASGLASPGC.
[0226] In some embodiments, the second RNA binding protein
comprises or consists of an RNAseT2. In some embodiments, the
RNAseT2 protein comprises or consists of:
TABLE-US-00089 (SEQ ID NO: 28)
VQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDL
LPEMRAYWPDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSL
ELYRELDLNSVLLKLGIKPSINYYQVADFKDALARVYGVIPKIQCLPPSQ
DEEVQTIGQIELCLTKQDQQLQNCTEPGEQPSPKQEVWLANGAAESRGLR
VCEDGPVFYPPPKKTKH.
[0227] In some embodiments, the second RNA binding protein
comprises or consists of an RNAse11. In some embodiments, the
RNAse1l11 protein comprises or consists of:
TABLE-US-00090 (SEQ ID NO: 29)
EASESTMKIIKEEFTDEEMQYDMAKSGQEKQTIEILMNPILLVKNTSLSM
SKDDMSSTLLTFRSLHYNDPKGNSSGNDKECCNDMTVWRKVSEANGSCKW
SNNFIRSSTEVMRRVHRAPSCKFVQNPGISCCESLELENTVCQFTTGKQF
PRCQYHSVTSLEKILTVLTGHSLMSWLVCGSKL.
[0228] In some embodiments, the second RNA binding protein
comprises or consists of an RNAseT2-like. In some embodiments, the
RNAseT2-like protein comprises or consists of:
TABLE-US-00091 (SEQ ID NO: 30)
XLGGADKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWP
DKSEGCNRSWPFNLEEIKDLLPEMRAYWPDVIHSFPNRSRFWKHEWEKHG
TCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGIKPSINYYQTTEED
LNLDVEPTTEDTAEEVTIHVLLHSALFGEIGPRRW.
[0229] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a mutated
RNAse.
[0230] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(K41R))
polypeptide. In some embodiments, the Rnase1(K41R) polypeptide
comprises or consists of:
TABLE-US-00092 (SEQ ID NO: 116)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVHFDASVEDST.
[0231] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E))
polypeptide. In some embodiments, the Rnase1 (Rnase1(K41R, D121E))
polypeptide comprises or consists of:
TABLE-US-00093 (SEQ ID NO: 117)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVHFEASVEDST.
[0232] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E,
H119N)) polypeptide. In some embodiments, the Rnase1 (Rnase1(K41R,
D121E, H119N)) polypeptide comprises or consists of:
TABLE-US-00094 (SEQ ID NO: 118)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVNFEASVEDST.
[0233] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1. In some embodiments, the
second RNA binding protein comprises or consists of a mutated
Rnase1 (Rnase1(H119N)) polypeptide. In some embodiments, the Rnase1
(Rnase1(H119N)) polypeptide comprises or consists of:
TABLE-US-00095 (SEQ ID NO: 119)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVNFDASVEDST.
[0234] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A,
G89D, R91D, H119N)) polypeptide. In some embodiments, the Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide comprises
or consists of:
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCKPVNTFVHEPLVDVQNV
CFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTSPKERHIIVACEGSPYV
[0235] PVNFDASVEDST (SEQ ID NO: 120). In some embodiments, the
second RNA binding protein comprises or consists of a mutated
Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
In some embodiments, the Rnase1 (Rnase1(R39D, N67D, N88A, G89D,
R91D, H119N, K41R, D121E)) polypeptide comprises or consists
of:
TABLE-US-00096 (SEQ ID NO: 121)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEP
LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS
PKERHIIVACEGSPYVPVNFEASVEDST.
[0236] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A,
G89D, R91D, H119N)) polypeptide. In some embodiments, the Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D)) polypeptide comprises or
consists of:
TABLE-US-00097 (SEQ ID NO: 122)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCKPVNTFVHEP
LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS
PKERHIIVACEGSPYVPVHFDASVEDST.
[0237] In some embodiments, the second RNA binding protein
comprises or consists of a mutated Rnase1 (Rnase1 (R39D, N67D,
N88A, G89D, R91D, H119N, K41R, D121E)) polypeptide that comprises
or consists of:
TABLE-US-00098 (SEQ ID NO: 208)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEP
LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS
PKERHIIVACEGSPYVPVNFEASVEDST.
[0238] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a NOB 1
polypeptide. In some embodiments, the NOB 1 polypeptide comprises
or consists of:
TABLE-US-00099 (SEQ ID NO: 31)
APVEHVVADAGAFLRHAALQDIGKNIYTIREVVTEIRDKATRRRLAVLPY
ELRFKEPLPEYVRLVTEFSKKTGDYPSLSATDIQVLALTYQLEAEFVGVS
HLKQEPQKVKVSSSIQHPETPLHISGFHLPYKPKPPQETEKGHSACEPEN
LEFSSFMFWRNPLPNIDHELQELLIDRGEDVPSEEEEEEENGFEDRKDDS
DDDGGGWITPSNIKQIQQELEQCDVPEDVRVGCLTTDFAMQNVLLQMGLH
VLAVNGMLIREARSYILRCHGCFKTTSDMSRVFCSHCGNKTLKKVSVTV.
[0239] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
endonuclease. In some embodiments, the second RNA binding protein
comprises or consists of an endonuclease V (ENDOV). In some
embodiments, the ENDOV protein comprises or consists of:
TABLE-US-00100 (SEQ ID NO: 32)
AFSGLQRVGGVDVSFVKGDSVRACASLVVLSFPELEVVYEESRMVSLTAP
YVSGFLAFREVPFLLELVQQLREKEPGLMPQVLLVDGNGVLHHRGFGVAC
HLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTRGDSFPLLGDSG
TVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQAD ICSREHIRKS.
[0240] In some embodiments, the second RNA binding protein
comprises or consists of an endonuclease G (ENDOG). In some
embodiments, the ENDOG protein comprises or consists of:
TABLE-US-00101 (SEQ ID NO: 33)
AELPPVPGGPRGPGELAKYGLPGLAQLKSRESYVLCYDPRTRGALWVVEQ
LRPERLRGDGDRRECDFREDDSVHAYHRATNADYRGSGFDRGHLAAAANH
RWSQKAMDDTFYLSNVAPQVPHLNQNAWNNLEKYSRSLTRSYQNVYVCTG
PLFLPRTEADGKSYVKYQVIGKNHVAVPTHFFKVLILEAAGGQIELRTYV
MPNAPVDEAIPLERFLVPIESIERASGLLFVPNILARAGSLKAITAGSK.
[0241] In some embodiments, the second RNA binding protein
comprises or consists of an endonuclease D1 (ENDOD1). In some
embodiments, the ENDOD1 protein comprises or consists of:
TABLE-US-00102 (SEQ ID NO: 34)
RLVGEEEAGFGECDKFFYAGTPPAGLAADSHVKICQRAEGAERFATLYST
RDRIPVYSAFRAPRPAPGGAEQRWLVEPQIDDPNSNLEEAINEAEAITSV
NSLGSKQALNTDYLDSDYQRGQLYPFSLSSDVQVATFTLTNSAPMTQSFQ
ERWYVNLHSLMDRALTPQCGSGEDLYILTGTVPSDYRVKDKVAVPEFVWL
AACCAVPGGGWAMGFVKHTRDSDIIEDVMVKDLQKLLPFNPQLFQNNCGE
TEQDTEKMKKILEVVNQIQDEERMVQSQKSSSPLSSTRSKRSTLLPPEAS
EGSSSFLGKLMGFIATPFIKLFQLIYYLVVAILKNIVYFLWCVTKQVING
IESCLYRLGSATISYFMAIGEELVSIPWKVLKVVAKVIRALLRILCCLLK
AICRVLSIPVRVLVDVATFPVYTMGAIPIVCKDIALGLGGTVSLLFDTAF
GTLGGLFQVVFSVCKRIGYKVTFDNSGEL.
[0242] In some embodiments, the second RNA binding protein
comprises or consists of a Human flap endonuclease-1 (hFEN1). In
some embodiments, the hFEN1 polypeptide comprises or consists
of:
TABLE-US-00103 (SEQ ID NO: 35)
MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGG
DVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKR
SERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI
PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAK
KLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLI
QKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSE
PNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSL
SSAKRKEPEPKGSTKKKAKTGAAGKFKRGK.
[0243] In some embodiments, the second RNA binding protein
comprises or consists of a DNA repair endonuclease XPF (ERCC4)
polypeptide. In some embodiments, the ERCC4 polypeptide comprises
or consists of:
TABLE-US-00104 (SEQ ID NO: 124)
MESGQPARRIAMAPLLEYERQLVLELLDTDGLVVCARGLGADRLLYHFLQ
LHCHPACLVLVLNTQPAEEEYFINQLKIEGVEHLPRRVTNEITSNSRYEV
YTQGGVIFATSRILVVDFLTDRIPSDLITGILVYRAHRIIESCQEAFILR
LFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLWPRFHVAV
NSFLEQHKPEVVEIHVSMTPTMLAIQTAILDILNACLKELKCHNPSLEVE
DLSLENAIGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQ
YDCVTFLNLLESLRATEKAFGQNSGWLFLDSSTSMFINARARVYHLPDAK
MSKKEKISEKMEIKEGEGILWG.
[0244] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
Endonuclease III-like protein 1 (NTHL) polypeptide. In some
embodiments, the NTHL polypeptide comprises or consists of:
TABLE-US-00105 (SEQ ID NO: 123)
CSPQESGMTALSARMLTRSRSLGPGAGPRGCREEPGPLRRREAAAEARKS
HSPVKRPRKAQRLRVAYEGSDSEKGEGAEPLKVPVWEPQDWQQQLVNIRA
MRNKKDAPVDHLGTEHCYDSSAPPKVRRYQVLLSLMLSSQTKDQVTAGAM
QRLRARGLTVDSILQTDDATLGKLIYPVGFWRSKVKYIKQTSAILQQHYG
GDIPASVAELVALPGVGPKMAHLAMAVAWGTVSGIAVDTHVHRIANRLRW
TKKATKSPEETRAALEEWLPRELWHEINGLLVGFGQQTCLPVHPRCHACL NQALCPAAQGL.
[0245] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a human
Schlafen 14 (hSLFN14) polypeptide. In some embodiments, the hSLFN14
polypeptide comprises or consists of:
TABLE-US-00106 (SEQ ID NO: 36)
ESTHVEFKRFTTKKVIPRIKEMLPHYVSAFANTQGGYVLIGVDDKSKEVV
GCKWEKVNPDLLKKEIENCIEKLPTFHFCCEKPKVNFTTKILNVYQKDVL
DGYVCVIQVEPFCCVVFAEAPDSWIMKDNSVTRLTAEQWVVMMLDTQSAP
PSLVTDYNSCLISSASSARKSPGYPIKVHKFKEALQ.
[0246] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a human
beta-lactamase-like protein 2 (hLACTB2) polypeptide.
[0247] In some embodiments, the hLACTB2 polypeptide comprises or
consists of:
TABLE-US-00107 (SEQ ID NO: 37)
TLQGTNTYLVGTGPRRILIDTGEPAIPEYISCLKQALTEFNTAIQEIVVT
HWHRDHSGGIGDICKSINNDTTYCIKKLPRNPQREEIIGNGEQQYVYLKD
GDVIKTEGATLRVLYTPGHTDDHMALLLEEENAIFSGDCILGEGTTVFED
LYDYMNSLKELLKIKADIIYPGHGPVIHNAEAKIQQYISHRNIREQQILT
LFRENFEKSFTVMELVKIIYKNTPENLHEMAKHNLLLHLKKLEKEGKIFS
NTDPDKKWKAHL.
[0248] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
apurinic/apyrimidinic (AP) endodeoxyribonuclease (APEX)
polypeptide. In some embodiments, the second RNA binding protein
comprises or consists of an apurinic/apyrimidinic (AP)
endodeoxyribonuclease (APEX2) polypeptide. In some embodiments, the
APEX2 polypeptide comprises or consists of:
TABLE-US-00108 (SEQ ID NO: 38)
MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETK
VTRDALTEPLAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLS
GLFATQNGDVGCYGNMDEFTQEELRALDSEGRALLTQHKIRTWEGKEKTL
TLINVYCPHADPGRPERLVFKMRFYRLLQIRAEALLAAGSHVIILGDLNT
AHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQSASHVGPFIDSYRCF
QPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASFLLPEVM
GSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQSP
VLEQSTLQHNNQTRVQTCQNKAQVRSTRPQPSQVGSSRGQKNLKSYFQPS
PSCPQASPDIELPSLPLMSALMTPKTPEEKAVAKVVKGQAKTSEAKDEKE
LRTSFWKSVLAGPLRTPLCGGHREPCVMRTVKKPGPNLGRRFYMCARPRG
PPTDPSSRCNFFLWSRPS.
[0249] In some embodiments, the APEX2 polypeptide comprises or
consists of:
TABLE-US-00109 (SEQ ID NO: 39)
MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETK
VTRDALTEPLAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLS
GLFATQNGDVGCYGNMDEFTQEELRALDSEGRALLTQHKIRTWEGKEKTL
TLINVYCPHADPGRPERLVFKMRFYRLLQIRAEALLAAGSHVIILGDLNT
AHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQSASHVGPFIDSYRCF
QPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASFLLPEVM
GSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQS P.
[0250] In some embodiments, the second RNA binding protein
comprises or consists of an apurinic or apyrimidinic site lyase
(APEX1) polypeptide. In some embodiments, the APEX1 polypeptide
comprises or consists of:
TABLE-US-00110 (SEQ ID NO: 125)
PKRGKKGAVAEDGDELRTEPEAKKSKTAAKKNDKEAAGEGPALYEDPPDQ
KTSPSGKPATLKICSWNVDGLRAWIKKKGLDWVKEEAPDILCLQETKCSE
NKLPAELQELPGLSHQYWSAPSDKEGYSGVGLLSRQCPLKVSYGIGDEEH
DQEGRVIVAEFDSFVLVTAYVPNAGRGLVRLEYRQRWDEAFRKFLKGLAS
RKPLVLCGDLNVAHEEIDLRNPKGNKKNAGFTPQERQGFGELLQAVPLAD
SFRHLYPNTPYAYTFWTYMMNARSKNVGWRLDYFLLS.
[0251] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
angiogenin (ANG) polypeptide. In some embodiments, the ANG
polypeptide comprises or consists of:
TABLE-US-00111 (SEQ ID NO: 40)
QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNK
RSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGF
RNVVVACENGLPVHLDQSIFRRP.
[0252] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a heat
responsive protein 12 (HRSP12) polypeptide. In some embodiments,
the HRSP12 polypeptide comprises or consists of:
TABLE-US-00112 (SEQ ID NO: 41)
SSLIRRVISTAKAPGAIGPYSQAVLVDRTIYISGQIGMDPSSGQLVSGGV
AEEAKQALKNMGEILKAAGCDFTNVVKTTVLLADINDFNTVNEIYKQYFK
SNFPARAAYQVAALPKGSRIEIEAVAIQGPLTTASL.
[0253] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Zinc
Finger CCCH-Type Containing 12A (ZC3H12A) polypeptide. In some
embodiments, the ZC3H12A polypeptide comprises or consists of:
TABLE-US-00113 (SEQ ID NO: 42)
GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGIL
LAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFT
PSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIE
ERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPLTLE.
[0254] In some embodiments, the ZC3H12A polypeptide comprises or
consists of:
TABLE-US-00114 (SEQ ID NO: 43)
SGPCGEKPVLEASPTMSLWEFEDSHSRQGTPRPGQELAAEEASALELQMK
VDFFRKLGYSSTEIHSVLQKLGVQADTNTVLGELVKHGTATERERQTSPD
PCPQLPLVPRGGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGN
KEVFSCRGILLAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRE
LEKKKILVFTPSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQG
ERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPLTLEH
RKQPCPYGRKCTYGIKCRFFHPERPSCPQRSVADELRANALLSPPRAPSK
DKNGRRPSPSSQSSSLLTESEQCSLDGKKLGAQASPGSRQEGLTQTYAPS
GRSLAPSGGSGSSFGPTDWLPQTLDSLPYVSQDCLDSGIGSLESQMSELW
GVRGGGPGEPGPPRAPYTGYSPYGSELPATAAFSAFGRAMGAGHFSVPAD
YPPAPPAFPPREYWSEPYPLPPPTSVLQEPPVQSPGAGRSPWGRAGSLAK
EQASVYTKLCGVFPPHLVEAVMGRFPQLLDPQQLAAEILSYKSQHPSE.
[0255] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Reactive
Intermediate Imine Deaminase A (RIDA) polypeptide. In some
embodiments, the RIDA polypeptidecomprises or consists of:
TABLE-US-00115 (SEQ ID NO: 44)
SSLIRRVISTAKAPGAIGPYSQAVLVDRTIYISGQIGMDPSSGQLVSGGV
AEEAKQALKNMGEILKAAGCDFTNVVKTTVLLADINDFNTVNEIYKQYFK
SNFPARAAYQVAALPKGSRIEIEAVAIQGPLTTASL.
[0256] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Phospholipase D Family Member 6 (PDL6) polypeptide. In some
embodiments, the PDL6 polypeptide comprises or consists of:
TABLE-US-00116 (SEQ ID NO: 126)
EALFFPSQVTCTEALLRAPGAELAELPEGCPCGLPHGESALSRLLRALLA
ARASLDLCLFAFSSPQLGRAVQLLHQRGVRVRVVTDCDYMALNGSQIGLL
RKAGIQVRHDQDPGYMHHKFAIVDKRVLITGSLNWTTQAIQNNRENVLIT
EDDEYVRLFLEEFERIWEQFNPTKYTFFPPKKSHGSCAPPVSRAGGRLLS
WHRTCGTSSESQT.
[0257] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
mitochondrial ribonuclease P catalytic subunit (KIAA0391)
polypeptide. In some embodiments, the KIAA0391 polypeptide
comprises or consists of:
TABLE-US-00117 (SEQ ID NO: 127)
KARYKTLEPRGYSLLIRGLIHSDRWREALLLLEDIKKVITPSKKNYNDCI
QGALLHQDVNTAWNLYQELLGHDIVPMLETLKAFFDFGKDIKDDNYSNKL
LDILSYLRNNQLYPGESFAHSIKTWFESVPGKQWKGQFTTVRKSGQCSGC
GKTIESIQLSPEEYECLKGKIMRDVIDGGDQYRKTTPQELKRFENFIKSR
PPFDVVIDGLNVAKMFPKVRESQLLLNVVSQLAKRNLRLLVLGRKHMLRR
SSQWSRDEMEEVQKQASCFFADDISEDDPFLLYATLHSGNHCRFITRDLM
RDHKACLPDAKTQRLFFKWQQGHQLAIVNRFPGSKLTFQRILSYDTVVQT
TGDSWHIPYDEDLVERCSCEVPTKWLCLHQKT.
[0258] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
argonaute 2 (AGO2) polypeptide.
[0259] In some embodiments of the compositions of the disclosure,
the AGO2 polypeptide comprises or consists of:
TABLE-US-00118 (SEQ ID NO: 128)
SVEPMFRHLKNTYAGLQLVVVILPGKTPVYAEVKRVGDTVLGMATQCVQM
KNVQRTTPQTLSNLCLKINVKLGGVNNILLPQGRPPVFQQPVIFLGADVT
HPPAGDGKKPSIAAVVGSMDAHPNRYCATVRVQQHRQEIIQDLAAMVREL
LIQFYKSTRFKPTRIIFYRDGVSEGQFQQVLHHELLAIREACIKLEKDYQ
PGITFIVVQKRHHTRLFCTDKNERVGKSGNIPAGTTVDTKITHPTEFDFY
LCSHAGIQGTSRPSHYHVLWDDNRFSSDELQILTYQLCHTYVRCTRSVSI
PAPAYYAHLVAFRARYHLVDKEHDSAEGSHTSGQSNGRDHQALAKAVQVH QDTLRTMYFA.
