RNA-TARGETING FUSION PROTEIN COMPOSITIONS AND METHODS FOR USE

Description

RELATED APPLICATIONS

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

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

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

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

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.

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.

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.

In some embodiments of the compositions of the disclosure, the target sequence comprises at least one repeated sequence.

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.

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.

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.

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

(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.

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

(SEQ ID NO: 14)

	GUGAUAAGUGGAAUGCCAUG,

(SEQ ID NO: 15)

	CUGGUGAACUUCCGAUAGUG,
	or

(SEQ ID NO: 16)

GAGATATAGCCTGGTGGTTC.

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.

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

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 13). In some embodiments, the scaffold sequence comprises or consists of the sequence

GGACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG

CACCGAGUCGGUGCUUUUU (SEQ ID NO: 17). In some embodiments, the scaffold sequence comprises or consists of the sequence

(SEQ ID NO: 82)

GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU

CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU

or

(SEQ ID NO: 83)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA

CUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

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.

In some embodiments of the compositions of the disclosure, an RNA genome or an RNA transcriptome comprises the RNA molecule.

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.

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.

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.

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.

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.

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.

In some embodiments of the compositions of the disclosure, an RNA genome or an RNA transcriptome comprises the RNA molecule.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

FIG. 2 is a schematic diagram depicting an exemplary modular therapeutic platform for treating genetic disease by targeting RNA molecules.

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.

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)

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.

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)

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.

FIG. 6 is a table providing a key to the endonucleases shown in FIGS. 4B, 5B, and 9.

FIG. 7A is a schematic diagram depicting an exemplary RNA Endonuclease-C. jejuni Cas9 fusion protein.

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.

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.

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.

FIG. 9 is a graph depicting the cleavage efficiencies of a variety of exemplary fusion proteins (SpyCas9 fused to the annotated endonuclease).

DETAILED DESCRIPTION

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

The terms guide RNA (gRNA) and single guide RNA (sgRNA) are used interchangeably throughout the disclosure.

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.

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 (Ψ), 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.

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).

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).

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.

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).

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.

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.

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.

In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof does not comprise a nuclear localization sequence (NLS).

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).

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′.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

Exemplary wild type S. pyogenes Cas9 proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

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):

(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.

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 ββα-metal fold or a portion thereof comprising a DNAse active site or any combination thereof.

Other exemplary Cas9 proteins or portions thereof may comprise or consist of the following amino acid sequences.

In some embodiments the Cas9 protein can be S. pyogenes Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be S. aureus Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be S. thermophiles CRISPR1 Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be N. meningitidis Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Parvibaculum. lavamentivorans Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Corynebacter diphtheria Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Streptococcus pasteurianus Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Neisseria cinerea Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Campylobacter lari Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be T7 denticola Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be S. mutans Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be S. thermophilus CRISPR 3 Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be C. jejuni Cas9 and may comprise or consist of the amino acid sequence:

(SEQ ID NO: 161)

MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRL

ARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLIS

PYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIK

QNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFL

KDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFT

DEKRAPKNSPLAFWVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKN

GTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDD

LNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKAL

KLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPV

VLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNEN

YKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQD

EKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKW

QKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYT

KDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDR

NNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKN

KRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQS

YGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYT

MDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDM

QEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKS

IGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK

In some embodiments the Cas9 protein can be P. multocida Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be F. novicida Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Lactobacillus buchneri Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be Listeria innocua Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be L. pneumophilia Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be N. lactamica Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be N. meningitides Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be B. longum Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be A. muciniphila Cas9 and may comprise or consist of the amino acid sequence:

(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

In some embodiments the Cas9 protein can be O. laneus Cas9 and may comprise or consist of the amino acid sequence:

(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.

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.

Exemplary wild type Francisella tularensis subsp. Novicida Cpf1 (FnCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

Exemplary wild type Lachnospiraceae bacterium sp. ND2006 Cpf1 (LbCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

Exemplary wild type Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

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.

Exemplary Cas13a proteins include, but are not limited to:

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)

Exemplary wild type Cas13a proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

Exemplary Cas13b proteins include, but are not limited to:

		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

Exemplary wild type Bergeyella zoohelcum ATCC 43767 Cas13b (BzCas13b) proteins of the disclosure may comprise or consist of the amino acid sequence:

(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.

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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig6049000251:

(SEQ ID NO: 54)

LYLTSFGKGN AAVIEQKIEP ENGYRVTGMQ ITPSITVNKA TDESVRFRVK RKIAQKDEFI	60
ADNPMHEGRH RIEPSAGSDM LGLKTKLEKY YFGKEFDDNL HIQIIYNILD IEKILAVYST	120
NITA.	124

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig546000275:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig4114000374:

(SEQ ID NO: 61)

DTKINPQTWL YQLENTPDLD NEYRDTLDHF FDERFNEINE HFVTQNATNL CIMKEVFPDE	60
DFKSIADLYY DFIVVKSYKN IGFSIKKLRE KMLELPEAKR VTSTEMDSVR SKLYKLIDFC	120
IFKHYHEKPE TVEMIVSMLR AYTSEDMKE.	149

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig721000619:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig2002000411:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contigl3552000311:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig10037000527:

(SEQ ID NO: 72)

YMDQNFANSD AWAIHVYRNK IQHLDAVRHA DMYIGDIREF HSWFELYHYI IQRRIIDQYA	60
YESTPGSSRD GSAIIDEERL NPATRRYFRL ITTYKT.	96

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig238000329:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut meta enome conti 2643000492:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig874000057:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig4781000489:

(SEQ ID NO: 86)

LDKQLDYEYI RTLNYMFNDI ADELTRTFSK NSAANVNYIA ETLNIDPNKF AEQYFRFSIM	60
KEQKNLGFNL TKLRESMLDR RELSDIRDNH NVFDSIRPKL YTMMDFVIYK HYIDEAKKTE	120
AENKSLPDDR KNLSEKD.	137

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig12144000352:

(SEQ ID NO: 87)

RMGEPVANTK RVMMIDAVKI LGTDLSDDEL KEMADSFFKD SDGNLLKKGK HGMRNFITNN	60
VIKNKRFHYL IRYGDPAHLH EIAKNEA.	87

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig5590000448:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig525000349:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig7229000302:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig3227000343:

(SEQ ID NO: 91)

IGDISEVNSY FQLYHYIMQR ILIDKIGSKT TGKAKEYFDS VIVNKKYDDR LLKLLCSPLG	60
YCLTRYKDLS IEALFDMNEA AKYDKLNKER KNKKK.	95

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Gut_metagenome_contig7030000469:

(SEQ ID NO: 92)

SIRSKLYTMM DFVIYRYYIE ESAKAAAENK PSESDSFVIR LRGSFNENQK EELYIEEAER	60
LWKKFGEIML KIKEFRGEKV KEYKKEVPRI ERILPHGKDI SAFSKLMYML SMFLD.	115

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d gut_metagenome_P17E0k2120140920, c87000043:

(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

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):

(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

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):

(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

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:

(SEQ ID NO: 96)

caactacaac cccgtaaaaa tacggggttc tgaaac.

36

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):

(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

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):

(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

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):

(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

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):

(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

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):

(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

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):

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Metagenomic hit (no protein accession): contig OIZX01000427.1:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Metagenomic hit (no protein accession): contig OCTW011587266.1:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Metagenomic hit (no protein accession): contig emb|OGNF01009141.1:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Metagenomic hit (no protein accession): contig emb|OIEN01002196.1:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Metagenomic hit (no protein accession): contig e-k87_11092736:

(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

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:

CasRX/Cas13d Direct repeat 1:

(SEQ ID NO: 108)

gtgagaagtc tccttatggg gagatgctac.