[0260] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
mitochondrial nuclease EXOG (EXOG) polypeptide. In some
embodiments, the EXOG polypeptide comprises or consists of:
TABLE-US-00119 (SEQ ID NO: 129)
QGAEGALTGKQPDGSAEKAVLEQFGFPLTGTEARCYTNHALSYDQAKRVP
RWVLEHISKSKIMGDADRKHCKFKPDPNIPPTFSAFNEDYVGSGWSRGHM
APAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYWNRIEMYCRELTERFED
VWVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVPSHLYKVILARRSSVST
EPLALGAFVVPNEAIGFQPQLTEFQVSLQDLEKLSGLVFFPHLDRTSDIR
NICSVDTCKLLDFQEFTLYLSTRKIEGARSVLRLEKIMENLKNAEIEPDD
YFMSRYEKKLEELKAKEQSGTQIRKPS.
[0261] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Zinc
Finger CCCH-Type Containing 12D (ZC3H12D) polypeptide. In some
embodiments, the ZC3H12D polypeptide comprises or consists of:
TABLE-US-00120 (SEQ ID NO: 130)
EHPSKMEFFQKLGYDREDVLRVLGKLGEGALVNDVLQELIRTGSRPGALE
HPAAPRLVPRGSCGVPDSAQRGPGTALEEDFRTLASSLRPIVIDGSNVAM
SHGNKETFSCRGIKLAVDWFRDRGHTYIKVFVPSWRKDPPRADTPIREQH
VLAELERQAVLVYTPSRKVHGKRLVCYDDRYIVKVAYEQDGVIVSNDNYR
DLQSENPEWKWFIEQRLLMFSFVNDRFMPPDDPLGRHGPSLSNFLSRKPK
PPEPSWQHCPYGKKCTYGIKCKFYHPERPHHAQLAVADELRAKTGARPGA
GAEEQRPPRAPGGSAGARAAPREPFAHSLPPARGSPDLAALRGSFSRLAF
SDDLGPLGPPLPVPACSLTPRLGGPDWVSAGGRVPGPLSLPSPESQFSPG
DLPPPPGLQLQPRGEHRPRDLHGDLLSPRRPPDDPWARPPRSDRFPGRSV
WAEPAWGDGATGGLSVYATEDDEGDARARARIALYSVFPRDQVDRVMAAF
PELSDLARLILLVQRCQSAGAPLGKP.
[0262] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
endoplasmic reticulum to nucleus signaling 2 (ERN2) polypeptide. In
some embodiments, the ERN2 polypeptide comprises or consists
of:
TABLE-US-00121 (SEQ ID NO: 131)
RQQQPQVVEKQQETPLAPADFAHISQDAQSLHSGASRRSQKRLQSPSKQA
QPLDDPEAEQLTVVGKISFNPKDVLGRGAGGTFVFRGQFEGRAVAVKRLL
RECFGLVRREVQLLQESDRHPNVLRYFCTERGPQFHYIALELCRASLQEY
VENPDLDRGGLEPEVVLQQLMSGLAHLHSLHIVHRDLKPGNILITGPDSQ
GLGRVVLSDFGLCKKLPAGRCSFSLHSGIPGTEGWMAPELLQLLPPDSPT
SAVDIFSAGCVFYYVLSGGSHPFGDSLYRQANILTGAPCLAHLEEEVHDK
VVARDLVGAMLSPLPQPRPSAPQVLAHPFFWSRAKQLQFFQDVSDWLEKE
SEQEPLVRALEAGGCAVVRDNWHEHISMPLQTDLRKFRSYKGTSVRDLLR
AVRNKKHHYRELPVEVRQALGQVPDGFVQYFTNRFPRLLLHTHRAMRSCA
SESLFLPYYPPDSEARRPCPGATGR.
[0263] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a pelota
mRNA surveillance and ribosome rescue factor (PELO) polypeptide. In
some embodiments, the PELO polypeptide comprises or consists
of:
TABLE-US-00122 (SEQ ID NO: 132)
KLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTES
STGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHT
IELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVAAVVMQEGLAHICL
VTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDV
VKCILVASPGFVREQFCDYLFQQAVKTDNKLLLENRSKFLQVHASSGHKY
SLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEK
ANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTVRIFSSLHV
SGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED.
[0264] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a YBEY
metallopeptidase (YBEY) polypeptide. In some embodiments, the YBEY
polypeptide comprises or consists of:
TABLE-US-00123 (SEQ ID NO: 133)
SLVIRNLQRVIPIRRAPLRSKIEIVRRILGVQKFDLGIICVDNKNIQHIN
RIYRDRNVPTDVLSFPFHEHLKAGEFPQPDFPDDYNLGDIFLGVEYIFHQ
CKENEDYNDVLTVTATHGLCHLLGFTHGTEAEWQQMFQKEKAVLDELGRR
TGTRLQPLTRGLFGGS.
[0265] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a cleavage
and polyadenylation specific factor 4 like (CPSF4L) polypeptide. In
some embodiments, the CPSF4L polypeptide comprises or consists
of:
TABLE-US-00124 (SEQ ID NO: 134)
QEVIAGLERFTFAFEKDVEMQKGTGLLPFQGMDKSASAVCNFFTKGLCEK
GKLCPFRHDRGEKMVVCKHWLRGLCKKGDHCKFLHQYDLTRMPECYFYSK
FGDCSNKECSFLHVKPAFKSQDCPWYDQGFCKDGPLCKYRHVPRIMCLNY
LVGFCPEGPKCQFAQKIREFKLLPGSKI.
[0266] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an
hCG_2002731 polypeptide. In some embodiments, the hCG_2002731
polypeptide comprises or consists of:
TABLE-US-00125 (SEQ ID NO: 135)
KLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTES
STGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHT
IELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVAAVVMQEGLAHICL
VTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDV
VKCILVASPGFVREQFCDYMFQQAVKTDNKLLLENRSKFLQVHASSGHKY
SLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEK
ANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTVRIFSSLHV
SGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED.
[0267] In some embodiments, the hCG_2002731 polypeptide comprises
or consists of:
TABLE-US-00126 (SEQ ID NO: 136)
DPAWSADVAAVVMQEGLAHICLVTPSMTLTRAKVEVNIPRKRKGNCSQHD
RALERFYEQVVQAIQRHIHFDVVKCILVASPGFVREQFCDYMFQQAVKTD
NKLLLENRSKFLQVHASSGHKYSLKEALCDPTVASRLSDTKAAGEVKALD
DFYKMLQHEPDRAFYGLKQVEKANEAMAIDTLLISDELFRHQDVATRSRY
VRLVDSVKENAGTVRIFSSLHVSGEQLSQLTGVAAILRFPVPELSDQEGD SSSEED.
[0268] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of an Excision
Repair Cross-Complementation Group 1 (ERCC1) polypeptide. In some
embodiments, the ERCC1 polypeptide comprises or consists of:
TABLE-US-00127 (SEQ ID NO: 137)
MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVRGNPVLKFVRNVPWE
FGDVIPDYVLGQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQ
VDVKDPQQALKELAKMCILADCTLILAWSPEEAGRYLETYKAYEQKPADL
LMEKLEQDFVSRVTECLTTVKSVNKTDSQTLLTTFGSLEQLIAASREDLA LCPGLGPQK.
[0269] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
ras-related C3 botulinum toxin substrate 1 isoform (RAC1)
polypeptide. In some embodiments, the RAC1 polypeptide comprises or
consists of:
TABLE-US-00128 (SEQ ID NO: 138)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEP
LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS
PKERHIIVACEGSPYVPVHFDASVEDST.
[0270] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Ribonuclease A A1 (RAA1) polypeptide. In some embodiments, the RAA1
polypeptide comprises or consists of:
TABLE-US-00129 (SEQ ID NO: 139)
QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNK
RSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGF
RNVVVACENGLPVHLDQSIFRRP.
[0271] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a Ras
Related Protein (RAB1) polypeptide. In some embodiments, the RAB1
polypeptide comprises or consists of:
TABLE-US-00130 (SEQ ID NO: 140)
GLGLVQPSYGQDGMYQRFLRQHVHPEETGGSDRYCNLMMQRRKMTLYHCK
RFNTFIHEDIWNIRSICSTTNIQCKNGKMNCHEGVVKVTDCRDTGSSRAP
NCRYRAIASTRRVVIACEGNPQVPVHFDG.
[0272] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a DNA
Replication Helicase/Nuclease 2 (DNA2) polypeptide. In some
embodiments, the DNA2 polypeptide comprises or consists of:
TABLE-US-00131 (SEQ ID NO: 141)
XSAVDNILLKLAKFKIGFLRLGQIQKVHPAIQQFTEQEICRSKSIKSLAL
LEELYNSQLIVATTCMGINHPIFSRKIFDFCIVDEASQISQPICLGPLFF
SRRFVLVGDHQQLPPLVLNREARALGMSESLFKRLEQNKSAVVQLTVQYR
MNSKIMSLSNKLTYEGKLECGSDKVANAVINLRHFKDVKLELEFYADYSD
NPWLMGVFEPNNPVCFLNTDKVPAPEQVEKGGVSNVTEAKLIVFLTSIFV
KAGCSPSDIGIIAPYRQQLKIINDLLARSIGMVEVNTVDKYQGRDKSIVL
VSFVRSNKDGTVGELLKDWRRLNVAITRAKHKLILLGCVPSLNCYPPLEK
LLNHLNSEKLISFFFCIWSHLIALL.
[0273] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a FLJ35220
polypeptide. In some embodiments, the FLJ35220 polypeptide
comprises or consists of:
TABLE-US-00132 (SEQ ID NO: 142)
MALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQADICSR
EHIRKSLGLPGPPTPRSPKAQRPVACPKGDSGESSALC.
[0274] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a FLJ13173
polypeptide. In some embodiments, the FLJ13173 polypeptide
comprises or consists of:
TABLE-US-00133 (SEQ ID NO: 143)
CYTNHALSYDQAKRVPRWVLEHISKSKIMGDADRKHCKFKPDPNIPPTFS
AFNEDYVGSGWSRGHMAPAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYW
NRIEMYCRELTERFEDVWVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVP
SHLYKVILARRSSVSTEPLALGAFVVPNEAIGFQPQLTEFQVSLQDLEKL
SGLVFFPHLDRT.
[0275] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of Teneurin
Transmembrane Protein (TENM) polypeptide. In some embodiments, the
second RNA binding protein comprises or consists of Teneurin
Transmembrane Protein 1 (TENM1) polypeptide. In some embodiments,
the TENM1 polypeptide comprises or consists of:
TABLE-US-00134 (SEQ ID NO: 144)
VTVSQMTSVLNGKTRRFADIQLQHGALCFNIRYGTTVEEEKNHVLEIARQ
RAVAQAWTKEQRRLQEGEEGIRAWTEGEKQQLLSTGRVQGYDGYFVLSVE
QYLELSDSANNIHFMRQSEIGRR.
[0276] In some embodiments, the second RNA binding protein
comprises or consists of Teneurin Transmembrane Protein 2 (TENM2)
polypeptide. In some embodiments, the TENM2 polypeptide comprises
or consists of:
TABLE-US-00135 (SEQ ID NO: 145)
TVSQPTLLVNGKTRRFTNIEFQYSTLLLSIRYGLTPDTLDEEKARVLDQA
RQRALGTAWAKEQQKARDGREGSRLWTEGEKQQLLSTGRVQGYEGYYVLP
VEQYPELADSSSNIQFLRQNEMGKR.
[0277] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
Ribonuclease Kappa (RNAseK) polypeptide. In some embodiments, the
RNAseK polypeptide comprises or consists of:
TABLE-US-00136 (SEQ ID NO: 204)
MGWLRPGPRPLCPPARASWAFSHRFPSPLAPRRSPTPFFMASLLCCGPKL
AACGIVLSAWGVIMLIMLGIFFNVHSAVLIEDVPFTEKDFENGPQNIYNL
YEQVSYNCFIAAGLYLLLGGFSFCQVRLNKRKEYMVR.
[0278] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a
transcription activator-like effector nuclease (TALEN) polypeptide
or a nuclease domain thereof. In some embodiments, the TALEN
polypeptide comprises or consists of:
TABLE-US-00137 (SEQ ID NO: 205) 1 MRIGKSSGWL NESVSLEYEH VSPPTRPRDT
RRRPRAAGDG GLAHLHRRLA VGYAEDTPRT 61 EARSPAPRRP LPVAPASAPP
APSLVPEPPM PVSLPAVSSP RFSAGSSAAI TDPFPSLPPT 121 PVLYAMAREL
EALSDATWQP AVPLPAEPPT DARRGNTVFD EASASSPVIA SACPQAFASP 181
PRAPRSARAR RARTGGDAWP APTFLSRPSS SRIGRDVFGK LVALGYSREQ IRKLKQESLS
241 EIAKYHTTLT GQGFTHADIC RISRRRQSLR VVARNYPELA AALPELTRAH
IVDIARQRSG 301 DLALQALLPV ATALTAAPLR LSASQIATVA QYGERPAIQA
LYRLRRKLTR APLHLTPQQV 361 VAIASNTGGK RALEAVCVQL PVLRAAPYRL
STEQVVAIAS NKGGKQALEA VKAHLLDLLG 421 APYVLDTEQV VAIASHNGGK
QALEAVKADL LDLRGAPYAL STEQVVAIAS HNGGKQALEA 481 VKADLLELRG
APYALSTEQV VAIASHNGGK QALEAVKAHL LDLRGVPYAL STEQVVAIAS 541
HNGGKQALEA VKAQLLDLRG APYALSTAQV VAIASNGGGK QALEGIGEQL LKLRTAPYGL
601 STEQVVAIAS HDGGKQALEA VGAQLVALRA APYALSTEQV VAIASNKGGK
QALEAVKAQL 661 LELRGAPYAL STAQVVAIAS HDGGNQALEA VGTQLVALRA
APYALSTEQV VAIASHDGGK 721 QALEAVGAQL VALRAAPYAL NTEQVVAIAS
SHGGKQALEA VRALFPDLRA APYALSTAQL 781 VAIASNPGGK QALEAVRALF
RELRAAPYAL STEQVVAIAS NHGGKQALEA VRALFRGLRA 841 APYGLSTAQV
VAIASSNGGK QALEAVWALL PVLRATPYDL NTAQIVAIAS HDGGKPALEA 901
VWAKLPVLRG APYALSTAQV VAIACISGQQ ALEAIEAHMP TLRQASHSLS PERVAAIACI
961 GGRSAVEAVR QGLPVKAIRR IRREKAPVAG PPPASLGPTP QELVAVLHFF
RAHQQPRQAF 1021 VDALAAFQAT RPALLRLLSS VGVTEIEALG GTIPDATERW
QRLLGRLGFR PATGAAAPSP 1081 DSLQGFAQSL ERTLGSPGMA GQSACSPHRK
RPAETAIAPR SIRRSPNNAG QPSEPWPDQL 1141 AWLQRRKRTA RSHIRADSAA
SVPANLHLGT RAQFTPDRLR AEPGPIMQAH TSPASVSFGS 1201 HVAFEPGLPD
PGTPTSADLA SFEAEPFGVG PLDFHLDWLL QILET.
[0279] In some embodiments, the TALEN polypeptide comprises or
consists of:
TABLE-US-00138 (SEQ ID NO: 206) 1 mdpirsrtps parellpgpq pdrvqptadr
ggappaggpl dglparrtms rtrlpsppap 61 spafsagsfs dllrqfdpsl
ldtslldsmp avgtphtaaa paecdevqsg lraaddpppt 121 vrvavtaarp
prakpaprrr aaqpsdaspa aqvdlrtlgy sqqqqekikp kvgstvaqhh 181
ealvghgfth ahivalsrhp aalgtvavky qdmiaalpea thedivgvgk qwsgaralea
241 lltvagelrg pplqldtgql vkiakrggvt aveavhasrn altgaplnlt
paqvvaiasn 301 nggkqaletv qrllpvlcqa hgltpaqvva iashdggkqa
letmqrllpv lcqahglppd 361 qvvaiasnig gkqaletvqr llpvlcqahg
ltpdqvvaia shgggkqale tvqrllpvlc 421 qahgltpdqv vaiashdggk
qaletvqrll pvlcqahglt pdqvvaiasn gggkqaletv 481 qrllpvlcqa
hgltpdqvva iasnggkqal etvqrllpvl cqahgltpdq vvaiashdgg 541
kqaletvqrl lpvlcqthgl tpaqvvaias hdggkqalet vqqllpvlcq ahgltpdqvv
601 aiasniggkq alatvqrllp vlcqahgltp dqvvaiasng ggkqaletvq
rllpvlcqah 661 gltpdqvvai asngggkqal etvqrllpvl cqahgltqvq
vvaiasnigg kqaletvqrl 721 lpvlcqahgl tpaqvvaias hdggkqalet
vqrllpvlcq ahgltpdqvv aiasngggkq 781 aletvqrllp vlcqahgltq
eqvvaiasnn ggkqaletvq rllpvlcqah gltpdqvvai 841 asngggkqal
etvqrllpvl cqahgltpaq vvaiasnigg kqaletvqrl lpvlcqdhgl 901
tlaqvvaias niggkqalet vqrllpvlcq ahgltqdqvv aiasniggkq aletvqrllp
961 vlcqdhgltp dqvvaiasni ggkqaletvq rllpvlcqdh gltldqvvai
asnggkqale 1021 tvqrllpvlc qdhgltpdqv vaiasnsggk qaletvqrll
pvlcqdhglt pnqvvaiasn 1081 ggkqalesiv aqlsrpdpal aaltndhlva
laclggrpam davkkglpha pelirrvnrr 1141 igertshrva dyaqvvrvle
ffqchshpay afdeamtqfg msrnglvqlf rrvgvtelea 1201 rggtlppasq
rwdrilqasg mkrakpspts aqtpdqaslh afadslerdl dapspmhegd 1261
qtgassrkrs rsdravtgps aqhsfevrvp eqrdalhlpl swrvkrprtr iggglpdpgt
1321 piaadlaass tvmweqdaap fagaaddfpa fneeelawlm ellpqsgsvg
gti.