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Ga0129306_1000735:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Ga0129317_1008067:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d Ga0224415_10048792:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence CasRX/Cas13d 160582958 gene49834:

(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

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:

(SEQ ID NO: 113)

gaactacacc cctctgttct tgtaggggtc taacac.

36

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d 250twins_35838_GL0110300:

(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

Exemplary CasRX/Cas13d proteins may comprise or consist of the sequence: CasRX/Cas13d 250twins_36050_GL0158985:

(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

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.

Exemplary wild type Cas13d proteins of the disclosure may comprise or consist of the amino acid sequence:

Cas13d (Ruminococcus flavefaciens XPD3002) Sequence:

(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.

Exemplary wild type Cas13d proteins of the disclosure may comprise or consist of the amino acid sequence:

Cas13d (contig e-k87_11092736):

(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.

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).

Exemplary wild type Cas13d proteins of the disclosure may comprise or consist of the amino acid sequence:

Cas13d (160582958_gene49834):

(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.

An exemplary direct repeat sequence of Cas13d (160582958_gene49834) (SEQ ID NO: 48) comprises or consists of the nucleic acid sequence:

Cas13d (160582958_gene49834) Direct Repeat Sequence:

(SEQ ID NO: 49)

GAACTACACCCCTCTGTTCTTGTAGGGGTCTAACAC.

Exemplary wild type Cas13d proteins of the disclosure may comprise or consist of the amino acid sequence:

Cas13d (contig tpg|DJXD01000002.1|; uncultivated Ruminococcus assembly, UBA7013, from sheep gut metagenome):

(SEQ ID NO: 50)

MKKQKSKKTVSKTSGLKEALSVQGTVIMTSFGKGNMANLSYKIPSSQKPQ

NLNSSAGLKNVEVSGKKIKFQGRHPKIATTDNPLFKPQPGMDLLCLKDKL

EMHYFGKTFDDNIHIQLIYQILDIEKILAVHVNNIVFTLDNVLHPQKEEL

TEDFIGAGGWRINLDYQTLRGQTNKYDRFKNYIKRKELLYFGEAFYHENE

RRYEEDIFAILTLLSALRQFCFHSDLSSDESDHVNSFWLYQLEDQLSDEF

KETLSILWEEVTERIDSEFLKTNTVNLHILCHVFPKESKETIVRAYYEFL

IKKSFKNMGFSIKKLREIMLEQSDLKSFKEDKYNSVRAKLYKLFDFIITY

YYDHHAFEKEALVSSLRSSLTEENKEEIYIKTARTLASALGADFKKAAAD

VNAKNIRDYQKKANDYRISFEDIKIGNTGIGYFSELIYMLTLLLDGKEIN

DLLTTLINKFDNIISFIDILKKLNLEFKFKPEYADFFNMTNCRYTLEELR

VINSIARMQKPSADARKIMYRDALRILGMDNRPDEEIDRELERTMPVGAD

GKFIKGKQGFRNFIASNVIESSRFHYLVRYNNPHKTRTLVKNPNVVKFVL

EGIPETQIKRYFDVCKGQEIPPTSDKSAQIDVLARIISSVDYKIFEDVPQ

SAKINKDDPSRNFSDALKKQRYQAIVSLYLTVMYLITKNLVYVNSRYVIA

FHCLERDAFLHGVTLPKMNKKIVYSQLTTHLLTDKNYTTYGHLKNQKGHR

KWYVLVKNNLQNSDITAVSSFRNIVAHISVVRNSNEYISGIGELHSYFEL

YHYLVQSMIAKNNWYDTSHQPKTAEYLNNLKKHHTYCKDFVKAYCIPFGY

VVPRYKNLTINELFDRNNPNPEPKEEV.

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

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.

In some embodiments of the compositions and methods of the disclosure, the sequence motif is a signature of a disease or disorder.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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)).

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).

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.

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.

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

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.

In some embodiments of the compositions of the disclosure, 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 protein comprises or consists of:

(SEQ ID NO: 20)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGLCKPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVHFDASVEDST.

In some embodiments, the second RNA binding protein comprises or consists of an RNAse4. In some embodiments, the RNAse4 protein comprises or consists of:

(SEQ ID NO: 21)

QDGMYQRFLRQHVHPEETGGSDRYCDLMMQRRKMTLYHCKRFNTFIHEDI

WNIRSICSTTNIQCKNGKMNCHEGVVKVTDCRDTGSSRAPNCRYRAIAST

RRVVIACEGNPQVPVHFDG.

In some embodiments, the second RNA binding protein comprises or consists of an RNAse6. In some embodiments, the RNAse6 protein comprises or consists of:

(SEQ ID NO: 22)

WPKRLTKAHWFEIQHIQPSPLQCNRAMSGINNYTQHCKHQNTFLHDSFQN

VAAVCDLLSIVCKNRRHNCHQSSKPVNMTDCRLTSGKYPQCRYSAAAQYK

FFIVACDPPQKSDPPYKLVPVHLDSIL.

In some embodiments, the second RNA binding protein comprises or consists of an RNAse7. In some embodiments, the RNAse7 protein comprises or consists of:

(SEQ ID NO: 23)

APARAGFCPLLLLLLLGLWVAEIPVSAKPKGMTSSQWFKIQHMQPSPQAC

NSAMKNINKHTKRCKDLNTFLHEPFSSVAATCQTPKIACKNGDKNCHQSH

GPVSLTMCKLTSGKYPNCRYKEKRQNKSYVVACKPPQKKDSQQFHLVPVH

LDRVL.

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)

TSSQWFKTQHVQPSPQACNSAMSIINKYTERCKDLNTFLHEPFSSVAITC

QTPNIACKNSCKNCHQSHGPMSLTMGELTSGKYPNCRYKEKHLNTPYIVA

CDPPQQGDPGYPLVPVHLDKVV.

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)

KPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFAN

VVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQ

TPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII.

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)

DKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEG

CNRSWPFNLEEIKKNWMEITDSSLPSPSMGPAPPRWMRSTPRRSTLAEAW

NSTGSWTSTGGCALPPAALPSGDLCCRPSLTAGSRGVGVDLTALHQLLHV

HYSATGIIPEECSEPTKPFQIILHHDHTEWVQSIGMPIWGTISSSESAIG

KNEESQPACAVLSHDS.

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)

AAVEDNHLLIKAVQNEDVDLVQQLLEGGANVNFQEEEGGWTPLHNAVQMS

REDIVELLLRHGADPVLRKKNGATPFILAAIAGSVKdLLKLFLSKGADVN

ECDFYGFTAFMEAAVYGKVKALKFLYKRGANVNLRRKTKEDQERLRKGGA

TALMDAAEKGHVEVLKILLDEMGADVNACDNMGRNALIHALLSSDDSDVE

AITHLLLDHGADVNVRGERGKTPLILAVEKKHLGLVQRLLEQEHIEINDT

DSDGKTALLLAVELKLKKIAELLCKRGASTDCGDLVMTARRNYDHSLVKV

LLSHGAKEDFHPPAEDWKPQSSHWGAALKDLHRIYRPMIGKLKFFIDEKY

KIADTSEGGIYLGFYEKQEVAVKTFCEGSPRAQREVSCLQSSRENSHLVT

FYGSESHRGHLFVCVTLCEQTLEACLDVHRGEDVENEEDEFARNVLSSIF

KAVQELHLSCGYTHQDLQPQNILIDSKKAAHLADFDKSIKWAGDPQEVKR

DLEDLGRLVLYVVKKGSISFEDLKAQSNEEVVQLSPDEETKDLIHRLFHP

GEHVRDCLSDLLGHPFFWTWESRYRTLRNVGNESDIKTRKSESEILRLLQ

PGPSEHSKSFDKWTTKINECVMKKMNKFYEKRGNFYQNTVGDLLKFIRNL

GEHIDEEKHKKMKLKIGDPSLYFQKTFPDLVIYVYTKLQNTEYRKHFPQT

HSPNKPQCDGAGGASGLASPGC.