[0280] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists a zinc finger
nuclease polypeptide or a nuclease domain thereof. In some
embodiments, the second RNA binding protein comprises or consists
of a ZNF638 polypeptide or a nuclease domain thereof. In some
embodiments, the ZNF638 polypeptide polypeptide comprises or
consists of:
TABLE-US-00139 (SEQ ID NO: 207) 1 MSRPRFNPRG DFPLQRPRAP NPSGMRPPGP
FMRPGSMGLP RFYPAGRARG IPHRFAGHES 61 YQNMGPQRMN VQVTQHRTDP
RLTKEKLDFH EAQQKKGKPH GSRWDDEPHI SASVAVKQSS 121 VTQVTEQSPK
VQSRYTKESA SSILASFGLS NEDLEELSRY PDEQLTPENM PLILRDIRMR 181
KMGRRLPNLP SQSRNKETLG SEAVSSNVID YGHASKYGYT EDPLEVRIYD PEIPTDEVEN
241 EFQSQQNISA SVPNPNVICN SMFPVEDVFR QMDFPGESSN NRSFFSVESG
TKMSGLHISG 301 GQSVLEPIKS VNQSINQTVS QTMSQSLIPP SMNQQPFSSE
LISSVSQQER IPHEPVINSS 361 NVHVGSRGSK KNYQSQADIP IRSPFGIVKA
SWLPKFSHAD AQKMKRLPTP SMMNDYYAAS 421 PRIFPHLCSL CNVECSHLKD
WIQHQNTSTH IESCRQLRQQ YPDWNPEILP SRRNEGNRKE 481 NETPRRRSHS
PSPRRSRRSS SSHRFRRSRS PMHYMYRPRS RSPRICHRFI SRYRSRSRSR 541
SPYRIRNPFR GSPKCFRSVS PERMSRRSVR SSDRKKALED VVQRSGHGTE FNKQKHLEAA
601 DKGHSPAQKP KTSSGTKPSV KPTSATKSDS NLGGHSIRCK SKNLEDDTLS
ECKQVSDKAV 661 SLQRKLRKEQ SLHYGSVLLI TELPEDGCTE EDVRKLFQPF
GKVNDVLIVP YRKEAYLEME 721 FKEAITAIMK YIETTPLTIK GKSVKICVPG
KKKAQNKEVK KKTLESKKVS ASTLKRDADA 781 SKAVEIVTST SAAKTGQAKA
SVAKVNKSTG KSASSVKSVV TVAVKGNKAS IKTAKSGGKK 841 SLEAKKTGNV
KNKDSNKPVT IPENSEIKTS IEVKATENCA KEAISDAALE ATENEPLNKE 901
TEEMCVMLVS NLPNKGYSVE EVYDLAKPFG GLKDILILSS HKKAYIEINR KAAESMVKFY
961 TCFPVLMDGN QLSISMAPEN MNIKDEEAIF ITLVKENDPE ANIDTIYDRF
VHLDNLPEDG 1021 LQCVLCVGLQ FGKVDHHVFI SNRNKAILQL DSPESAQSMY
SFLKQNPQNI GDHMLTCSLS 1081 PKIDLPEVQI EHDPELEKES PGLKNSPIDE
SEVQTATDSP SVKPNELEEE STPSIQTETL 1141 VQQEEPCEEE AEKATCDSDF
AVETLELETQ GEEVKEEIPL VASASVSIEQ FTENAEECAL 1201 NQQMFNSDLE
KKGAEIINPK TALLPSDSVF AEERNLKGIL EESPSEAEDF ISGITQTMVE 1261
AVAEVEKNET VSEILPSTCI VTLVPGIPTG DEKTVDKKNI SEKKGNMDEK EEKEFNTKET
1321 RMDLQIGTEK AEKNEGRMDA EKVEKMAAMK EKPAENTLFK AYPNKGVGQA
NKPDETSKTS 1381 ILAVSDVSSS KPSIKAVIVS SPKAKATVSK TENQKSFPKS
VPRDQINAEK KLSAKEFGLL 1441 KPTSARSGLA ESSSKFKPTQ SSLTRGGSGR
ISALQGKLSK LDYRDITKQS QETEARPSIM 1501 KRDDSNNKTL AEQNTKNPKS
TTGRSSKSKE EPLFPFNLDE FVTVDEVIEE VNPSQAKQNP 1561 LKGKRKETLK
NVPFSELNLK KKKGKTSTPR GVEGELSFVT LDEIGEEEDA AAHLAQALVT 1621
VDEVIDEEEL NMEEMVKNSN SLFTLDELID QDDCISHSEP KDVTVLSVAE EQDLLKQERL
1681 VTVDEIGEVE ELPLNESADI TFATLNTKGN EGDTVRDSIG FISSQVPEDP
STLVTVDEIQ 1741 DDSSDLHLVT LDEVTEEDED SLADFNNLKE ELNFVTVDEV
GEEEDGDNDL KVELAQSKND 1801 HPTDKKGNRK KRAVDTKKTK LESLSQVGPV
NENVMEEDLK TMIERHLTAK TPTKRVRIGK 1861 TLPSEKAVVT EPAKGEEAFQ
MSEVDEESGL KDSEPERKRK KTEDSSSGKS VASDVPEELD 1921 FLVPKAGFFC
PICSLFYSGE KAMTNHCKST RHKQNTEKFM AKQRKEKEQN EAEERSSR.
[0281] In some embodiments of the compositions of the disclosure,
the second RNA binding protein comprises or consists of a PIN
domain derived from the human SMG6 protein, also commonly known as
telomerase-binding protein EST1A isoform 3, NCBI Reference
Sequence: NP 001243756.1. In some embodiments, the PIN from hSMG6
is used herein in the form of a Cas fusion protein and as an
internal control, for example, and without limitation, see FIG. 9,
which shows PIN-dSauCas9, PIN-dSauCas9dHNH, PIN-dSPCas9, and
dcjeCas9-PIN.
[0282] In some embodiments of the compositions of the disclosure,
the composition further comprises (a) a sequence comprising a gRNA
that specifically binds within an RNA molecule and (b) a sequence
encoding a nuclease. In some embodiments, a nuclease comprises a
sequence isolated or derived from a CRISPR/Cas protein. In some
embodiments, the CRISPR/Cas protein is isolated or derived from any
one of a type I, a type IA, a type IB, a type IC, a type ID, a type
IE, a type IF, a type IU, a type III, a type IIIA, a type IIIB, a
type IIIC, a type IIID, a type IV, a type IVA, a type IVB, a type
II, a type IIA, a type IIB, a type IIC, a type V, or a type VI
CRISPR/Cas protein. In some embodiments, a nuclease comprises a
sequence isolated or derived from a TALEN or a nuclease domain
thereof. In some embodiments, a nuclease comprises a sequence
isolated or derived from a zinc finger nuclease or a nuclease
domain thereof.
Fusion Proteins
[0283] In some embodiments of the compositions and methods of the
disclosure, the composition comprises a sequence encoding a target
RNA-binding fusion protein comprising (a) a sequence encoding a
first RNA-binding polypeptide or portion thereof; and (b) a
sequence encoding a second RNA-binding polypeptide, wherein the
first RNA-biding polypeptide binds a target RNA, and wherein the
second RNA-binding polypeptide comprises RNA-nuclease activity.
[0284] In some embodiments, a target RNA-binding fusion protein is
an RNA-guided target RNA-binding fusion protein. RNA-guided target
RNA-binding fusion proteins comprise at least one RNA-binding
polypeptide which corresponds to a gRNA which guides the
RNA-binding polypeptide to target RNA. RNA-guided target
RNA-binding fusion proteins include without limitation, RNA-binding
polypeptides which are CRISPR/Cas-based RNA-binding polypeptides or
portions thereof.
[0285] In some embodiments, a target RNA-binding fusion protein is
not an RNA-guided target RNA-binding fusion protein and as such
comprises at least one RNA-binding polypeptide which is capable of
binding a target RNA without a corresponding gRNA sequence. Such
non-guided RNA-binding polypeptides include, without limitation, at
least one RNA-binding protein or RNA-binding portion thereof which
is a PUF (Pumilio and FBF homology family). This type RNA-binding
polypeptide can be used in place of a gRNA-guided RNA binding
protein such as CRISPR/Cas. The unique RNA recognition mode of PUF
proteins (named for Drosophila Pumilio and C. elegans fem-3 binding
factor) that are involved in mediating mRNA stability and
translation are well known in the art. The PUF domain of human
Pumiliol, also known in the art, binds tightly to cognate RNA
sequences and its specificity can be modified. It contains eight
PUF repeats that recognize eight consecutive RNA bases with each
repeat recognizing a single base. Since two amino acid side chains
in each repeat recognize the Watson-Crick edge of the corresponding
base and determine the specificity of that repeat, a PUF domain can
be designed to specifically bind most 8-nt RNA. Wang et al., Nat
Methods. 2009; 6(11): 825-830. See also WO2012/068627 which is
incorporated by reference herein in its entirety.
[0286] In some embodiments of the non-guided RNA-binding fusion
proteins of the disclosure, the fusion protein comprises at least
one RNA-binding protein or RNA-binding portion thereof which is a
PUMBY (Pumilio-based assembly) protein. RNA-binding protein PumHD
(Pumilio homology domain, a member of the PUF family), which has
been widely used in native and modified form for targeting RNA, has
been engineered to yield a set of four canonical protein modules,
each of which targets one RNA base. These modules (i.e., Pumby, for
Pumilio-based assembly) can be concatenated in chains of varying
composition and length, to bind desired target RNAs. The
specificity of such Pumby-RNA interactions is high, with
undetectable binding of a Pumby chain to RNA sequences that bear
three or more mismatches from the target sequence. Katarzyna et
al., PNAS, 2016; 113(19): E2579-E2588. See also US 2016/0238593
which is incorporated by reference herein in its entirety.
[0287] In some embodiments of the compositions of the disclosure,
the first RNA binding protein comprises a Pumilio and FBF (PUF)
protein. In some embodiments, the first RNA binding protein
comprises a Pumilio-based assembly (PUMBY) protein. In some
embodiments, a PUF1 protein of the disclosure comprises or consists
of the amino acid sequence of
TABLE-US-00140 (SEQ ID NO: 209) MDKSKQMNIN NLSNIPEVID PGITIPIYEE
EYENNGESNS QLQQQPQKLG SYRSRAGKFS 60 NTLSNLLPSI SAKLHHSKKN
SHGKNGAEFS SSNNSSQSTV ASKTPRASPS RSKMMESSID 120 GVTMDRPGSL
TPPQDMEKLV HFPDSSNNFL IPAPRGSSDS FNLPHQISRT RNNTMSSQIT 180
SISSIAPKPR TSSGIWSSNA SANDPMQQHL LQQLQPTTSN NTTNSNTLND YSTKTAYFDN
240 MVSTSGSQMA DNKMNTNNLA IPNSVWSNTR QRSQSNASSI YTDAPLYEQP
ARASISSHYT 300 IPTQESPLIA DEIDPQSINW VTMDPTVPSI NQISNLLPTN
TISISNVFPL QHQQPQLNNA 360 INLTSTSLAT LCSKYGEVIS ARTLRNLNMA
LVEFSSVESA VKALDSLQGK EVSMIGAPSK 420 ISFAKILPMH QQPPQFLLNS
QGLPLGLENN NLQPQPLLQE QLFNGAVTFQ QQGNVSIPVF 480 NQQSQQSQHQ
NHSSGSAGFS NVLHGYNNNN SMHGNNNNSA NEKEQCPFPL PPPNVNEKED 540
LLREIIELFE ANSDEYQINS LIKKSLNHKG TSDTQNFGPL PEPLSGREFD PPKLRELRKS
600 IDSNAFSDLE IEQLAIAMLD ELPELSSDYL GNTIVQKLFE HSSDIIKDIM
LRKTSKYLTS 660 MGVHKNGTWA CQKMITMAHT PRQIMQVTQG VKDYCTPLIN
DQFGNYVIQC VLKFGFPWNQ 720 FIFESIIANF WVIVQNRYGA RAVRACLEAH
DIVTPEQSIV LSAMIVTYAE YLSTNSNGAL 780 LVTWFLDTSV LPNRHSILAP
RLTKRIVELC GHRLASLTIL KVLNYRGDDN ARKIILDSLF 840 GNVNAHDSSP
PKELTKLLCE TNYGPTFVHK VLAMPLLEDD LRAHIIKQVR KVLTDSTQIQ 900
PSRRLLEEVG LASPSSTHNK TKQQQQQHHN SSISHMFATP DTSGQHMRGL SVSSVKSGGS
960 KHTTMNTTTT NGSSASTLSP GQPLNANSNS SMGYFSYPGV FPVSGFSGNA
SNGYAMNNDD 1020 LSSQFDMLNF NNGTRLSLPQ LSLTNHNNTT MELVNNVGSS
QPHTNNNNNN NNTNYNDDNT 1080 VFETLTLHSA N. 1091
[0288] In some embodiments, a PUF3 protein of the disclosure
comprises or consists of the amino acid sequence of [0289] 1
MEMNMDMDMD MELASIVSSL SALSHSNNNG GQAAAAGIVi GGAAGSQQIG GFRRSSFTTA
[0290] 61 NEVDSEILLL HGSSESSPIF KKTALSVGTA PPFSTNSKKF FGNGGNYYQY
RSTDTASLSS [0291] 121 ASYNNYHTHH TAANLGKNNK VNHLLGQYSA SIAGPVYYNG
NDNNNSGGEG FFEKFGKSLI [0292] 181 DGTRELESQD RPDANTQSQ FITKSVSNAS
LDTQNTFEQN VESDKNFNKL NRNTTNSGSL [0293] 241 YHSSSNSGSS ASLESENAHY
PKRNIWNVAN TPVFRPSNNP AAVGATNVAL PNQQDGPANN [0294] 301 NFPPYMNGFP
PNQFHQGPHY QNFPNYLIGS PSNFISQMIS VQIPANEDTE DSNGKKKKKA [0295] 361
NRPSSVSSPS SPPNNSPFPF AYPNPMMFMP PPPLSAPQQQ QQQQQQQQQE DQQQQQQQEN
[0296] 421 PYTYYPTPNP IPVKMPKDEK TFKKRNNKNH PANNNNANK QANPIYLENSI
PTKNTSKKNA [0297] 481 SSKSNESTAN NHKSHSHSHP HSQSLQQQQQ TYHRSPLLEQ
LRNSSSDKNS NSNMSLKDIF [0298] 541 GHSLEFCKDQ HGSRFIQREL ATSPASEKEV I
FNEIRDDAI ELSNDVFGNY VIQKFFEFGS [0299] 601 KIQKNTLVDQ FKGNMKIQLSL
QMYACRVQK ALEYIDSNQR IELVLELSDS VLQMIKDQNG [0300] 661 NHVIQKAIE
IPiEKLPFIL SSLTGHIYHL STHSYGCRVI QRLLEFGSSE DQESILNELK [0301] 721
DFIPYLIQDQ YGNYVIQYVL QQDQFTNKEM VDIKQEIIET VANNVVEYSK HKEASNVVEK
[0302] 781 SILYGSKNQK DLIISKILPR DKNHALNLED DSPMILMIKD QFANYVIQKL
VNVSEGEGKK [0303] 841 LIVIAIRAYL DKLNKSNSLG NRHLASVEKL AALVENAEV
(SEQ ID NO: 210). In some embodiments, a PUF4 protein of the
disclosure comprises or consists of the amino acid sequence of
TABLE-US-00141 [0303] (SEQ ID NO: 211) 1 MSTKGLKEEI DDVPSVDPVV
SETVNSALEQ LQLDDPEENA TSNAFANKVS QDSQFANGPP 61 SQMFPHPQMM
GGMGFMPYSQ MMQVPHNPCP FFPPPDFNDP TAPLSSSPLN AGGPPMLFKN 121
DSLPFQMLSS GAAVATQGGQ NLNPLINDNS MKVLPIASAD PLWTHSNVPG SASVAIEETT
181 ATLQESLPSK GRESNNKASS FRRQTFHALS PTDLINAANN VTLSKDFQSD
MQNFSKAKKP 241 SVGANNTAKT RTQSISFDNT PSSTSFIPPT NSVSEKLSDF
KIETSKEDLI NKTAPAKKES 301 PTTYGAAYPY GGPLLQPNPI MPGHPHNISS
PIYGIRSPFP NSYEMGAQFQ PFSPILNPTS 361 HSLNANSPIP LTQSPIHLAP
VLNPSSNSVA FSDMKNDGGK PTTDNDKAGP NVRMDLINPN 421 LGPSMQPFHI
LPPQQNTPPP PWLYSTPPPF NAMVPPHLLA QNHMPLMNSA NNKHHGRNNN 481
SMSSHNDNDN IGNSNYNNKD TGRSNVGKMK NMKNSYHGYY NNNNNNNNNN NNNNNSNATN
541 SNSAEKQRKI EESSRFADAV LDQYIGSIHS LCKDQHGCRF LQKQLDILGS
KAADAIFEET 601 KDYTVELMTD SFGNYLIQKL LEEVTTEQRI VLTKISSPHF
VEISLNPHGT RALQKLIECI 661 KTDEEAQIVV DSLRPYTVQL SKDLNGNHVI
QKCLQRLKPE NFQFIFDAIS DSCIDIATHR 721 HGCCVLQRCL DHGTTEQCDN
LCDKLLALVD KLTLDPFGNY VVQYIITKEA EKNKYDYTHK 781 IVHLLKPRAI
ELSIHKFGSN VIEKILKTAI VSEPMILEIL NNGGETGIQS LLNDSYGNYV 841
LQTALDISHK QNDYLYKRLS EIVAPLLVGP IRNTPHGKRI IGMLHLDS.
[0304] In some embodiments, a PUF5 protein of the disclosure
comprises or consists of the amino acid sequence of [0305] 1
MSDSTGRINS KASDSSSISD HQTADLSIFN GSFDGGAFSS SNIPLFNFMG TGNQRFQYSP
[0306] 61 HPFAKSSDPC RLAALTPSTP KGPLNLTPAD FGLADFSVGN ESFADFTANN
TSFVGNVQSN [0307] 121 VRSTRLLPAW AVDNSGNIRD DLTLQDVVSN GSLIDFAMDR
TGVKFLERHF PEDHDNEMHF [0308] 181 VLFDKLTEQG AVFTSLCRSA AGNFIIQKFV
EHATLDEQER LVRKMCDNGL IEMCLDKFAC [0309] 241 RVVQMSIQKF DVSIAMKLVE
KISSLDFLPL CTDQCAIHVL QKVVKLLPIS AWSFFVKFLC [0310] 301 RDDNLMTVCQ
DKYGCRLVQQ TIDKLSDNPK LHCFNTRLQL LHGLMTSVAR NCFRLSSNEF [0311] 361
ANYVVQYVIK SSGVMEMYRD TIIEKCLLRN ILSMSQDKYA SHVVEGAFLF APPLLLSEMM
[0312] 421 DEIFDGYVKD QETNRDALDI LLFHQYGNYV VQQMISICIS ALLGKEERKM
VASEMRLYAK [0313] 481 WFDRIKNRVN RHSGRLERFS SGKKITIESLQ KLNVPMTMTN
EPMPYWAMPT PLMDISAHFM [0314] 541 NKLNFQKNSV FDE (SEQ ID NO: 212).
In some embodiments, a PUF6 protein of the disclosure comprises or
consists of the amino acid sequence of [0315] 1 MTPNRRSTDS
YNMLGASFDF DPDFSLLSNK THKNKNPKPP VKLLPYRHGS NTTSSDLDNY [0316] 61
IFNSGSGSSD DETPPPAAPI FISLEEVLLN GLLIDFAIDP SGVKFLEANY PLDSEDQIRPK
[0317] 121 AVFEKLTEST TLFVGLCHSR NGNFIVQKLV ELATPAEQRE LLRQMIDGGL
LVMCKDKFAC [0318] 181 RVVQLALQKF DHSNVFQLIQ ELSTFDLAAM CTDQISIHVI
QRVVKQLPVD MWTFFVHFLS [0319] 241 SGDSLMAVCQ DKYGC:LVQQ VIDRLAENPK
LPCFKFRIQL LHSLMTCIVR NCYRLSSNEF [0320] 301 ANYVIQYVIK SSGIMEMYRD
TIIDKCLLRN LLSMSQDKYA SHVIEGAFLF APPALLHEMM [0321] 361 EEIFSGYVKD
VELNRDALDI LLFHQYGNYV VQQMISICTA ALIGKEEPQL PPAILLLYSG [0322] 421
WYEKMKQRVL QHASRLERFS SGKKIIDSVM RHGVPTAAAI NAQAAPSLME LTAQFDAFP
[0323] 4181 SFLAR (SEQ ID NO: 213). In some embodiments, a PUF7
protein of the disclosure comprises or consists of the amino acid
sequence of [0324] 1 MTPNRRSTDS YNMLGASFDF DPDFSLLSNK THKNKNPKPP
VKLLPYRIGS NTTSSDSDSY [0325] 61 IFNSGSGSSD AETPAPVAPI FISLEDVLLN
GQLIDFAIDP SGVKFLEANY PLDSEDQIRK [0326] 121 AVFEKFTEST TLFVGLCHSR
NGNFIVQKLV ELATPAEQRE LLRQMIDGGL LAMCKDKFAC [0327] 181 RVVQLALQKF
DHSNVFQLIQ ELSTFDLAAM CTDQISIHVI QRVVKQLPVD MWTFFVHFLS [0328] 241
SGDSLMAVCQ DKYGCRLVQQ VIDRLAENPK LPCFKFEPRIQL LHSLMTCIVR NCYRLSSNEF
[0329] 301 ANYVIQYVIK SSGIMEMYRD TIIDKCLLRN LLSMSQDKYA SHVIEGAFLF
APPALLHEMM [0330] 361 EEIFSGYVKD VESNRDALDI LLFHQYGNYV VQQMISICTA
ALIGKEEREL PPAILLLYSG [0331] 421 WYEKMKQRVL QHASRLERFS SGKKIIDSVM
RHGVPTAAAV NAQAAPSLME LTAQFDAMFP [0332] 481 SFLAR (SEQ ID NO: 214).