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)

VQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDL

LPEMRAYWPDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSL

ELYRELDLNSVLLKLGIKPSINYYQVADFKDALARVYGVIPKIQCLPPSQ

DEEVQTIGQIELCLTKQDQQLQNCTEPGEQPSPKQEVWLANGAAESRGLR

VCEDGPVFYPPPKKTKH.

In some embodiments, the second RNA binding protein comprises or consists of an RNAse11. In some embodiments, the RNAse1l11 protein comprises or consists of:

(SEQ ID NO: 29)

EASESTMKIIKEEFTDEEMQYDMAKSGQEKQTIEILMNPILLVKNTSLSM

SKDDMSSTLLTFRSLHYNDPKGNSSGNDKECCNDMTVWRKVSEANGSCKW

SNNFIRSSTEVMRRVHRAPSCKFVQNPGISCCESLELENTVCQFTTGKQF

PRCQYHSVTSLEKILTVLTGHSLMSWLVCGSKL.

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)

XLGGADKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWP

DKSEGCNRSWPFNLEEIKDLLPEMRAYWPDVIHSFPNRSRFWKHEWEKHG

TCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGIKPSINYYQTTEED

LNLDVEPTTEDTAEEVTIHVLLHSALFGEIGPRRW.

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)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVHFDASVEDST.

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: 117)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVHFEASVEDST.

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)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVNFEASVEDST.

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:

(SEQ ID NO: 119)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVNFDASVEDST.

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

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:

(SEQ ID NO: 121)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEP

LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS

PKERHIIVACEGSPYVPVNFEASVEDST.

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)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCKPVNTFVHEP

LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS

PKERHIIVACEGSPYVPVHFDASVEDST.

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:

(SEQ ID NO: 208)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEP

LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS

PKERHIIVACEGSPYVPVNFEASVEDST.

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)

APVEHVVADAGAFLRHAALQDIGKNIYTIREVVTEIRDKATRRRLAVLPY

ELRFKEPLPEYVRLVTEFSKKTGDYPSLSATDIQVLALTYQLEAEFVGVS

HLKQEPQKVKVSSSIQHPETPLHISGFHLPYKPKPPQETEKGHSACEPEN

LEFSSFMFWRNPLPNIDHELQELLIDRGEDVPSEEEEEEENGFEDRKDDS

DDDGGGWITPSNIKQIQQELEQCDVPEDVRVGCLTTDFAMQNVLLQMGLH

VLAVNGMLIREARSYILRCHGCFKTTSDMSRVFCSHCGNKTLKKVSVTV.

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)

AFSGLQRVGGVDVSFVKGDSVRACASLVVLSFPELEVVYEESRMVSLTAP

YVSGFLAFREVPFLLELVQQLREKEPGLMPQVLLVDGNGVLHHRGFGVAC

HLGVLTDLPCVGVAKKLLQVDGLENNALHKEKIRLLQTRGDSFPLLGDSG

TVLGMALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQAD

ICSREHIRKS.

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)

AELPPVPGGPRGPGELAKYGLPGLAQLKSRESYVLCYDPRTRGALWVVEQ

LRPERLRGDGDRRECDFREDDSVHAYHRATNADYRGSGFDRGHLAAAANH

RWSQKAMDDTFYLSNVAPQVPHLNQNAWNNLEKYSRSLTRSYQNVYVCTG

PLFLPRTEADGKSYVKYQVIGKNHVAVPTHFFKVLILEAAGGQIELRTYV

MPNAPVDEAIPLERFLVPIESIERASGLLFVPNILARAGSLKAITAGSK.

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)

RLVGEEEAGFGECDKFFYAGTPPAGLAADSHVKICQRAEGAERFATLYST

RDRIPVYSAFRAPRPAPGGAEQRWLVEPQIDDPNSNLEEAINEAEAITSV

NSLGSKQALNTDYLDSDYQRGQLYPFSLSSDVQVATFTLTNSAPMTQSFQ

ERWYVNLHSLMDRALTPQCGSGEDLYILTGTVPSDYRVKDKVAVPEFVWL

AACCAVPGGGWAMGFVKHTRDSDIIEDVMVKDLQKLLPFNPQLFQNNCGE

TEQDTEKMKKILEVVNQIQDEERMVQSQKSSSPLSSTRSKRSTLLPPEAS

EGSSSFLGKLMGFIATPFIKLFQLIYYLVVAILKNIVYFLWCVTKQVING

IESCLYRLGSATISYFMAIGEELVSIPWKVLKVVAKVIRALLRILCCLLK

AICRVLSIPVRVLVDVATFPVYTMGAIPIVCKDIALGLGGTVSLLFDTAF

GTLGGLFQVVFSVCKRIGYKVTFDNSGEL.

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:

(SEQ ID NO: 35)

MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGG

DVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKR

SERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGI

PYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAK

KLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLI

QKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSE

PNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSL

SSAKRKEPEPKGSTKKKAKTGAAGKFKRGK.

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:

(SEQ ID NO: 124)

MESGQPARRIAMAPLLEYERQLVLELLDTDGLVVCARGLGADRLLYHFLQ

LHCHPACLVLVLNTQPAEEEYFINQLKIEGVEHLPRRVTNEITSNSRYEV

YTQGGVIFATSRILVVDFLTDRIPSDLITGILVYRAHRIIESCQEAFILR

LFRQKNKRGFIKAFTDNAVAFDTGFCHVERVMRNLFVRKLYLWPRFHVAV

NSFLEQHKPEVVEIHVSMTPTMLAIQTAILDILNACLKELKCHNPSLEVE

DLSLENAIGKPFDKTIRHYLDPLWHQLGAKTKSLVQDLKILRTLLQYLSQ

YDCVTFLNLLESLRATEKAFGQNSGWLFLDSSTSMFINARARVYHLPDAK

MSKKEKISEKMEIKEGEGILWG.

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)

CSPQESGMTALSARMLTRSRSLGPGAGPRGCREEPGPLRRREAAAEARKS

HSPVKRPRKAQRLRVAYEGSDSEKGEGAEPLKVPVWEPQDWQQQLVNIRA

MRNKKDAPVDHLGTEHCYDSSAPPKVRRYQVLLSLMLSSQTKDQVTAGAM

QRLRARGLTVDSILQTDDATLGKLIYPVGFWRSKVKYIKQTSAILQQHYG

GDIPASVAELVALPGVGPKMAHLAMAVAWGTVSGIAVDTHVHRIANRLRW

TKKATKSPEETRAALEEWLPRELWHEINGLLVGFGQQTCLPVHPRCHACL

NQALCPAAQGL.

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)

ESTHVEFKRFTTKKVIPRIKEMLPHYVSAFANTQGGYVLIGVDDKSKEVV

GCKWEKVNPDLLKKEIENCIEKLPTFHFCCEKPKVNFTTKILNVYQKDVL

DGYVCVIQVEPFCCVVFAEAPDSWIMKDNSVTRLTAEQWVVMMLDTQSAP

PSLVTDYNSCLISSASSARKSPGYPIKVHKFKEALQ.

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)

TLQGTNTYLVGTGPRRILIDTGEPAIPEYISCLKQALTEFNTAIQEIVVT

HWHRDHSGGIGDICKSINNDTTYCIKKLPRNPQREEIIGNGEQQYVYLKD

GDVIKTEGATLRVLYTPGHTDDHMALLLEEENAIFSGDCILGEGTTVFED

LYDYMNSLKELLKIKADIIYPGHGPVIHNAEAKIQQYISHRNIREQQILT

LFRENFEKSFTVMELVKIIYKNTPENLHEMAKHNLLLHLKKLEKEGKIFS

NTDPDKKWKAHL.