In some embodiments, a PUF8 protein of the disclosure comprises or
consists of the amino acid sequence of [0333] 1 MSRPISIGNT
CTFDPSASPI ESLGRSIGAQ KIVDSVCGSP IRSYGRHIST NPKNERLPDT [0334] 61
PEFQFATYMH QGGKVIGQNT LHMFGTPPSC YCAQENIPIS SNVGHVLSTI NNNYMNHQYN
[0335] 121 GSNMFSNQMT QMLQAQAYND LQMHQAIHSQS IRVPVQPSAT GIFSNPYPREP
TTTDDLLTRY [0336] 181 RANPAMMKNL KLSDIRGALL KFAKDQVGSR FIQQELASSK
DRFEKDSIFD EVVSNADELV [0337] 241 DDIFGNYVVQ KFFEYGEERH WARLVDAIID
RVPEYAFQMY ACRVLQKALE KINEPLQIKI [0338] 301 LSQIRHVIHR CMKDCNGNHV
VOKAIEKVSP QYVQFIVDTL LESSNTIYEM SVDPYGCRVV [0339] 361 QRCLEHCSPS
QTKPVIGQIH KRFDEIANNQ YGNYVQIIHVI EHGSEEDRMV IVTRVSNNLF [0340] 421
EFATHKYSSN VIEKCLEQGA VYHKSMIVGA ACHHQEGSVP IVVQMMKDQY ANYVVQKMFD
[0341] 481 QVTSEQRREL ILTVNRPHPV LRQFPHGKHI LAKLEKYFQK PANMSYPYQD
MQGSH (SEQ ID NO: 215). In some embodiments, a PUF9 protein of the
disclosure comprises or consists of the amino acid sequence of
TABLE-US-00142 [0341] (SEQ ID NO: 216) 1 MADPNWAYAP PTNYYADHSI
AKPIMISGGH PSQDQGHSPK SESFGQSVTT AFNGMVDNLV 61 GSPSSSVQQR
NYFTTTPFPI SRSPNDRNDD KIMGNGSYGV PIPIPQDGVP QGTPDFQMTP 121
FLQQGGHLIG GSPNGPVQVS GNWYSGGAGI FSTMQQADPS NGMPGMAAEF VNNENGMPGP
181 NGMHQQAMIS GSPPFPYQNM MNLTTSFGAM GLGPQQIQQR DPQMFQQPIL
HEPIQGMAQN 241 GFGQQVFFTQ MQNQQHPQGQ AQQQLQQLAQ QHQQQQNSQQ
FFGQGPNGMG NGGVMNDWSQ 301 RSFGMPQQQA QQNGLPPNFS QNPPRRRGPE
DPNGQTPKTL QDIKNNVIEF AKDQHGSRFI 361 QQKLERASLR DKAAIFTPVL
ENAEELMTDV FGNYVIQKFF EFGNNEQRNQ LVGTIRGNVM 421 KLALQMYGCR
VIQKALEYVE EKYQHEILGE MEGQVLKCVK DQNGNHVIQK VIERVEPERL 481
QFIIDAFTKN NSDNVYTLSV HPYGCRVIQR VLEYCNEEQK QPVLDALQIH LKQLVLDQYG
541 NYVIQHVIEH GSPSDKEQIV QDVISDDLLK FAQHKFASNV IEKCLTFGGH
AERNLIIDKV 601 CGDPNDPSPP LLQMMKDPFA NYVVQKMLDV ADPQHRKKIT
LTIKPHIATL RKYNFGKHIL 661 LKLEKYFAKQ APANSSNSSS NDQIYEHSPF
DIPLGADFSN HPF.
[0342] In some embodiments of the compositions of the disclosure,
at least one of the RNA-binding proteins or RNA-binding portions
thereof is a PPR protein. PPR proteins (proteins with
pentatricopeptide repeat (PPR) motifs derived from plants) are
nuclear-encoded and exclusively controlled at the RNA level
organelles (chloroplasts and mitochondria), cutting, translation,
splicing, RNA editing, genes specifically acting on RNA stability.
PPR proteins are typically a motif of 35 amino acids and have a
structure in which a PPR motif is about 10 contiguous amino acids.
The combination of PPR motifs can be used for sequence-selective
binding to RNA. PPR proteins are often comprised of PPR motifs of
about 10 repeat domains. PPR domains or RNA-binding domains may be
configured to be catalytically inactive. WO 2013/058404
incorporated herein by reference in its entirety.
[0343] In some embodiments, the fusion protein disclosed herein
comprises a linker between the at least two RNA-binding
polypeptides. In some embodiments, the linker is a peptide linker.
In some embodiments, the peptide linker comprises one or more
repeats of the tri-peptide GGS. In other embodiments, the linker is
a non-peptide linker. In some embodiments, the non-peptide linker
comprises polyethylene glycol (PEG), polypropylene glycol (PPG),
co-poly(ethylene/propylene) glycol, polyoxyethylene (POE),
polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl
alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl
amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins,
hyaluronic acid, heparin, or an alkyl linker.
[0344] In some embodiments, the at least one RNA-binding protein
does not require multimerization for RNA-binding activity. In some
embodiments, the at least one RNA-binding protein is not a monomer
of a multimer complex. In some embodiments, a multimer protein
complex does not comprise the RNA binding protein. In some
embodiments, the at least one of RNA-binding protein selectively
binds to a target sequence within the RNA molecule. In some
embodiments, the at least one RNA-binding protein does not comprise
an affinity for a second sequence within the RNA molecule. In some
embodiments, the at least one RNA-binding protein does not comprise
a high affinity for or selectively bind a second sequence within
the RNA molecule. In some embodiments, the at least one RNA-binding
protein comprises between 2 and 1300 amino acids, inclusive of the
endpoints.
[0345] In some embodiments, the at least one RNA-binding protein of
the fusion proteins disclosed herein further comprises a sequence
encoding a nuclear localization signal (NLS). In some embodiments,
a nuclear localization signal (NLS) is positioned 3' to the RNA
binding protein. In some embodiments, the at least one RNA-binding
protein comprises an NLS at a C-terminus of the protein. In some
embodiments, the at least one RNA-binding protein further comprises
a first sequence encoding a first NLS and a second sequence
encoding a second NLS. In some embodiments, the first NLS or the
second NLS is positioned 3' to the RNA-binding protein. In some
embodiments, the at least one RNA-binding protein comprises the
first NLS or the second NLS at a C-terminus of the protein. In some
embodiments, the at least one RNA-binding protein further comprises
an NES (nuclear export signal) or other peptide tag or secretory
signal.
[0346] In some embodiments, a fusion protein disclosed herein
comprises the at least one RNA-binding protein as a first
RNA-binding protein together with a second RNA-binding protein
comprising or consisting of a nuclease domain.
[0347] In some embodiments, the second RNA-binding polypeptide is
operably configured to the first RNA-binding polypeptide at the
C-terminus of the first RNA-binding polypeptide. In some
embodiments, the second RNA-binding polypeptide is operably
configured to the first RNA-binding polypeptide at the N-terminus
of the first RNA-binding polypeptide. For example, one such
exemplary fusion protein is E99 which is configured so that
RNAse1(R39D, N67D, N88A, G89D, R19D, H119N, K41R) is located at the
N-terminus of SpyCas9 whereas another exemplary fusion protein,
E100, is configured so that RNAse1(R39D, N67D, N88A, G89D, R19D,
H119N, K41R) is located at the C-terminus of SpyCas9. See FIG.
6.
Vectors
[0348] In some embodiments of the compositions and methods of the
disclosure, a vector comprises a guide RNA of the disclosure. In
some embodiments, the vector comprises at least one guide RNA of
the disclosure. In some embodiments, the vector comprises one or
more guide RNA(s) of the disclosure. In some embodiments, the
vector comprises two or more guide RNAs of the disclosure. In some
embodiments, the vector further comprises a fusion protein of the
disclosure. In some embodiments, the fusion protein comprises a
first RNA binding protein and a second RNA binding protein.
[0349] In some embodiments of the compositions and methods of the
disclosure, a first vector comprises a guide RNA of the disclosure
and a second vector comprises a fusion protein of the disclosure.
In some embodiments, the first vector comprises at least one guide
RNA of the disclosure. In some embodiments, the first vector
comprises one or more guide RNA(s) of the disclosure. In some
embodiments, the first vector comprises two or more guide RNA(s) of
the disclosure. In some embodiments, the fusion protein comprises a
first RNA binding protein and a second RNA binding protein. In some
embodiments, the first vector and the second vector are identical.
In some embodiments, the first vector and the second vector are not
identical.
[0350] In some embodiments of the compositions and methods of the
disclosure, the vector is or comprises a component of a
"2-component RNA targeting system" comprising (a) nucleic acid
sequence encoding a RNA-targeted fusion protein of the disclosure;
and (b) a single guide RNA (sgRNA) sequence comprising: on its 5'
end, an RNA sequence (or spacer sequence) that hybridizes to or
binds to a target RNA sequence; and on its 3' end, an RNA sequence
(or scaffold sequence) capable of binding to or associating with
the CRISPR/Cas protein of the fusion protein; and wherein the
2-component RNA targeting system recognizes and alters the target
RNA in a cell in the absence of a PAMmer. In some embodiments, the
sequences of the 2-component system are in a single vector. In some
embodiments, the spacer sequence of the 2-component system targets
a repeat sequence selected from the group consisting of CUG, CCUG,
CAG, and GGGGCC.
[0351] In some embodiments of the compositions and methods of the
disclosure, a vector of the disclosure is a viral vector. In some
embodiments, the viral vector comprises a sequence isolated or
derived from a retrovirus. In some embodiments, the viral vector
comprises a sequence isolated or derived from a lentivirus. In some
embodiments, the viral vector comprises a sequence isolated or
derived from an adenovirus. In some embodiments, the viral vector
comprises a sequence isolated or derived from an adeno-associated
virus (AAV). In some embodiments, the viral vector is replication
incompetent. In some embodiments, the viral vector is isolated or
recombinant. In some embodiments, the viral vector is
self-complementary.
[0352] In some embodiments of the compositions and methods of the
disclosure, the viral vector comprises a sequence isolated or
derived from an adeno-associated virus (AAV). In some embodiments,
the viral vector comprises an inverted terminal repeat sequence or
a capsid sequence that is isolated or derived from an AAV of
serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11 or AAV12. In some embodiments, the viral vector is
replication incompetent. In some embodiments, the viral vector is
isolated or recombinant (rAAV). In some embodiments, the viral
vector is self-complementary (scAAV).
[0353] In some embodiments of the compositions and methods of the
disclosure, a vector of the disclosure is a non-viral vector. In
some embodiments, the vector comprises or consists of a
nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a
polyplex or a dendrimer. In some embodiments, the vector is an
expression vector or recombinant expression system. As used herein,
the term "recombinant expression system" refers to a genetic
construct for the expression of certain genetic material formed by
recombination.
[0354] In some embodiments of the compositions and methods of the
disclosure, an expression vector, viral vector or non-viral vector
provided herein, includes without limitation, an expression control
element. An "expression control element" as used herein refers to
any sequence that regulates the expression of a coding sequence,
such as a gene. Exemplary expression control elements include but
are not limited to promoters, enhancers, microRNAs,
post-transcriptional regulatory elements, polyadenylation signal
sequences, and introns. Expression control elements may be
constitutive, inducible, repressible, or tissue-specific, for
example. A "promoter" is a control sequence that is a region of a
polynucleotide sequence at which initiation and rate of
transcription are controlled. It may contain genetic elements at
which regulatory proteins and molecules may bind such as RNA
polymerase and other transcription factors. In some embodiments,
expression control by a promoter is tissue-specific. Non-limiting
exemplary promoters include CMV, CBA, CAG, Cbh, EF-1a, PGK, UBC,
GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF,
MecP2, CaMKII, mGluR2, NFL, NFH, n.beta.2, PPE, ENK, EAAT2, GFAP,
MBP, and U6 promoters. An "enhancer" is a region of DNA that can be
bound by activating proteins to increase the likelihood or
frequency of transcription. Non-limiting exemplary enhancers and
posttranscriptional regulatory elements include the CMV enhancer
and WPRE.
[0355] In some embodiments of the compositions and methods of the
disclosure, an expression vector, viral vector or non-viral vector
provided herein, includes without limitation, vector elements such
as an IRES or 2A peptide sites for configuration of
"multicistronic" or "polycistronic" or "bicistronic" or
tricistronic" constructs, i.e., having double or triple or multiple
coding areas or exons, and as such will have the capability to
express from mRNA two or more proteins from a single construct.
Multicistronic vectors simultaneously express two or more separate
proteins from the same mRNA. The two strategies most widely used
for constructing multicistronic configurations are through the use
of an IRES or a 2A self-cleaving site. An "IRES" refers to an
internal ribosome entry site or portion thereof of viral,
prokaryotic, or eukaryotic origin which are used within
polycistronic vector constructs. In some embodiments, an IRES is an
RNA element that allows for translation initiation in a
cap-independent manner. The term "self-cleaving peptides" or
"sequences encoding self-cleaving peptides" or "2A self-cleaving
site" refer to linking sequences which are used within vector
constructs to incorporate sites to promote ribosomal skipping and
thus to generate two polypeptides from a single promoter, such
self-cleaving peptides include without limitation, T2A, and P2A
peptides or sequences encoding the self-cleaving peptides.
[0356] In some embodiments, the vector is a viral vector. In some
embodiments, the vector is an adenoviral vector, an
adeno-associated viral (AAV) vector, or a lentiviral vector. In
some embodiments, the vector is a retroviral vector, an
adenoviral/retroviral chimera vector, a herpes simplex viral I or
II vector, a parvoviral vector, a reticuloendotheliosis viral
vector, a polioviral vector, a papillomaviral vector, a vaccinia
viral vector, or any hybrid or chimeric vector incorporating
favorable aspects of two or more viral vectors. In some
embodiments, the vector further comprises one or more expression
control elements operably linked to the polynucleotide. In some
embodiments, the vector further comprises one or more selectable
markers. In some embodiments, the AAV vector has low toxicity. In
some embodiments, the AAV vector does not incorporate into the host
genome, thereby having a low probability of causing insertional
mutagenesis. In some embodiments, the AAV vector can encode a range
of total polynucleotides from 4.5 kb to 4.75 kb. In some
embodiments, exemplary AAV vectors that may be used in any of the
herein described compositions, systems, methods, and kits can
include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a
modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an
AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified
AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7
vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an
AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33 vector,
a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified
AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1
vector and any combinations or equivalents thereof. In some
embodiments, the lentiviral vector is an integrase-competent
lentiviral vector (ICLV). In some embodiments, the lentiviral
vector can refer to the transgene plasmid vector as well as the
transgene plasmid vector in conjunction with related plasmids
(e.g., a packaging plasmid, a rev expressing plasmid, an envelope
plasmid) as well as a lentiviral-based particle capable of
introducing exogenous nucleic acid into a cell through a viral or
viral-like entry mechanism. Lentiviral vectors are well-known in
the art (see, e.g., Trono D. (2002) Lentiviral vectors, New York:
Spring-Verlag Berlin Heidelberg and Durand et al. (2011) Viruses
3(2):132-159 doi: 10.3390/v3020132). In some embodiments, exemplary
lentiviral vectors that may be used in any of the herein described
compositions, systems, methods, and kits can include a human
immunodeficiency virus (HIV) 1 vector, a modified human
immunodeficiency virus (HIV) 1 vector, a human immunodeficiency
virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV)
2 vector, a sooty mangabey simian immunodeficiency virus
(SIV.sub.SM) vector, a modified sooty mangabey simian
immunodeficiency virus (SIV.sub.SM) vector, a African green monkey
simian immunodeficiency virus (SIV.sub.AGM) vector, a modified
African green monkey simian immunodeficiency virus (SIV.sub.AGM)
vector, an equine infectious anemia virus (EIAV) vector, a modified
equine infectious anemia virus (EIAV) vector, a feline
immunodeficiency virus (FIV) vector, a modified feline
immunodeficiency virus (FIV) vector, a Visna/maedi virus (VNV/VMV)
vector, a modified Visna/maedi virus (VNV/VMV) vector, a caprine
arthritis-encephalitis virus (CAEV) vector, a modified caprine
arthritis-encephalitis virus (CAEV) vector, a bovine
immunodeficiency virus (BIV), or a modified bovine immunodeficiency
virus (BIV).
Nucleic Acids
[0357] Provided herein are the nucleic acid sequences encoding the
fusion proteins disclosed herein for use in gene transfer and
expression techniques described herein. It should be understood,
although not always explicitly stated that the sequences provided
herein can be used to provide the expression product as well as
substantially identical sequences that produce a protein that has
the same biological properties. These "biologically equivalent" or
"biologically active" or "equivalent" polypeptides are encoded by
equivalent polynucleotides as described herein. They may possess at
least 60%, or alternatively, at least 65%, or alternatively, at
least 70%, or alternatively, at least 75%, or alternatively, at
least 80%, or alternatively at least 85%, or alternatively at least
90%, or alternatively at least 95% or alternatively at least 98%,
identical primary amino acid sequence to the reference polypeptide
when compared using sequence identity methods run under default
conditions. Specific polypeptide sequences are provided as examples
of particular embodiments. Modifications to the sequences to amino
acids with alternate amino acids that have similar charge.
Additionally, an equivalent polynucleotide is one that hybridizes
under stringent conditions to the reference polynucleotide or its
complement or in reference to a polypeptide, a polypeptide encoded
by a polynucleotide that hybridizes to the reference encoding
polynucleotide under stringent conditions or its complementary
strand. Alternatively, an equivalent polypeptide or protein is one
that is expressed from an equivalent polynucleotide.