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)

MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETK

VTRDALTEPLAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLS

GLFATQNGDVGCYGNMDEFTQEELRALDSEGRALLTQHKIRTWEGKEKTL

TLINVYCPHADPGRPERLVFKMRFYRLLQIRAEALLAAGSHVIILGDLNT

AHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQSASHVGPFIDSYRCF

QPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASFLLPEVM

GSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQSP

VLEQSTLQHNNQTRVQTCQNKAQVRSTRPQPSQVGSSRGQKNLKSYFQPS

PSCPQASPDIELPSLPLMSALMTPKTPEEKAVAKVVKGQAKTSEAKDEKE

LRTSFWKSVLAGPLRTPLCGGHREPCVMRTVKKPGPNLGRRFYMCARPRG

PPTDPSSRCNFFLWSRPS.

In some embodiments, the APEX2 polypeptide comprises or consists of:

(SEQ ID NO: 39)

MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETK

VTRDALTEPLAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLS

GLFATQNGDVGCYGNMDEFTQEELRALDSEGRALLTQHKIRTWEGKEKTL

TLINVYCPHADPGRPERLVFKMRFYRLLQIRAEALLAAGSHVIILGDLNT

AHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQSASHVGPFIDSYRCF

QPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASFLLPEVM

GSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQS

P.

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)

PKRGKKGAVAEDGDELRTEPEAKKSKTAAKKNDKEAAGEGPALYEDPPDQ

KTSPSGKPATLKICSWNVDGLRAWIKKKGLDWVKEEAPDILCLQETKCSE

NKLPAELQELPGLSHQYWSAPSDKEGYSGVGLLSRQCPLKVSYGIGDEEH

DQEGRVIVAEFDSFVLVTAYVPNAGRGLVRLEYRQRWDEAFRKFLKGLAS

RKPLVLCGDLNVAHEEIDLRNPKGNKKNAGFTPQERQGFGELLQAVPLAD

SFRHLYPNTPYAYTFWTYMMNARSKNVGWRLDYFLLS.

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:

(SEQ ID NO: 40)

QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNK

RSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGF

RNVVVACENGLPVHLDQSIFRRP.

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)

SSLIRRVISTAKAPGAIGPYSQAVLVDRTIYISGQIGMDPSSGQLVSGGV

AEEAKQALKNMGEILKAAGCDFTNVVKTTVLLADINDFNTVNEIYKQYFK

SNFPARAAYQVAALPKGSRIEIEAVAIQGPLTTASL.

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)

GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGIL

LAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFT

PSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIE

ERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPLTLE.

In some embodiments, the ZC3H12A polypeptide comprises or consists of:

(SEQ ID NO: 43)

SGPCGEKPVLEASPTMSLWEFEDSHSRQGTPRPGQELAAEEASALELQMK

VDFFRKLGYSSTEIHSVLQKLGVQADTNTVLGELVKHGTATERERQTSPD

PCPQLPLVPRGGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGN

KEVFSCRGILLAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRE

LEKKKILVFTPSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQG

ERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPLTLEH

RKQPCPYGRKCTYGIKCRFFHPERPSCPQRSVADELRANALLSPPRAPSK

DKNGRRPSPSSQSSSLLTESEQCSLDGKKLGAQASPGSRQEGLTQTYAPS

GRSLAPSGGSGSSFGPTDWLPQTLDSLPYVSQDCLDSGIGSLESQMSELW

GVRGGGPGEPGPPRAPYTGYSPYGSELPATAAFSAFGRAMGAGHFSVPAD

YPPAPPAFPPREYWSEPYPLPPPTSVLQEPPVQSPGAGRSPWGRAGSLAK

EQASVYTKLCGVFPPHLVEAVMGRFPQLLDPQQLAAEILSYKSQHPSE.