[0358] The nucleic acid sequences (e.g., polynucleotide sequences)
disclosed herein may be codon-optimized which is a technique well
known in the art. In some embodiments disclosed herein, exemplary
Cas sequences, such as e.g., SEQ ID NO: 46 (Cas13d), are codon
optimized for expression in human cells. Codon optimization refers
to the fact that different cells differ in their usage of
particular codons. This codon bias corresponds to a bias in the
relative abundance of particular tRNAs in the cell type. By
altering the codons in the sequence to match with the relative
abundance of corresponding tRNAs, it is possible to increase
expression. It is also possible to decrease expression by
deliberately choosing codons for which the corresponding tRNAs are
known to be rare in a particular cell type. Codon usage tables are
known in the art for mammalian cells, as well as for a variety of
other organisms. Based on the genetic code, nucleic acid sequences
coding for, e.g., a Cas protein, can be generated. In some
embodiments, such a sequence is optimized for expression in a host
or target cell, such as a host cell used to express the Cas protein
or a cell in which the disclosed methods are practiced (such as in
a mammalian cell, e.g., a human cell). Codon preferences and codon
usage tables for a particular species can be used to engineer
isolated nucleic acid molecules encoding a Cas protein (such as one
encoding a protein having at least 80%, at least 85%, at least 90%,
at least 92%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% sequence identity to its corresponding
wild-type protein) that takes advantage of the codon usage
preferences of that particular species. For example, the Cas
proteins disclosed herein can be designed to have codons that are
preferentially used by a particular organism of interest. In one
example, an Cas nucleic acid sequence is optimized for expression
in human cells, such as one having at least 70%, at least 80%, at
least 85%, at least 90%, at least 92%, at least 95%, at least 98%,
or at least 99% sequence identity to its corresponding wild-type or
originating nucleic acid sequence. In some embodiments, an isolated
nucleic acid molecule encoding at least one Cas protein (which can
be part of a vector) includes at least one Cas protein coding
sequence that is codon optimized for expression in a eukaryotic
cell, or at least one Cas protein coding sequence codon optimized
for expression in a human cell. In one embodiment, such a codon
optimized Cas coding sequence has at least 80%, at least 85%, at
least 90%, at least 92%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% sequence identity to its
corresponding wild-type or originating sequence. In another
embodiment, a eukaryotic cell codon optimized nucleic acid sequence
encodes a Cas protein having at least 85%, at least 90%, at least
92%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to its corresponding wild-type
or originating protein. In another embodiment, a variety of clones
containing functionally equivalent nucleic acids may be routinely
generated, such as nucleic acids which differ in sequence but which
encode the same Cas protein sequence. Silent mutations in the
coding sequence result from the degeneracy (i.e., redundancy) of
the genetic code, whereby more than one codon can encode the same
amino acid residue. Thus, for example, leucine can be encoded by
CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC,
TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC;
aspartic acid can be encoded by GAT or GAC; cysteine can be encoded
by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG;
glutamine can be encoded by CAA or CAG; tyrosine can be encoded by
TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA.
Tables showing the standard genetic code can be found in various
sources (see, for example, Stryer, 1988, Biochemistry, 3.sup.rd
Edition, W.H. 5 Freeman and Co., NY).
[0359] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of a PC reaction, or the enzymatic cleavage of a
polynucleotide by a ribozyme.
[0360] Examples of stringent hybridization conditions include:
incubation temperatures of about 25.degree. C. to about 37.degree.
C.; hybridization buffer concentrations of about 6.times.SSC to
about 10.times.SSC; formamide concentrations of about 0% to about
25%; and wash solutions from about 4.times.SSC to about
8.times.SSC. Examples of moderate hybridization conditions include:
incubation temperatures of about 40.degree. C. to about 50.degree.
C.; buffer concentrations of about 9.times.SSC to about
2.times.SSC; formamide concentrations of about 30% to about 50%;
and wash solutions of about 5.times.SSC to about 2.times.SSC.
Examples of high stringency conditions include: incubation
temperatures of about 55.degree. C. to about 68.degree. C.; buffer
concentrations of about 1.times.SSC to about 0.1.times.SSC;
formamide concentrations of about 55% to about 75%; and wash
solutions of about 1.times.SSC, 0.1.times.SSC, or deionized water.
In general, hybridization incubation times are from 5 minutes to 24
hours, with 1, 2, or more washing steps, and wash incubation times
are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate
buffer. It is understood that equivalents of SSC using other buffer
systems can be employed.
[0361] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, or alternatively less than 25% identity, with one of the
sequences of the present invention.
Cells
[0362] In some embodiments of the compositions and methods of the
disclosure, a cell of the disclosure is a prokaryotic cell.
[0363] In some embodiments of the compositions and methods of the
disclosure, a cell of the disclosure is a eukaryotic cell. In some
embodiments, the cell is a mammalian cell. In some embodiments, the
cell is a bovine, murine, feline, equine, porcine, canine, simian,
or human cell. In some embodiments, the cell is a non-human
mammalian cell such as a non-human primate cell.
[0364] In some embodiments, a cell of the disclosure is a somatic
cell. In some embodiments, a cell of the disclosure is a germline
cell. In some embodiments, a germline cell of the disclosure is not
a human cell.
[0365] In some embodiments of the compositions and methods of the
disclosure, a cell of the disclosure is a stem cell. In some
embodiments, a cell of the disclosure is an embryonic stem cell. In
some embodiments, an embryonic stem cell of the disclosure is not a
human cell. In some embodiments, a cell of the disclosure is a
multipotent stem cell or a pluripotent stem cell. In some
embodiments, a cell of the disclosure is an adult stem cell. In
some embodiments, a cell of the disclosure is an induced
pluripotent stem cell (iPSC). In some embodiments, a cell of the
disclosure is a hematopoietic stem cell (HSC).
[0366] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is an immune cell. In
some embodiments, an immune cell of the disclosure is a lymphocyte.
In some embodiments, an immune cell of the disclosure is a T
lymphocyte (also referred to herein as a T-cell). Exemplary T-cells
of the disclosure include, but are not limited to, naive T cells,
effector T cells, helper T cells, memory T cells, regulatory T
cells (Tregs) and Gamma delta T cells. In some embodiments, an
immune cell of the disclosure is a B lymphocyte. In some
embodiments, an immune cell of the disclosure is a natural killer
cell. In some embodiments, an immune cell of the disclosure is an
antigen-presenting cell.
[0367] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is a muscle cell. In
some embodiments, a muscle cell of the disclosure is a myoblast or
a myocyte. In some embodiments, a muscle cell of the disclosure is
a cardiac muscle cell, skeletal muscle cell or smooth muscle cell.
In some embodiments, a muscle cell of the disclosure is a striated
cell.
[0368] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is an epithelial cell.
In some embodiments, an epithelial cell of the disclosure forms a
squamous cell epithelium, a cuboidal cell epithelium, a columnar
cell epithelium, a stratified cell epithelium, a pseudostratified
columnar cell epithelium or a transitional cell epithelium. In some
embodiments, an epithelial cell of the disclosure forms a gland
including, but not limited to, a pineal gland, a thymus gland, a
pituitary gland, a thyroid gland, an adrenal gland, an apocrine
gland, a holocrine gland, a merocrine gland, a serous gland, a
mucous gland and a sebaceous gland. In some embodiments, an
epithelial cell of the disclosure contacts an outer surface of an
organ including, but not limited to, a lung, a spleen, a stomach, a
pancreas, a bladder, an intestine, a kidney, a gallbladder, a
liver, a larynx or a pharynx. In some embodiments, an epithelial
cell of the disclosure contacts an outer surface of a blood vessel
or a vein.
[0369] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is a neuronal cell. In
some embodiments, a neuron cell of the disclosure is a neuron of
the central nervous system. In some embodiments, a neuron cell of
the disclosure is a neuron of the brain or the spinal cord. In some
embodiments, a neuron cell of the disclosure is a neuron of the
retina. In some embodiments, a neuron cell of the disclosure is a
neuron of a cranial nerve or an optic nerve. In some embodiments, a
neuron cell of the disclosure is a neuron of the peripheral nervous
system. In some embodiments, a neuron cell of the disclosure is a
neuroglial or a glial cell. In some embodiments, a glial of the
disclosure is a glial cell of the central nervous system including,
but not limited to, oligodendrocytes, astrocytes, ependymal cells,
and microglia. In some embodiments, a glial of the disclosure is a
glial cell of the peripheral nervous system including, but not
limited to, Schwann cells and satellite cells.
[0370] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is a primary cell.
[0371] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is a cultured
cell.
[0372] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is in vivo, in vitro,
ex vivo or in situ.
[0373] In some embodiments of the compositions and methods of the
disclosure, a somatic cell of the disclosure is autologous or
allogeneic.
Methods of Use
[0374] The disclosure provides a method of modifying level of
expression of an RNA molecule of the disclosure or a protein
encoded by the RNA molecule comprising contacting the composition
and the RNA molecule under conditions suitable for binding of one
or more of the guide RNA or the fusion protein (or a portion
thereof) to the RNA molecule.
[0375] The disclosure provides a method of modifying an activity of
a protein encoded by an RNA molecule comprising contacting the
composition and the RNA molecule under conditions suitable for
binding of one or more of the guide RNA or the fusion protein (or a
portion thereof) to the RNA molecule.
[0376] The disclosure provides a method of modifying level of
expression of an RNA molecule of the disclosure or a protein
encoded by the RNA molecule comprising contacting the composition
and a cell comprising the RNA molecule under conditions suitable
for binding of one or more of the guide RNA or the fusion protein
(or a portion thereof) to the RNA molecule. In some embodiments,
the cell is in vivo, in vitro, ex vivo or in situ. In some
embodiments, the composition comprises a vector comprising
composition comprising a guide RNA of the disclosure and a fusion
protein of the disclosure. In some embodiments, the vector is an
AAV.
[0377] The disclosure provides a method of modifying an activity of
a protein encoded by an RNA molecule comprising contacting the
composition and a cell comprising the RNA molecule under conditions
suitable for binding of one or more of the guide RNA or the fusion
protein (or a portion thereof) to the RNA molecule. In some
embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In
some embodiments, the composition comprises a vector comprising
composition comprising a guide RNA or a single guide RNA of the
disclosure and a fusion protein of the disclosure. In some
embodiments, the vector is an AAV.
[0378] The disclosure provides a method of modifying level of
expression of an RNA molecule of the disclosure or a protein
encoded by the RNA molecule comprising contacting the composition
and the RNA molecule under conditions suitable for RNA nuclease
activity wherein the fusion protein induces a break in the RNA
molecule.
[0379] The disclosure provides a method of modifying an activity of
a protein encoded by an RNA molecule comprising contacting the
composition and the RNA molecule under conditions suitable for RNA
nuclease activity wherein the fusion protein induces a break in the
RNA molecule.
[0380] The disclosure provides a method of modifying a level of
expression of an RNA molecule of the disclosure or a protein
encoded by the RNA molecule comprising contacting the composition
and a cell comprising the RNA molecule under conditions suitable
for RNA nuclease activity wherein the fusion protein induces a
break in the RNA molecule. In some embodiments, the cell is in
vivo, in vitro, ex vivo or in situ. In some embodiments, the
composition comprises a vector comprising composition comprising a
guide RNA of the disclosure and a fusion protein of the disclosure.
In some embodiments, the vector is an AAV.
[0381] The disclosure provides a method of modifying an activity of
a protein encoded by an RNA molecule comprising contacting the
composition and a cell comprising the RNA molecule under conditions
suitable for RNA nuclease activity wherein the fusion protein
induces a break in the RNA molecule. In some embodiments, the cell
is in vivo, in vitro, ex vivo or in situ. In some embodiments, the
composition comprises a vector comprising composition comprising a
guide RNA or a single guide RNA of the disclosure and a fusion
protein of the disclosure. In some embodiments, the vector is an
AAV.
[0382] The disclosure provides a method of treating a disease or
disorder comprising administering to a subject a therapeutically
effective amount of a composition of the disclosure.
[0383] The disclosure provides a method of treating a disease or
disorder comprising administering to a subject a therapeutically
effective amount of a composition of the disclosure, wherein the
composition comprises a vector comprising composition comprising a
guide RNA of the disclosure and a fusion protein of the disclosure
and wherein the composition modifies a level of expression of an
RNA molecule of the disclosure or a protein encoded by the RNA
molecule.
[0384] The disclosure provides a method of treating a disease or
disorder comprising administering to a subject a therapeutically
effective amount of a composition of the disclosure, wherein the
composition comprises a vector comprising composition comprising a
guide RNA of the disclosure and a fusion protein of the disclosure
and wherein the composition modifies an activity of a protein
encoded by an RNA molecule.
[0385] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a genetic disease or disorder. In some
embodiments, the genetic disease or disorder is a single-gene
disease or disorder. In some embodiments, the single-gene disease
or disorder is an autosomal dominant disease or disorder, an
autosomal recessive disease or disorder, an X-chromosome linked
(X-linked) disease or disorder, an X-linked dominant disease or
disorder, an X-linked recessive disease or disorder, a Y-linked
disease or disorder or a mitochondrial disease or disorder. In some
embodiments, the genetic disease or disorder is a multiple-gene
disease or disorder. In some embodiments, the genetic disease or
disorder is a multiple-gene disease or disorder. In some
embodiments, the single-gene disease or disorder is an autosomal
dominant disease or disorder including, but not limited to,
Huntington's disease, neurofibromatosis type 1, neurofibromatosis
type 2, Marfan syndrome, hereditary nonpolyposis colorectal cancer,
hereditary multiple exostoses, Von Willebrand disease, and acute
intermittent porphyria. In some embodiments, the single-gene
disease or disorder is an autosomal recessive disease or disorder
including, but not limited to, Albinism, Medium-chain acyl-CoA
dehydrogenase deficiency, cystic fibrosis, sickle-cell disease,
Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy,
and Roberts syndrome. In some embodiments, the single-gene disease
or disorder is X-linked disease or disorder including, but not
limited to, muscular dystrophy, Duchenne muscular dystrophy,
Hemophilia, Adrenoleukodystrophy (ALD), Rett syndrome, and
Hemophilia A. In some embodiments, the single-gene disease or
disorder is a mitochondrial disorder including, but not limited to,
Leber's hereditary optic neuropathy.
[0386] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, an immune disease or disorder. In some
embodiments, the immune disease or disorder is an immunodeficiency
disease or disorder including, but not limited to, B-cell
deficiency, T-cell deficiency, neutropenia, asplenia, complement
deficiency, acquired immunodeficiency syndrome (AIDS) and
immunodeficiency due to medical intervention (immunosuppression as
an intended or adverse effect of a medical therapy). In some
embodiments, the immune disease or disorder is an autoimmune
disease or disorder including, but not limited to, Achalasia,
Addison's disease, Adult Still's disease, Agammaglobulinemia,
Alopecia areata, Amyloidosis, Anti-GBM/Anti-TBM nephritis,
Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune
dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis,
Autoimmune inner ear disease (AIED), Autoimmune myocarditis,
Autoimmune oophoritis, Autoimmune orchitis, Autoimmune
pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal
& neuronal neuropathy (AMAN), Balo disease, Behcet's disease,
Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease
(CD), Celiac disease, Chagas disease, Chronic inflammatory
demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal
osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic
Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome,
Cold agglutinin disease, Congenital heart block, Coxsackie
myocarditis, CREST syndrome, Crohn's disease, Dermatitis
herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis
optica), Discoid lupus, Dressler's syndrome, Endometriosis,
Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema
nodosum, Essential mixed cryoglobulinemia, Evans syndrome,
Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal
arteritis), Giant cell myocarditis, Glomerulonephritis,
Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves'
disease, Guillain-Barre syndrome, Hashimoto's thyroiditis,
Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes
gestationis or pemphigoid gestationis (PG), Hidradenitis
Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA
Nephropathy, IgG4-related sclerosing disease, Immune
thrombocytopenic purpura (ITP), Inclusion body myositis (IBM),
Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes
(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,
Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,
Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease
(LAD), Lupus, Lyme disease chronic, Meniere's disease, Microscopic
polyangiitis (MPA), Mixed connective tissue disease (MCTD),
Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor
Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis,
Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica,
Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis,
Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar
degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH),
Parry Romberg syndrome, Pars planitis (peripheral uveitis),
Parsonnage-Turner syndrome, Pemphigus, Peripheral neuropathy,
Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS
syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II,
III, Polymyalgia rheumatica, Polymyositis, Postmyocardial
infarction syndrome, Postpericardiotomy syndrome, Primary biliary
cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis,
Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA),
Pyoderma gangrenosum, Raynaud's phenomenon, Reactive Arthritis,
Reflex sympathetic dystrophy, Relapsing polychondritis, Restless
legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever,
Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis,
Scleroderma, Sjogren's syndrome, Sperm & testicular
autoimmunity, Stiff person syndrome (SPS), Subacute bacterial
endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),
Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,
Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS),
Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC),
Undifferentiated connective tissue disease (UCTD), Uveitis,
Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, or Wegener's
granulomatosis.
[0387] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, an inflammatory disease or disorder.
[0388] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a metabolic disease or disorder.
[0389] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a degenerative or a progressive disease or
disorder. In some embodiments, the degenerative or a progressive
disease or disorder includes, but is not limited to, amyotrophic
lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease,
and aging.
[0390] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, an infectious disease or disorder.
[0391] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a pediatric or a developmental disease or
disorder.
[0392] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a cardiovascular disease or disorder.
[0393] In some embodiments of the compositions and methods of the
disclosure, a disease or disorder of the disclosure includes, but
is not limited to, a proliferative disease or disorder. In some
embodiments, the proliferative disease or disorder is a cancer. In
some embodiments, the cancer includes, but is not limited to, Acute
Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),
Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma
(Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary
CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer,
Gastrointestinal Carcinoid Tumors, Astrocytomas, Atypical
Teratoid/Rhabdoid Tumor, Central Nervous System (Brain Cancer),
Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Ewing Sarcoma, Osteosarcoma, Malignant Fibrous
Histiocytoma, Brain Tumors, Breast Cancer, Burkitt Lymphoma,
Carcinoid Tumor, Carcinoma, Cardiac (Heart) Tumors, Embryonal
Tumors, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer,
Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL),
Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative
Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell
Lymphoma, Ductal Carcinoma In Situ, Embryonal Tumors, Endometrial
Cancer (Uterine Cancer), Ependymoma, Esophageal Cancer,
Esthesioneuroblastoma (Head and Neck Cancer), Ewing Sarcoma (Bone
Cancer), Extracranial Germ Cell Tumor, Extragonadal Germ Cell
Tumor, Eye Cancer, Childhood Intraocular Melanoma, Intraocular
Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous
Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder
Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor,
Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma),
Childhood Gastrointestinal Stromal Tumors, Germ Cell Tumors,
Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell
Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational
Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer,
Heart Tumors, Hepatocellular (Liver) Cancer, Histiocytosis, Hodgkin
Lymphoma, Hypopharyngeal Cancer (Head and Neck Cancer), Intraocular
Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors,
Kaposi Sarcoma (Soft Tissue Sarcoma), Kidney (Renal Cell) Cancer,
Langerhans Cell Histiocytosis, Laryngeal Cancer (Head and Neck
Cancer), Leukemia, Lip and Oral Cavity Cancer (Head and Neck
Cancer), Liver Cancer, Lung Cancer (Non-Small Cell and Small Cell),
Childhood Lung Cancer, Lymphoma, Male Breast Cancer, Malignant
Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel
Cell Carcinoma (Skin Cancer), Mesothelioma, Metastatic Squamous
Neck Cancer with Occult Primary (Head and Neck Cancer), Midline
Tract Carcinoma With NUT Gene Changes, Mouth Cancer (Head and Neck
Cancer), Multiple Endocrine Neoplasia Syndromes, Multiple
Myeloma/Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma),
Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative
Neoplasms, Nasal Cavity and Paranasal Sinus Cancer (Head and Neck
Cancer), Nasopharyngeal Cancer (Head and Neck Cancer),
Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer,
Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer,
Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian
Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet
Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer,
Penile Cancer, Pharyngeal Cancer (Head and Neck Cancer),
Pheochromocytoma, Plasma Cell Neoplasm/Multiple Myeloma,
Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary
Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer,
Prostate Cancer, Rectal Cancer, Recurrent Cancer, Renal Cell
(Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Childhood (Soft
Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer),
Sarcoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma),
Childhood Vascular Tumors (Soft Tissue Sarcoma), Ewing Sarcoma
(Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma
(Bone Cancer), Uterine Sarcoma, Sezary Syndrome, Lymphoma, Skin
Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue
Sarcoma, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer,
Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer,
Throat Cancer (Head and Neck Cancer), Nasopharyngeal Cancer,
Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic
Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal
Pelvis and Ureter, Renal Cell Cancer, Urethral Cancer, Uterine
Sarcoma, Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma),
Vulvar Cancer, Wilms Tumor and Other Childhood Kidney Tumors.
[0394] In some embodiments of the methods of the disclosure, a
subject of the disclosure has been diagnosed with the disease or
disorder. In some embodiments, the subject of the disclosure
presents at least one sign or symptom of the disease or disorder.
In some embodiments, the subject has a biomarker predictive of a
risk of developing the disease or disorder. In some embodiments,
the biomarker is a genetic mutation.
[0395] In some embodiments of the methods of the disclosure, a
subject of the disclosure is female. In some embodiments of the
methods of the disclosure, a subject of the disclosure is male. In
some embodiments, a subject of the disclosure has two XX or XY
chromosomes. In some embodiments, a subject of the disclosure has
two XX or XY chromosomes and a third chromosome, either an X or a
Y.
[0396] In some embodiments of the methods of the disclosure, a
subject of the disclosure is a neonate, an infant, a child, an
adult, a senior adult, or an elderly adult. In some embodiments of
the methods of the disclosure, a subject of the disclosure is at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days old.
In some embodiments of the methods of the disclosure, a subject of
the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12
months old. In some embodiments of the methods of the disclosure, a
subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100 or any number of years or partial years in between of
age.
[0397] In some embodiments of the methods of the disclosure, a
subject of the disclosure is a mammal. In some embodiments, a
subject of the disclosure is a non-human mammal.
[0398] In some embodiments of the methods of the disclosure, a
subject of the disclosure is a human.
[0399] In some embodiments of the methods of the disclosure, a
therapeutically effective amount comprises a single dose of a
composition of the disclosure. In some embodiments, a
therapeutically effective amount comprises a therapeutically
effective amount comprises at least one dose of a composition of
the disclosure. In some embodiments, a therapeutically effective
amount comprises a therapeutically effective amount comprises one
or more dose(s) of a composition of the disclosure.
[0400] In some embodiments of the methods of the disclosure, a
therapeutically effective amount eliminates a sign or symptom of
the disease or disorder. In some embodiments, a therapeutically
effective amount reduces a severity of a sign or symptom of the
disease or disorder.
[0401] In some embodiments of the methods of the disclosure, a
therapeutically effective amount eliminates the disease or
disorder.
[0402] In some embodiments of the methods of the disclosure, a
therapeutically effective amount prevents an onset of a disease or
disorder. In some embodiments, a therapeutically effective amount
delays the onset of a disease or disorder. In some embodiments, a
therapeutically effective amount reduces the severity of a sign or
symptom of the disease or disorder. In some embodiments, a
therapeutically effective amount improves a prognosis for the
subject.
[0403] In some embodiments of the methods of the disclosure, a
composition of the disclosure is administered to the subject
systemically. In some embodiments, the composition of the
disclosure is administered to the subject by an intravenous route.
In some embodiments, the composition of the disclosure is
administered to the subject by an injection or an infusion.
[0404] In some embodiments of the methods of the disclosure, a
composition of the disclosure is administered to the subject
locally. In some embodiments, the composition of the disclosure is
administered to the subject by an intraosseous, intraocular,
intracerebrospinal or intraspinal route. In some embodiments, the
composition of the disclosure is administered directly to the
cerebral spinal fluid of the central nervous system. In some
embodiments, the composition of the disclosure is administered
directly to a tissue or fluid of the eye and does not have
bioavailability outside of ocular structures. In some embodiments,
the composition of the disclosure is administered to the subject by
an injection or an infusion.
[0405] In some embodiments, the compositions comprising the
RNA-binding fusion proteins disclosed herein are formulated as
pharmaceutical compositions. Briefly, pharmaceutical compositions
for use as disclosed herein may comprise a fusion protein(s) or a
polynucleotide encoding the fusion protein(s), optionally comprised
in an AAV, which is optionally also immune orthogonal, in
combination with one or more pharmaceutically or physiologically
acceptable carriers, diluents or excipients. Such compositions may
comprise buffers such as neutral buffered saline, phosphate
buffered saline and the like; carbohydrates such as glucose,
mannose, sucrose or dextrans, mannitol; proteins; polypeptides or
amino acids such as glycine; antioxidants; chelating agents such as
EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and
preservatives. Compositions of the disclosure may be formulated for
oral, intravenous, topical, enteral, intraocular, and/or parenteral
administration. In certain embodiments, the compositions of the
present disclosure are formulated for intravenous
administration.
EXAMPLE EMBODIMENTS
Embodiment 1
[0406] A composition comprising:
[0407] (a) a sequence comprising a guide RNA (gRNA) that
specifically binds a target sequence within an RNA molecule and
[0408] (b) a sequence encoding a fusion protein, the sequence
comprising a sequence encoding a first RNA-binding polypeptide and
a sequence encoding a second RNA-binding polypeptide,
[0409] wherein neither the first RNA-binding polypeptide nor the
second RNA-binding polypeptide comprises a significant DNA-nuclease
activity,
[0410] wherein the first RNA-binding polypeptide and the second
RNA-binding polypeptide are not identical, and
[0411] wherein the second RNA-binding polypeptide comprises an
RNA-nuclease activity; or
[0412] a composition comprising nucleic acid sequence encoding a
fusion protein, the fusion protein comprising a first RNA-binding
polypeptide and a second RNA-binding polypeptide, wherein the first
RNA-binding polypeptide is not a guided RNA-binding polypeptide,
wherein the first RNA-binding polypeptide and the second
RNA-binding polypeptide are not identical, and wherein the second
RNA-binding polypeptide comprises an RNA-nuclease activity.
Embodiment 2
[0413] The composition of embodiment 1, wherein the target sequence
comprises at least one repeated sequence.
Embodiment 3
[0414] The composition of embodiment 1 or 2, wherein the sequence
comprising the gRNA comprises a promoter capable of expressing the
gRNA in a eukaryotic cell.
Embodiment 4
[0415] The composition of embodiment 3, wherein the eukaryotic cell
is an animal cell.
Embodiment 5
[0416] The composition of embodiment 4, wherein the animal cell is
a mammalian cell.
Embodiment 6
[0417] The composition of embodiment 5, wherein the animal cell is
a human cell.
Embodiment 7
[0418] The composition of any one of embodiments 1-6, wherein the
promoter is a constitutively active promoter.
Embodiment 8
[0419] The composition of any one of embodiments 1-7, wherein the
promoter is isolated or derived from a promoter capable of driving
expression of an RNA polymerase.
Embodiment 9
[0420] The composition of embodiment 8, wherein the promoter is
isolated or derived from a U6 promoter.
Embodiment 10
[0421] The composition of any one of embodiments 1-7, wherein the
promoter is isolated or derived from a promoter capable of driving
expression of a transfer RNA (tRNA).
Embodiment 11
[0422] The composition of embodiment 10, wherein the promoter is
isolated or derived from an alanine tRNA promoter, an arginine tRNA
promoter, an asparagine tRNA promoter, an aspartic acid tRNA
promoter, a cysteine tRNA promoter, a glutamine tRNA promoter, a
glutamic acid tRNA promoter, a glycine tRNA promoter, a histidine
tRNA promoter, an isoleucine tRNA promoter, a leucine tRNA
promoter, a lysine tRNA promoter, a methionine tRNA promoter, a
phenylalanine tRNA promoter, a proline tRNA promoter, a serine tRNA
promoter, a threonine tRNA promoter, a tryptophan tRNA promoter, a
tyrosine tRNA promoter, or a valine tRNA promoter.
Embodiment 12
[0423] The composition of embodiment 10, wherein the promoter is
isolated or derived from a valine tRNA promoter.
Embodiment 13
[0424] The composition of any one of embodiments 1-12, wherein the
sequence comprising the gRNA comprises a spacer sequence that
specifically binds to the target RNA sequence.
Embodiment 14
[0425] The composition of embodiment 13, wherein the spacer
sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%,
95%, 97%, 99% or any percentage in between of complementarity to
the target RNA sequence.
Embodiment 15
[0426] The composition of embodiment 13, wherein the spacer
sequence has 100% complementarity to the target RNA sequence.
Embodiment 16
[0427] The composition of any one of embodiments 13-15, wherein the
spacer sequence comprises or consists of 20 nucleotides.
Embodiment 17
[0428] The composition of any one of embodiments 13-15, wherein the
spacer sequence comprises or consists of 21 nucleotides.
Embodiment 18
[0429] The composition of embodiment 17, wherein the spacer
sequence comprises the sequence UGGAGCGAGCAUCCCCCAAA (SEQ ID NO:
1), GUUUGGGGGAUGCUCGCUCCA (SEQ ID NO: 2), CCCUCACUGCUGGGGAGUCC (SEQ
ID NO: 3), GGACUCCCCAGCAGUGAGGG (SEQ ID NO: 4),
GCAACUGGAUCAAUUUGCUG (SEQ ID NO: 5), GCAGCAAAUUGAUCCAGUUGC (SEQ ID
NO: 6), GCAUUCUUAUCUGGUCAGUGC (SEQ ID NO: 7), GCACUGACCAGAUAAGAAUG
(SEQ ID NO: 8), GAGCAGCAGCAGCAGCAGCAG (SEQ ID NO: 9),
GCAGGCAGGCAGGCAGGCAGG (SEQ ID NO: 10), GCCCCGGCCCCGGCCCCGGC (SEQ ID
NO: 11), or GCTGCTGCTGCTGCTGCTGC (SEQ ID NO: 12),
GGGGCCGGGGCCGGGGCCGG (SEQ ID NO: 74), GGGCCGGGGCCGGGGCCGGG (SEQ ID
NO: 75), GGCCGGGGCCGGGGCCGGGG (SEQ ID NO: 76), GCCGGGGCCGGGGCCGGGGC
(SEQ ID NO: 77), CCGGGGCCGGGGCCGGGGCC (SEQ ID NO: 78),
CGGGGCCGGGGCCGGGGCCG (SEQ ID NO: 79).
Embodiment 19
[0430] The composition of any one of embodiments 1-18, wherein the
sequence comprising the gRNA comprises a scaffold sequence that
specifically binds to the first RNA binding protein.
Embodiment 20
[0431] The composition of embodiment 19, wherein the scaffold
sequence comprises a stem-loop structure.
Embodiment 21
[0432] The composition of embodiment 19 or 20, wherein the scaffold
sequence comprises or consists of 90 nucleotides.
Embodiment 22
[0433] The composition of embodiment 19 or 20, wherein the scaffold
sequence comprises or consists of 93 nucleotides.
Embodiment 23
[0434] The composition of embodiment 22, wherein the scaffold
sequence comprises the sequence
TABLE-US-00143 (SEQ ID NO: 13)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC
CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.
Embodiment 24
[0435] The composition of embodiment 16, wherein the spacer
sequence comprises the sequence GUGAUAAGUGGAAUGCCAUG (SEQ ID NO:
14), CUGGUGAACUUCCGAUAGUG (SEQ ID NO: 15), or GAGATATAGCCTGGTGGTTC
(SEQ ID NO: 16).
Embodiment 25
[0436] The composition of embodiment 19 or 24, wherein the scaffold
sequence comprises a step-loop structure.
Embodiment 26
[0437] The composition of embodiment 25, wherein the scaffold
sequence comprises or consists of 85 nucleotides.
Embodiment 27
[0438] The composition of embodiment 26, wherein the scaffold
sequence comprises the sequence
TABLE-US-00144 (SEQ ID NO: 17)
GGACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA
GUGGCACCGAGUCGGUGCUUUUU.
Embodiment 28
[0439] The composition of embodiment 16, wherein the spacer
sequence comprises the sequence at least 1, 2, 3, 4, 5, 6, or 7
repeats of the sequence CUG (SEQ ID NO: 18), CCUG (SEQ ID NO: 19),
CAG (SEQ ID NO: 80), GGGGCC (SEQ ID NO: 81) or any combination
thereof.
Embodiment 29
[0440] The composition of embodiment 28, wherein the sequence
comprising the gRNA comprises a scaffold sequence that specifically
binds to the first RNA binding protein.
Embodiment 30
[0441] The composition of embodiment 29, wherein the scaffold
sequence comprises a stem-loop structure.
Embodiment 31
[0442] The composition of embodiment 29 or 30, wherein the scaffold
sequence comprises or consists of 90 nucleotides.
Embodiment 32
[0443] The composition of embodiment 30 or 31, wherein the scaffold
sequence comprises or consists of 93 nucleotides.
Embodiment 33
[0444] The composition of embodiment 32, wherein the scaffold
sequence comprises the sequence
TABLE-US-00145 (SEQ ID NO: 82)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC
CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU or (SEQ ID NO: 83)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.
Embodiment 34
[0445] The composition of any one of embodiments 1-33, wherein the
gRNA does not bind or does not selectively bind to a second
sequence within the RNA molecule.
Embodiment 35
[0446] The composition of embodiment 34, wherein an RNA genome or
an RNA transcriptome comprises the RNA molecule.
Embodiment 36
[0447] The composition of any one of embodiments 1-35, wherein the
first RNA binding protein comprises a CRISPR-Cas protein.
Embodiment 37
[0448] The composition of embodiment 36, wherein the CRISPR-Cas
protein is a Type II CRISPR-Cas protein.
Embodiment 38
[0449] The composition of embodiment 37, wherein the first RNA
binding protein comprises a Cas9 polypeptide or an RNA-binding
portion thereof.
Embodiment 39
[0450] The composition of embodiment 36, wherein the CRISPR-Cas
protein is a Type V CRISPR-Cas protein.
Embodiment 40
[0451] The composition of embodiment 39, wherein the first RNA
binding protein comprises a Cpf1 polypeptide or an RNA-binding
portion thereof.
Embodiment 41
[0452] The composition of embodiment 36, wherein the CRISPR-Cas
protein is a Type VI CRISPR-Cas protein.
Embodiment 42
[0453] The composition of embodiment 41, wherein the first RNA
binding protein comprises a Cas13 polypeptide or an RNA-binding
portion thereof.
Embodiment 43
[0454] The composition of any one of embodiments 36-42, wherein the
CRISPR-Cas protein comprises a native RNA nuclease activity.
Embodiment 44
[0455] The composition of embodiment 43, wherein the native RNA
nuclease activity is reduced or inhibited.
Embodiment 45
[0456] The composition of embodiment 43, wherein the native RNA
nuclease activity is increased or induced.
Embodiment 46
[0457] The composition of any one of embodiments 36-45, wherein the
CRISPR-Cas protein comprises a native DNA nuclease activity and
wherein the native DNA nuclease activity is inhibited.
Embodiment 47
[0458] The composition of embodiment 46, wherein the CRISPR-Cas
protein comprises a mutation.
Embodiment 48
[0459] The composition of embodiment 47, wherein a nuclease domain
of the CRISPR-Cas protein comprises the mutation.
Embodiment 49
[0460] The composition of embodiment 47, wherein the mutation
occurs in a nucleic acid encoding the CRISPR-Cas protein.
Embodiment 50
[0461] The composition of embodiment 47, wherein the mutation
occurs in an amino acid encoding the CRISPR-Cas protein.
Embodiment 51
[0462] The composition of any one of embodiments 47-50, wherein the
mutation comprises a substitution, an insertion, a deletion, a
frameshift, an inversion, or a transposition.
Embodiment 52
[0463] The composition of any one of embodiments 47-50, wherein the
mutation comprises a deletion of a nuclease domain, a binding site
within the nuclease domain, an active site within the nuclease
domain, or at least one essential amino acid residue within the
nuclease domain.
Embodiment 53
[0464] The composition of any one of embodiments 1-35, wherein the
first RNA binding protein comprises a Pumilio and FBF (PUF)
protein.
Embodiment 54
[0465] The composition of embodiment 53, wherein the first RNA
binding protein comprises a Pumilio-based assembly (PUMBY)
protein.
Embodiment 55
[0466] The composition of any one of embodiments 1-54, wherein the
first RNA binding protein does not require multimerization for
RNA-binding activity.
Embodiment 56
[0467] The composition of embodiment 55, wherein the first RNA
binding protein is not a monomer of a multimer complex Embodiment
57. The composition of embodiment 55, wherein a multimer protein
complex does not comprise the first RNA binding protein.
Embodiment 58
[0468] The composition of any one of embodiments 1-57, wherein the
first RNA binding protein selectively binds to a target sequence
within the RNA molecule.
Embodiment 59
[0469] The composition of embodiment 58, wherein the first RNA
binding protein does not comprise an affinity for a second sequence
within the RNA molecule.
Embodiment 60
[0470] The composition of embodiment 58 or 59, wherein the first
RNA binding protein does not comprise a high affinity for or
selectively bind a second sequence within the RNA molecule.
Embodiment 61
[0471] The composition of embodiment 60, wherein an RNA genome or
an RNA transcriptome comprises the RNA molecule.
Embodiment 62
[0472] The composition of any one of embodiments 1-61, wherein the
first RNA binding protein comprises between 2 and 1300 amino acids,
inclusive of the endpoints.
Embodiment 63
[0473] The composition of any one of embodiments 1-62, wherein the
sequence encoding the first RNA binding protein further comprises a
sequence encoding a nuclear localization signal (NLS).
Embodiment 64
[0474] The composition of embodiment 63, wherein the sequence
encoding a nuclear localization signal (NLS) is positioned 3' to
the sequence encoding the first RNA binding protein.
Embodiment 65
[0475] The composition of embodiment 64, wherein the first RNA
binding protein comprises an NLS at a C-terminus of the
protein.
Embodiment 66
[0476] The composition of any one of embodiments 1-62, wherein the
sequence encoding the first RNA binding protein further comprises a
first sequence encoding a first NLS and a second sequence encoding
a second NLS.
Embodiment 67
[0477] The composition of embodiment 66, wherein the sequence
encoding the first NLS or the second NLS is positioned 3' to the
sequence encoding the first RNA binding protein.
Embodiment 68
[0478] The composition of embodiment 67, wherein the first RNA
binding protein comprises the first NLS or the second NLS at a
C-terminus of the protein.
Embodiment 69
[0479] The composition of any one of embodiments 1-68, wherein the
second RNA binding protein comprises or consists of a nuclease
domain.
Embodiment 70
[0480] The composition of embodiment 69, wherein the sequence
encoding the second RNA binding protein comprises or consists of an
RNAse.
Embodiment 71
[0481] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse1.
Embodiment 72
[0482] The composition of embodiment 71, wherein the RNAse1 protein
comprises or consists of SEQ ID NO: 20.
Embodiment 73
[0483] The composition of embodiment 72, wherein the second RNA
binding protein comprises or consists of an RNAse4.
Embodiment 74
[0484] The composition of embodiment 73, wherein the RNAse4 protein
comprises or consists of: (SEQ ID NO: 21.
Embodiment 75
[0485] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse6.
Embodiment 76
[0486] The composition of embodiment 75, wherein the RNAse6 protein
comprises or consists of SEQ ID NO: 22.
Embodiment 77
[0487] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse7.
Embodiment 78
[0488] The composition of embodiment 77, wherein the RNAse7 protein
comprises or consists of SEQ ID NO: 23.
Embodiment 79
[0489] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse8.
Embodiment 80
[0490] The composition of embodiment 79, wherein the RNAse8 protein
comprises or consists of SEQ ID NO: 24.
Embodiment 81
[0491] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse2.
Embodiment 82
[0492] The composition of embodiment 81, wherein the RNAse2 protein
comprises or consists of SEQ ID NO: 25.
Embodiment 83
[0493] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse6PL.
Embodiment 84
[0494] The composition of embodiment 83, wherein the RNAse6PL
protein comprises or consists of SEQ ID NO: 26.
Embodiment 85
[0495] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAseL.
Embodiment 86
[0496] The composition of embodiment 85, wherein the RNAseL protein
comprises or consists of SEQ ID NO: 27.
Embodiment 87
[0497] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAseT2.
Embodiment 88
[0498] The composition of embodiment 87, wherein the RNAseT2
protein comprises or consists of SEQ ID NO: 28.
Embodiment 89
[0499] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAse11.
Embodiment 90
[0500] The composition of embodiment 89, wherein the RNAse11
comprises or consists of SEQ ID NO: 29.
Embodiment 91
[0501] The composition of embodiment 70, wherein the second RNA
binding protein comprises or consists of an RNAseT2-like.
Embodiment 92
[0502] The composition of embodiment 91, wherein the RNAseT2-like
protein comprises or consists of SEQ ID NO: 30.
Embodiment 93
[0503] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a NOB 1 polypeptide.
Embodiment 94
[0504] The composition of embodiment 93, wherein the NOB 1
polypeptide comprises or consists of SEQ ID NO: 31.
Embodiment 95
[0505] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an endonuclease.
Embodiment 96
[0506] The composition of embodiment 95, wherein the second RNA
binding protein comprises or consists of an endonuclease V
(ENDOV).
Embodiment 97
[0507] The composition of embodiment 96, wherein the ENDOV protein
comprises or consists of SEQ ID NO: 32.
Embodiment 98
[0508] The composition of embodiment 95, wherein the second RNA
binding protein comprises or consists of an endonuclease G
(ENDOG).
Embodiment 99
[0509] The composition of embodiment 98, wherein the ENDOG protein
comprises or consists of SEQ ID NO: 33.
Embodiment 100
[0510] The composition of embodiment 95, wherein the second RNA
binding protein comprises or consists of an endonuclease D1
(ENDOD1).
Embodiment 101
[0511] The composition of embodiment 100, wherein the ENDOD1
protein comprises or consists of SEQ ID NO: 34.
Embodiment 102
[0512] The composition of embodiment 95, wherein the second RNA
binding protein comprises or consists of a Human flap
endonuclease-1 (hFEN1).
Embodiment 103
[0513] The composition of embodiment 102, wherein the hFEN1 protein
comprises or consists of SEQ ID NO: 35.
Embodiment 104
[0514] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a human Schlafen 14
(hSLFN14) polypeptide.
Embodiment 105
[0515] The composition of embodiment 104, wherein the hSLFN14
polypeptide comprises or consists of SEQ ID NO: 36.
Embodiment 106
[0516] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a human
beta-lactamase-like protein 2 (hLACTB2) polypeptide.
Embodiment 107
[0517] The composition of embodiment 106, wherein the hLACTB2
polypeptide comprises or consists of SEQ ID NO: 37.
Embodiment 108
[0518] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an apurinic/apyrimidinic
(AP) endodeoxyribonuclease (APEX2) polypeptide.
Embodiment 109
[0519] The composition of embodiment 108, wherein the APEX2
polypeptide comprises or consists of SEQ ID NO: 38.
Embodiment 110
[0520] The composition of embodiment 108, wherein the APEX2
polypeptide comprises or consists of: SEQ ID NO: 39.
Embodiment 111
[0521] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an angiogenin (ANG)
polypeptide.
Embodiment 112
[0522] The composition of embodiment 111, wherein the ANG
polypeptide comprises or consists of SEQ ID NO: 40.
Embodiment 113
[0523] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a heat responsive protein
12 (HRSP12) polypeptide.
Embodiment 114
[0524] The composition of embodiment 113, wherein the HRSP12
polypeptide comprises or consists of SEQ ID NO: 41.
Embodiment 115
[0525] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Zinc Finger CCCH-Type
Containing 12A (ZC3H12A) polypeptide.
Embodiment 116
[0526] The composition of embodiment 115, wherein the ZC3H12A
polypeptide comprises or consists of SEQ ID NO: 42.
Embodiment 117
[0527] The composition of embodiment 115, wherein the ZC3H12A
polypeptide comprises or consists of SEQ ID NO: 43.
Embodiment 118
[0528] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Reactive Intermediate
Imine Deaminase A (RIDA) polypeptide.
Embodiment 119
[0529] The composition of embodiment 118, wherein the RIDA
polypeptide comprises or consists of SEQ ID NO: 44.
Embodiment 120
[0530] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Phospholipase D Family
Member 6 (PDL6) polypeptide.
Embodiment 121
[0531] The composition of embodiment 120, wherein the PDL6
polypeptide comprises or consists of: (SEQ ID NO: 126.
Embodiment 122
[0532] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Endonuclease III-like
protein 1 (NTHL) polypeptide.
Embodiment 123
[0533] The composition of embodiment 122, wherein the NTHL
polypeptide comprises or consists of SEQ ID NO: 123.
Embodiment 124
[0534] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Mitochondrial
ribonuclease P catalytic subunit (KIAA0391) polypeptide.
Embodiment 125
[0535] The composition of embodiment 124, wherein the KIAA0391
polypeptide comprises or consists of SEQ ID NO: 127.
Embodiment 126
[0536] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an apurinic or
apyrimidinic site lyase (APEX1) polypeptide.
Embodiment 127
[0537] The composition of embodiment 126, wherein the APEX1
polypeptide comprises or consists of SEQ ID NO: 125.
Embodiment 128
[0538] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an argonaute 2 (AGO2)
polypeptide.
Embodiment 129
[0539] The composition of embodiment 128, wherein the AGO2
polypeptide comprises or consists of SEQ ID NO: 128.
Embodiment 130
[0540] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mitochondrial nuclease
EXOG (EXOG) polypeptide.
Embodiment 131
[0541] The composition of embodiment 130, wherein the EXOG
polypeptide comprises or consists of SEQ ID NO: 129.
Embodiment 132
[0542] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Zinc Finger CCCH-Type
Containing 12D (ZC3H12D) polypeptide.
Embodiment 133
[0543] The composition of embodiment 132, wherein the ZC3H12D
polypeptide comprises or consists of SEQ ID NO: 130.
Embodiment 134
[0544] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an endoplasmic reticulum
to nucleus signaling 2 (ERN2) polypeptide.
Embodiment 135
[0545] The composition of embodiment 134, wherein the ERN2
polypeptide comprises or consists of SEQ ID NO: 131.
Embodiment 136
[0546] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a pelota mRNA surveillance
and ribosome rescue factor (PELO) polypeptide.
Embodiment 137
[0547] The composition of embodiment 136, wherein the PELO
polypeptide comprises or consists of SEQ ID NO: 132.
Embodiment 138
[0548] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a YBEY metallopeptidase
(YBEY) polypeptide.
Embodiment 139
[0549] The composition of embodiment 138, wherein the YBEY
polypeptide comprises or consists of SEQ ID NO: 133.
Embodiment 140
[0550] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a cleavage and
polyadenylation specific factor 4 like (CPSF4L) polypeptide.
Embodiment 141
[0551] The composition of embodiment 140, wherein the CPSF4L
comprises or consists of SEQ ID NO: 134.
Embodiment 142
[0552] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an hCG_200273
ipolypeptide.
Embodiment 143
[0553] The composition of embodiment 142, wherein the hCG_2002731
polypeptide comprises or consists of SEQ ID NO: 135.
Embodiment 144
[0554] The composition of embodiment 142, wherein the hCG_2002731
polypeptide comprises or consists of SEQ ID NO: 136.
Embodiment 145
[0555] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of an Excision Repair
Cross-Complementation Group 1 (ERCC1) polypeptide.
Embodiment 146
[0556] The composition of embodiment 145, wherein the ERCC1
polypeptide comprises or consists of SEQ ID NO: 137.
Embodiment 147
[0557] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a ras-related C3 botulinum
toxin substrate 1 isoform (RAC1) polypeptide.
Embodiment 148
[0558] The composition of embodiment 147, wherein the RAC1
polypeptide comprises or consists of SEQ ID NO: 138.
Embodiment 149
[0559] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Ribonuclease A A1 (RAA1)
polypeptide.
Embodiment 150
[0560] The composition of embodiment 149, wherein the RAA1
polypeptide comprises or consists of SEQ ID NO: 139.
Embodiment 151
[0561] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a Ras Related Protein
(RAB1) polypeptide.
Embodiment 152
[0562] The composition of embodiment 151, wherein the RAB1
polypeptide comprises or consists of SEQ ID NO: 140.
Embodiment 153
[0563] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a DNA Replication
Helicase/Nuclease 2 (DNA2) polypeptide.
Embodiment 154
[0564] The composition of embodiment 153, wherein the DNA2
polypeptide comprises or consists of SEQ ID NO: 141.
Embodiment 155
[0565] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a FLJ35220
polypeptide.
Embodiment 156
[0566] The composition of embodiment 155, wherein the FLJ35220
polypeptide comprises or consists of SEQ ID NO: 142.
Embodiment 157
[0567] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a FLJ13173
polypeptide.
Embodiment 158
[0568] The composition of embodiment 157, wherein the FLJ13173
polypeptide comprises or consists of: (SEQ ID NO: 143.
Embodiment 159
[0569] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a DNA repair endonuclease
XPF (ERCC4) polypeptide.
Embodiment 160
[0570] The composition of embodiment 159, wherein the ERCC4
polypeptide comprises or consists of SEQ ID NO: 64.
Embodiment 161
[0571] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(K41R)) polypeptide.
Embodiment 162
[0572] The composition of embodiment 161, wherein the Rnase1(K41R)
polypeptide comprises or consists of SEQ ID NO: 116.
Embodiment 163
[0573] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(K41R, D121E)) polypeptide.
Embodiment 164
[0574] The composition of embodiment 163, wherein the Rnase1
(Rnase1(K41R, D121E)) polypeptide comprises or consists of SEQ ID
NO: 117.
Embodiment 165
[0575] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(K41R, D121E, H119N)) polypeptide.
Embodiment 166
[0576] The composition of embodiment 165, wherein the Rnase1
(Rnase1(K41R, D121E, H119N)) polypeptide comprises or consists of
SEQ ID NO: 118.
Embodiment 167
[0577] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(H119N)) polypeptide.
Embodiment 168
[0578] The composition of embodiment 167, wherein the Rnase1
(Rnase1(H119N)) polypeptide comprises or consists of SEQ ID NO:
119.
Embodiment 169
[0579] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
Embodiment 170
[0580] The composition of embodiment 169, wherein the Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide comprises
or consists of SEQ ID NO: 120.
Embodiment 171
[0581] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
Embodiment 172
[0582] The composition of embodiment 171, wherein the Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E))
polypeptide comprises or consists of SEQ ID NO: 121.
Embodiment 173
[0583] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of a mutated Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
Embodiment 174
[0584] The composition of embodiment 173, wherein the Rnase1
(Rnase1(R39D, N67D, N88A, G89D, R91D)) polypeptide comprises or
consists of SEQ ID NO: 122.
Embodiment 175
[0585] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of Teneurin Transmembrane
Protein 1 (TENM1) polypeptide.
Embodiment 176
[0586] The composition of embodiment 175, wherein the TENM1
polypeptide comprises or consists of SEQ ID NO: 144.
Embodiment 177
[0587] The composition of embodiment 69, wherein the second RNA
binding protein comprises or consists of Teneurin Transmembrane
Protein 2 (TENM2) polypeptide.
Embodiment 178
[0588] The composition of embodiment 177, wherein the TENM2
polypeptide comprises or consists of SEQ ID NO: 145.
Embodiment 179
[0589] A composition comprising a sequence encoding a target
RNA-binding fusion protein comprising (a) a sequence encoding a
first RNA-binding polypeptide or portion thereof; and (b) a
sequence encoding a second RNA-binding polypeptide, wherein the
first RNA-biding polypeptide binds a target RNA not guided by a
gRNA sequence, and wherein the second RNA-binding polypeptide
comprises RNA-nuclease activity.
Embodiment 180
[0590] The composition of embodiment 179, wherein the first
RNA-binding polypeptide or portion thereof is a PUF, PUMBY, or PPR
polypeptide or portion thereof.
Embodiment 181
[0591] A method for modifying the level of expression of an RNA
molecule or a protein encoded by the RNA molecule, the method
comprising contacting the composition of embodiments 1 or 179 and
the RNA molecule under conditions suitable for binding of the
fusion protein or a portion thereof to the RNA molecule.
EXAMPLES
Example 1: Methods
[0592] HEK-293 cells were cultured in DMEM with 10% FBS and 1%
penicillin/streptomycin (GIBCO) and passaged at 90%-100%
confluency. Cells were seeded at 1.times.10{circumflex over ( )}5
cells per well of a 24-well plate for RNA isolation or
0.5.times.10{circumflex over ( )}5 cells per well of a 96-well
plate for luciferase assays. RNA isolations were carried out with
RNAeasy columns (Qiagen) according to the manufacturer's protocol.
RNA quality and concentrations were estimated using the Nanodrop
spectrophotometer. cDNA preparation was done using Superscript III
(Thermo) with random primers according to the manufacturer's
protocol. qPCR was carried out with primers in a sequence adjacent
to the CTG repeat in the reporter plasmid using the following
primers:
TABLE-US-00146 Forward Primer TetCTG_DMPK_E15_F SEQ ID NO: 83
TCGGAGCGGTTGTGAACT Reverse Primer TetCTG_DMPK_E15_R SEQ ID NO: 84
GTTCGCCGTTGTTCTGTC
[0593] Relative abundance of the CTG repeat reporter was determined
by normalization to GAPDH. Next, levels of the CTG-targeting sgRNA
were normalized to a non-targeting sgRNA to generate a final value
reported in the associated data package.
TABLE-US-00147 CTG-targeting spacer SEQ ID NO: 85
AGCAGCAGCAGCAGCAGCAG Non-targeting control spacer (.lamda.2) SEQ ID
NO: 86 GTGATAAGTGGAATGCCATG sgRNA scaffold (N's indicate spacer)
SEQ ID NO: 87 GNNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAUGCUGGAAACAGCAUAG
CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG
UCGGUGCUUUUUUU
[0594] Luciferase assays were conducted with the Promega Dual
Luciferase kit according to manufacturer's directions. Reported
values are a ratio of firefly and renilla luciferase luminescence
readings.
Example 2: RNA-Guided Cleavage of Repetitive RNA Molecules and mRNA
Molecules
[0595] Experimental Design: Various fusions of human proteins with
annotated RNA endonuclease activity and Cas9 (Streptococcus
pyogenes or Campylobacter jejuni) were constructed. Plasmids
encoding the above fusions were co-transfected with either a
repeat-containing plasmid or a luciferase assay plasmid (comprising
an mRNA sequence encoding a luciferase protein). A level of CTG
repeat-containing RNA was measured with qPCR in the condition in
which an RNA endonuclease/Cas9 fusion was co-transfected with a
repetitive RNA. A level of luciferase protein was measured using a
luminescence assay in the condition in which an RNA
endonuclease/Cas9 fusion was co-transfected with a luciferase assay
plasmid. All measurements were normalized to a non-targeting sgRNA
control construct (FIGS. 3A-5 and FIG. 9).
Example 3: RNA-Guided Cleavage of Viral RNA Molecules
[0596] A549 cells were cultured in DMEM with 10% FBS and 1%
penicillin/streptomycin (GIBCO) and passaged at 90%-100%
confluency. Cells were seeded at 1.times.10{circumflex over ( )}5
cells per well of a 24-well plate for RNA isolation or
0.5.times.10{circumflex over ( )}5 cells per well. Cells were
transfected with plasmids encoding Campylobacter jejuni Cas9
(CjeCas9) fused to the gene NTHL 1 (residues 31-312, E43) or CPSF4L
(full length, E67) with plasmids encoding one of four sites in Zika
NS5 RNA. CjeCas9 was driven by an EFS promoter while the guide RNAs
were driven by U6 promoter. The sequences of the sgRNAs are
presented in Table 1. The sequences of the constructs used in this
study are presented below.
[0597] RNA isolations were carried out with RNAeasy columns
(Qiagen) according to the manufacturer's protocol. RNA quality and
concentrations were estimated using the Nanodrop spectrophotometer.
cDNA preparation was done using Superscript III (Thermo) with
random primers according to the manufacturer's protocol. qPCR was
carried out with the following primers as listed in Table 2.
[0598] FIG. 7 shows expression levels of Zika NS5 assessed in the
presence of both E43 and E67 endonucleases with sgRNAs containing
the various NS5-targeting spacer sequences as indicated in Table 2.
Zika NS5 expression is displayed as fold change relative to the
endonuclease loaded with an sgRNA containing a control (Lambda)
spacer sequence.
[0599] Immunofluorescence microscopy was used to visualize Zika NS5
expression in the presence of E43 or E67 endonucleases fused to
CjeCas9. FIG. 8A shows a fluorescence microscopy image of cells
transfected with CjeCas9-endonuclease fusions loaded with an sgRNA
containing a Zika NS5-targeting spacer sequence. Expression of Zika
NS5 is markedly decreased in the presence of CjeCas9-endonuclease
fusions loaded with the appropriate Zika NS5-targeting sgRNA as
compared to CjeCas9-endonuclease fusions loaded with a non-Zika NS5
targeting sgRNA (FIGS. 8A and 8B). FIG. 6 is a list of exemplary
endonucleases for use in the compositions of the disclosure.
TABLE-US-00148 TABLE 1 qPCR primers GAPDH_F CAGCCTCAAGATCATCAGCAA
(SEQ ID NO: 192) GAPDH_R TGTGGTCATGAGTCCTTCCA (SEQ ID NO: 193)
NS5_F GAGGAGAGTGCCAGAGTTGT (SEQ ID NO: 194) NS5_R
TCTCTCTCCCCATCCAGTGA (SEQ ID NO: 195)
TABLE-US-00149 TABLE 2 sgRNA sequences NS5-targeting spacer 1
gcaatgatcttcatgttgggagc (SEQ ID NO: 196) NS5-targeting spacer 2
gaaccttgttgatgaactcttc (SEQ ID NO: 197) NS5-targeting spacer 3
gttggtgattagagcttcattc (SEQ ID NO: 198) NS5-targeting spacer 4
gagtgatcctcgttcaagaatcc (SEQ ID NO: 199) Non-targeting control
GTGATAAGTGGAATGCCATG (SEQ ID NO: 200) spacer (.lamda.2) sgRNA
scaffold (N's GNNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAUG indicate spacer)
CUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU (SEQ ID NO: 201)
TABLE-US-00150 (SEQ ID NO: 202)
gtttattacagggacagcagagatccagtttggttaattaaggtaccgag
ggcctatttcccatgattccttcatatttgcatatacgatacaaggctgt
tagagagataattagaattaatttgactgtaaacacaaagatattagtac
aaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagta
tttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCNNNN
NNNNNNNNNNNNNNNGTTTTAGTCCCTGAAGGGACTAAAATAAAGAGTTT
GCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTTTTCCTGCAG
CCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCA
GCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGAATTCGCTAGCTAGGT
CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCA
CATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCC
GGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA
CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAG
TAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
GACCGGTTCTAGAGCGCTATTTAGAACCatgTGTTCTCCCCAAGAATCTG
GCATGACCGCTCTTTCAGCGAGGATGTTGACGCGAAGCAGATCCCTGGGA
CCTGGGGCCGGGCCACGAGGGTGTCGGGAAGAACCAGGACCGTTGCGACG
GAGGGAAGCAGCAGCGGAAGCTCGGAAATCCCATTCTCCGGTTAAACGAC
CCCGCAAGGCACAACGGCTCAGGGTTGCTTACGAGGGGAGCGATTCCGAA
AAGGGTGAAGGAGCAGAGCCCTTGAAGGTTCCAGTATGGGAACCCCAGGA
TTGGCAGCAGCAGCTTGTAAACATCCGAGCAATGAGGAACAAAAAAGATG
CACCTGTTGATCACCTCGGAACCGAACATTGTTATGATTCTAGTGCGCCG
CCAAAAGTCCGCCGGTATCAGGTTCTGTTGAGTTTGATGCTGAGTAGTCA
GACTAAGGACCAGGTTACGGCCGGAGCAATGCAACGGCTTCGGGCACGGG
GACTCACGGTCGATAGCATTTTGCAGACCGATGACGCAACATTGGGTAAA
CTCATATATCCAGTTGGCTTCTGGCGGAGCAAAGTGAAGTACATCAAGCA
GACCTCAGCCATTCTCCAACAACATTACGGAGGTGATATACCCGCAAGCG
TAGCTGAACTGGTAGCACTGCCGGGCGTCGGTCCCAAAATGGCACATCTG
GCTATGGCGGTTGCTTGGGGAACGGTGTCTGGTATCGCAGTTGATACGCA
TGTCCACCGCATCGCCAATCGGCTGAGGTGGACTAAAAAAGCCACTAAGT
CTCCTGAAGAAACACGGGCTGCTCTGGAAGAGTGGCTTCCACGAGAGCTG
TGGCATGAAATCAATGGATTGCTGGTTGGTTTCGGGCAGCAGACATGCTT
GCCCGTGCACCCCCGGTGTCATGCTTGCTTGAACCAGGCTTTGTGCCCAG
CTGCCCAGGGCCTGAGTGGAAGTGAGACACCGGGAACATCTGAGTCTGCG
ACCCCGGAGAGCacaaacGCGCGAATCCTGGCCTTCGcgATTGGCATTAG
CAGCATCGGCTGGGCATTCTCTGAAAACGACGAACTGAAGGATTGCGGCG
TGCGAATTTTCACTAAGGTCGAAAATCCCAAAACTGGTGAATCACTCGCT
CTCCCTAGACGACTGGCACGCTCCGCACGAAAGAGGCTTGCCCGCCGCAA
GGCACGCTTGAACCATCTTAAACACCTTATTGCAAATGAGTTTAAACTGA
ATTATGAGGACTACCAATCCTTTGACGAGTCTCTTGCTAAAGCCTACAAA
GGGAGCCTTATATCCCCGTATGAGCTCCGGTTCAGAGCACTCAACGAACT
GCTGTCCAAACAGGATTTTGCTCGCGTGATTCTCCACATAGCGAAGAGGC
GAGGATACGATGACATTAAAAACAGTGATGATAAGGAAAAAGGGGCCATA
CTCAAAGCGATTAAGCAAAATGAAGAGAAGCTCGCTAACTATCAATCAGT
AGGGGAGTATCTCTATAAAGAGTACTTCCAGAAGTTCAAAGAAAATAGCA
AGGAATTTACTAATGTCCGGAATAAAAAGGAGTCTTACGAAAGATGTATT
GCGCAATCTTTCCTCAAGGACGAGCTCAAATTGATTTTCAAGAAACAAAG
GGAATTTGGGTTCAGCTTCTCAAAAAAATTTGAGGAAGAGGTTCTGAGCG
TTGCCTTTTACAAACGCGCCCTTAAGGACTTCTCACATCTCGTAGGGAAT
TGTAGTTTCTTCACCGATGAAAAACGGGCGCCAAAAAATAGCCCTTTGGC
TTTTATGTTTGTCGCTCTGACTCGCATCATTAATCTGCTCAACAACCTTA
AAAACACGGAAGGGATTCTGTACACAAAGGATGATCTGAACGCTCTGCTT
AACGAAGTTTTGAAGAACGGGACTTTGACCTACAAACAAACCAAAAAGCT
TCTTGGTCTCAGTGATGACTACGAATTCAAGGGAGAAAAAGGGACATATT
TCATCGAATTCAAGAAGTATAAGGAGTTCATCAAAGCCTTGGGCGAGCAC
AACTTGTCTCAAGATGATCTCAACGAAATTGCTAAGGATATCACTCTGAT
TAAAGACGAGATCAAGCTCAAAAAGGCGTTGGCGAAGTATGACCTTAACC
AAAACCAAATAGATAGCCTCAGCAAGTTGGAATTTAAAGATCACTTGAAT
ATAAGTTTCAAGGCCCTTAAGTTGGTCACCCCCTTGATGCTTGAAGGAAA
GAAATATGATGAGGCATGTAATGAGCTGAATCTCAAGGTTGCTATTAACG
AAGACAAAAAAGATTTCCTCCCAGCTTTCAATGAGACTTACTATAAGGAC
GAGGTTACCAATCCTGTGGTGCTCCGAGCCATCAAAGAGTATCGAAAGGT
CCTGAATGCTTTGCTCAAAAAATACGGTAAGGTACACAAAATAAATATTG
AGCTCGCAAGGGAGGTCGGTAAGAACCACTCCCAGCGCGCCAAAATAGAA
AAGGAACAGAATGAAAATTACAAAGCGAAAAAGGACGCCGAGCTCGAGTG
CGAAAAGCTGGGCCTGAAAATAAACAGCAAGAACATTCTCAAACTCCGCC
TCTTCAAAGAACAAAAAGAATTTTGTGCTTATAGTGGTGAGAAAATAAAA
ATCTCCGATCTTCAAGACGAGAAGATGCTCGAAATAGACgcgATATATCC
ATATAGCAGGTCTTTTGACGATTCTTACATGAATAAAGTGCTTGTTTTCA
CTAAGCAGAATCAGGAAAAGTTGAATCAGACCCCCTTTGAGGCCTTTGGC
AACGACTCAGCAAAGTGGCAGAAGATCGAGGTCTTGGCTAAGAATCTTCC
TACTAAGAAACAGAAAAGGATATTGGATAAGAACTATAAAGACAAAGAAC
AAAAGAACTTTAAAGACCGCAACCTCAATGACACCAGATACATAGCAAGA
TTGGTTCTGAACTACACAAAAGATTATTTGGACTTCTTGCCGCTGTCTGA
TGATGAGAACACGAAACTCAACGACACGCAAAAGGGGTCTAAAGTCCACG
TCGAAGCTAAATCTGGGATGCTCACCTCAGCATTGAGGCATACGTGGGGA
TTCTCAGCAAAGGACCGAAACAATCACCTGCACCATGCCATTGACGCAGT
TATCATAGCGTATGCCAATAATTCAATAGTAAAAGCGTTTAGCGACTTCA
AGAAGGAACAAGAGTCCAACAGCGCCGAGCTCTACGCAAAAAAGATTAGT
GAACTCGACTACAAAAACAAAAGAAAATTCTTTGAGCCGTTCAGCGGATT
TCGACAGAAGGTATTGGATAAAATAGATGAAATTTTCGTGAGCAAACCCG
AAAGGAAAAAGCCCTCAGGCGCCTTGCACGAAGAGACTTTCAGGAAGGAA
GAGGAATTCTACCAAAGCTACGGCGGAAAAGAGGGAGTTTTGAAGGCTCT
CGAACTTGGAAAGATTAGGAAGGTGAACGGCAAGATAGTGAAAAACGGCG
ATATGTTCCGGGTTGATATCTTCAAACATAAAAAAACGAATAAATTTTAT
GCTGTGCCTATATACACTATGGACTTCGCACTTAAGGTCCTGCCGAATAA
GGCGGTAGCCCGATCTAAAAAAGGCGAAATTAAGGACTGGATTTTGATGG
ATGAAAATTACGAGTTCTGCTTTTCTCTCTACAAGGATTCCCTTATATTG
ATACAGACGAAAGATATGCAGGAACCGGAATTCGTGTATTACAACGCTTT
TACTTCCTCTACGGTATCTTTGATTGTCTCCAAACATGACAACAAATTCG
AAACACTCAGTAAAAACCAAAAGATTCTCTTTAAAAATGCGAACGAGAAA
GAAGTAATTGCAAAATCAATTGGCATCCAAAATTTGAAAGTTTTTGAAAA
ATATATAGTATCTGCCCTCGGAGAGGTTACTAAAGCGGAATTTAGACAGC
GAGAGGACTTCAAAAAATCAGGTCCACCCAAGAAAAAACGCAAGGTGGAA
GATCCGAAGAAAAAGCGAAAAGTGGATGTGtaaCGTTTTCCGGGACGCCG
GCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCAC
CCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT
CACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT
TGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCG.
[0600] A E67-CjeCas9 and sgRNA plasmid may comprise or consist of
the sequence (U6: N's=sgRNA spacer, E67, CieCas9):
TABLE-US-00151 (SEQ ID NO: 203)
gtttattacagggacagcagagatccagtttggttaattaaggtaccgag
ggcctatttcccatgattccttcatatttgcatatacgatacaaggctgt
tagagagataattagaattaatttgactgtaaacacaaagatattagtac
aaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta
aaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagta
tttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCNNNN
NNNNNNNNNNNNNNNGTTTTAGTCCCTGAAGGGACTAAAATAAAGAGTTT
GCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTTTTCCTGCAG
CCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCA
GCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGAATTCGCTAGCTAGGT
CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCA
CATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCC
GGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA
CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAG
TAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG
GACCGGTTCTAGAGCGCTATTTAGAACCatgCAGGAGGTAATAGCGGGGC
TTGAGCGATTTACCTTTGCCTTCGAAAAAGACGTAGAGATGCAGAAGGGA
ACCGGCCTGCTCCCATTTCAAGGTATGGACAAATCAGCATCTGCCGTGTG
CAATTTTTTCACCAAGGGTCTGTGTGAAAAGGGGAAGCTCTGTCCATTTC
GCCATGATCGCGGAGAGAAGATGGTGGTGTGTAAGCACTGGCTGAGAGGG
CTTTGCAAAAAAGGCGACCACTGCAAATTTCTTCACCAATATGACCTGAC
TCGAATGCCTGAGTGTTATTTTTACAGTAAGTTCGGTGACTGTAGCAACA
AAGAATGCAGCTTCTTGCATGTCAAACCAGCATTCAAGTCACAGGATTGC
CCGTGGTACGATCAGGGTTTTTGCAAGGACGGTCCCCTCTGCAAATATCG
ACACGTACCCAGAATTATGTGCCTTAATTACCTGGTCGGCTTCTGTCCTG
AAGGGCCAAAATGTCAGTTTGCTCAAAAAATTCGCGAGTTCAAATTGCTC
CCTGGGTCTAAAATTTGGGAACCCCAGGATTGGCAGCAGCAGCTTGTAAA
CATCCGAGCAATGAGGAACAAAAAAGATGCACCTGTTGATCACCTCGGAA
CCGAACATTGTTATGATTCTAGTGCGCCGCCAAAAGTCCGCCGGTATCAG
GTTCTGTTGAGTTTGATGCTGAGTAGTCAGACTAAGGACCAGGTTACGGC
CGGAGCAATGCAACGGCTTCGGGCACGGGGACTCACGGTCGATAGCATTT
TGCAGACCGATGACGCAACATTGGGTAAACTCATATATCCAGTTGGCTTC
TGGCGGAGCAAAGTGAAGTACATCAAGCAGACCTCAGCCATTCTCCAACA
ACATTACGGAGGTGATATACCCGCAAGCGTAGCTGAACTGGTAGCACTGC
CGGGCGTCGGTCCCAAAATGGCACATCTGGCTATGGCGGTTGCTTGGGGA
ACGGTGTCTGGTATCGCAGTTGATACGCATGTCCACCGCATCGCCAATCG
GCTGAGGTGGACTAAAAAAGCCACTAAGTCTCCTGAAGAAACACGGGCTG
CTCTGGAAGAGTGGCTTCCACGAGAGCTGTGGCATGAAATCAATGGATTG
CTGGTTGGTTTCGGGCAGCAGACATGCTTGCCCGTGCACCCCCGGTGTCA
TGCTTGCTTGAACCAGGCTTTGTGCCCAGCTGCCCAGGGCCTGAGTGGAA
GTGAGACACCGGGAACATCTGAGTCTGCGACCCCGGAGAGCacaaacGCG
CGAATCCTGGCCTTCGcgATTGGCATTAGCAGCATCGGCTGGGCATTCTC
TGAAAACGACGAACTGAAGGATTGCGGCGTGCGAATTTTCACTAAGGTCG
AAAATCCCAAAACTGGTGAATCACTCGCTCTCCCTAGACGACTGGCACGC
TCCGCACGAAAGAGGCTTGCCCGCCGCAAGGCACGCTTGAACCATCTTAA
ACACCTTATTGCAAATGAGTTTAAACTGAATTATGAGGACTACCAATCCT
TTGACGAGTCTCTTGCTAAAGCCTACAAAGGGAGCCTTATATCCCCGTAT
GAGCTCCGGTTCAGAGCACTCAACGAACTGCTGTCCAAACAGGATTTTGC
TCGCGTGATTCTCCACATAGCGAAGAGGCGAGGATACGATGACATTAAAA
ACAGTGATGATAAGGAAAAAGGGGCCATACTCAAAGCGATTAAGCAAAAT
GAAGAGAAGCTCGCTAACTATCAATCAGTAGGGGAGTATCTCTATAAAGA
GTACTTCCAGAAGTTCAAAGAAAATAGCAAGGAATTTACTAATGTCCGGA
ATAAAAAGGAGTCTTACGAAAGATGTATTGCGCAATCTTTCCTCAAGGAC
GAGCTCAAATTGATTTTCAAGAAACAAAGGGAATTTGGGTTCAGCTTCTC
AAAAAAATTTGAGGAAGAGGTTCTGAGCGTTGCCTTTTACAAACGCGCCC
TTAAGGACTTCTCACATCTCGTAGGGAATTGTAGTTTCTTCACCGATGAA
AAACGGGCGCCAAAAAATAGCCCTTTGGCTTTTATGTTTGTCGCTCTGAC
TCGCATCATTAATCTGCTCAACAACCTTAAAAACACGGAAGGGATTCTGT
ACACAAAGGATGATCTGAACGCTCTGCTTAACGAAGTTTTGAAGAACGGG
ACTTTGACCTACAAACAAACCAAAAAGCTTCTTGGTCTCAGTGATGACTA
CGAATTCAAGGGAGAAAAAGGGACATATTTCATCGAATTCAAGAAGTATA
AGGAGTTCATCAAAGCCTTGGGCGAGCACAACTTGTCTCAAGATGATCTC
AACGAAATTGCTAAGGATATCACTCTGATTAAAGACGAGATCAAGCTCAA
AAAGGCGTTGGCGAAGTATGACCTTAACCAAAACCAAATAGATAGCCTCA
GCAAGTTGGAATTTAAAGATCACTTGAATATAAGTTTCAAGGCCCTTAAG
TTGGTCACCCCCTTGATGCTTGAAGGAAAGAAATATGATGAGGCATGTAA
TGAGCTGAATCTCAAGGTTGCTATTAACGAAGACAAAAAAGATTTCCTCC
CAGCTTTCAATGAGACTTACTATAAGGACGAGGTTACCAATCCTGTGGTG
CTCCGAGCCATCAAAGAGTATCGAAAGGTCCTGAATGCTTTGCTCAAAAA
ATACGGTAAGGTACACAAAATAAATATTGAGCTCGCAAGGGAGGTCGGTA
AGAACCACTCCCAGCGCGCCAAAATAGAAAAGGAACAGAATGAAAATTAC
AAAGCGAAAAAGGACGCCGAGCTCGAGTGCGAAAAGCTGGGCCTGAAAAT
AAACAGCAAGAACATTCTCAAACTCCGCCTCTTCAAAGAACAAAAAGAAT
TTTGTGCTTATAGTGGTGAGAAAATAAAAATCTCCGATCTTCAAGACGAG
AAGATGCTCGAAATAGACgcgATATATCCATATAGCAGGTCTTTTGACGA
TTCTTACATGAATAAAGTGCTTGTTTTCACTAAGCAGAATCAGGAAAAGT
TGAATCAGACCCCCTTTGAGGCCTTTGGCAACGACTCAGCAAAGTGGCAG
AAGATCGAGGTCTTGGCTAAGAATCTTCCTACTAAGAAACAGAAAAGGAT
ATTGGATAAGAACTATAAAGACAAAGAACAAAAGAACTTTAAAGACCGCA
ACCTCAATGACACCAGATACATAGCAAGATTGGTTCTGAACTACACAAAA
GATTATTTGGACTTCTTGCCGCTGTCTGATGATGAGAACACGAAACTCAA
CGACACGCAAAAGGGGTCTAAAGTCCACGTCGAAGCTAAATCTGGGATGC
TCACCTCAGCATTGAGGCATACGTGGGGATTCTCAGCAAAGGACCGAAAC
AATCACCTGCACCATGCCATTGACGCAGTTATCATAGCGTATGCCAATAA
TTCAATAGTAAAAGCGTTTAGCGACTTCAAGAAGGAACAAGAGTCCAACA
GCGCCGAGCTCTACGCAAAAAAGATTAGTGAACTCGACTACAAAAACAAA
AGAAAATTCTTTGAGCCGTTCAGCGGATTTCGACAGAAGGTATTGGATAA
AATAGATGAAATTTTCGTGAGCAAACCCGAAAGGAAAAAGCCCTCAGGCG
CCTTGCACGAAGAGACTTTCAGGAAGGAAGAGGAATTCTACCAAAGCTAC
GGCGGAAAAGAGGGAGTTTTGAAGGCTCTCGAACTTGGAAAGATTAGGAA
GGTGAACGGCAAGATAGTGAAAAACGGCGATATGTTCCGGGTTGATATCT
TCAAACATAAAAAAACGAATAAATTTTATGCTGTGCCTATATACACTATG
GACTTCGCACTTAAGGTCCTGCCGAATAAGGCGGTAGCCCGATCTAAAAA
AGGCGAAATTAAGGACTGGATTTTGATGGATGAAAATTACGAGTTCTGCT
TTTCTCTCTACAAGGATTCCCTTATATTGATACAGACGAAAGATATGCAG
GAACCGGAATTCGTGTATTACAACGCTTTTACTTCCTCTACGGTATCTTT
GATTGTCTCCAAACATGACAACAAATTCGAAACACTCAGTAAAAACCAAA
AGATTCTCTTTAAAAATGCGAACGAGAAAGAAGTAATTGCAAAATCAATT
GGCATCCAAAATTTGAAAGTTTTTGAAAAATATATAGTATCTGCCCTCGG
AGAGGTTACTAAAGCGGAATTTAGACAGCGAGAGGACTTCAAAAAATCAG
GTCCACCCAAGAAAAAACGCAAGGTGGAAGATCCGAAGAAAAAGCGAAAA
GTGGATGTGtaaCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCG
GGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCT
TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGC
ATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTAT
CTTATCATGTCTGTATACCG.
INCORPORATION BY REFERENCE
[0601] Every document cited herein, including any cross referenced
or related patent or application is hereby incorporated herein by
reference in its entirety unless expressly excluded or otherwise
limited. The citation of any document is not an admission that it
is prior art with respect to any invention disclosed or
embodimented herein or that it alone, or in any combination with
any other reference or references, teaches, suggests or discloses
any such invention. Further, to the extent that any meaning or
definition of a term in this document conflicts with any meaning or
definition of the same term in a document incorporated by
reference, the meaning or definition assigned to that term in this
document shall govern.
Other Embodiments
[0602] While particular embodiments of the disclosure have been
illustrated and described, various other changes and modifications
can be made without departing from the spirit and scope of the
disclosure. The scope of the appended claims includes all such
changes and modifications that are within the scope of this
disclosure.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200071718A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200071718A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References