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:

(SEQ ID NO: 44)

SSLIRRVISTAKAPGAIGPYSQAVLVDRTIYISGQIGMDPSSGQLVSGGV

AEEAKQALKNMGEILKAAGCDFTNVVKTTVLLADINDFNTVNEIYKQYFK

SNFPARAAYQVAALPKGSRIEIEAVAIQGPLTTASL.

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)

EALFFPSQVTCTEALLRAPGAELAELPEGCPCGLPHGESALSRLLRALLA

ARASLDLCLFAFSSPQLGRAVQLLHQRGVRVRVVTDCDYMALNGSQIGLL

RKAGIQVRHDQDPGYMHHKFAIVDKRVLITGSLNWTTQAIQNNRENVLIT

EDDEYVRLFLEEFERIWEQFNPTKYTFFPPKKSHGSCAPPVSRAGGRLLS

WHRTCGTSSESQT.

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)

KARYKTLEPRGYSLLIRGLIHSDRWREALLLLEDIKKVITPSKKNYNDCI

QGALLHQDVNTAWNLYQELLGHDIVPMLETLKAFFDFGKDIKDDNYSNKL

LDILSYLRNNQLYPGESFAHSIKTWFESVPGKQWKGQFTTVRKSGQCSGC

GKTIESIQLSPEEYECLKGKIMRDVIDGGDQYRKTTPQELKRFENFIKSR

PPFDVVIDGLNVAKMFPKVRESQLLLNVVSQLAKRNLRLLVLGRKHMLRR

SSQWSRDEMEEVQKQASCFFADDISEDDPFLLYATLHSGNHCRFITRDLM

RDHKACLPDAKTQRLFFKWQQGHQLAIVNRFPGSKLTFQRILSYDTVVQT

TGDSWHIPYDEDLVERCSCEVPTKWLCLHQKT.

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)

SVEPMFRHLKNTYAGLQLVVVILPGKTPVYAEVKRVGDTVLGMATQCVQM

KNVQRTTPQTLSNLCLKINVKLGGVNNILLPQGRPPVFQQPVIFLGADVT

HPPAGDGKKPSIAAVVGSMDAHPNRYCATVRVQQHRQEIIQDLAAMVREL

LIQFYKSTRFKPTRIIFYRDGVSEGQFQQVLHHELLAIREACIKLEKDYQ

PGITFIVVQKRHHTRLFCTDKNERVGKSGNIPAGTTVDTKITHPTEFDFY

LCSHAGIQGTSRPSHYHVLWDDNRFSSDELQILTYQLCHTYVRCTRSVSI

PAPAYYAHLVAFRARYHLVDKEHDSAEGSHTSGQSNGRDHQALAKAVQVH

QDTLRTMYFA.

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)

QGAEGALTGKQPDGSAEKAVLEQFGFPLTGTEARCYTNHALSYDQAKRVP

RWVLEHISKSKIMGDADRKHCKFKPDPNIPPTFSAFNEDYVGSGWSRGHM

APAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYWNRIEMYCRELTERFED

VWVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVPSHLYKVILARRSSVST

EPLALGAFVVPNEAIGFQPQLTEFQVSLQDLEKLSGLVFFPHLDRTSDIR

NICSVDTCKLLDFQEFTLYLSTRKIEGARSVLRLEKIMENLKNAEIEPDD

YFMSRYEKKLEELKAKEQSGTQIRKPS.

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)

EHPSKMEFFQKLGYDREDVLRVLGKLGEGALVNDVLQELIRTGSRPGALE

HPAAPRLVPRGSCGVPDSAQRGPGTALEEDFRTLASSLRPIVIDGSNVAM

SHGNKETFSCRGIKLAVDWFRDRGHTYIKVFVPSWRKDPPRADTPIREQH

VLAELERQAVLVYTPSRKVHGKRLVCYDDRYIVKVAYEQDGVIVSNDNYR

DLQSENPEWKWFIEQRLLMFSFVNDRFMPPDDPLGRHGPSLSNFLSRKPK

PPEPSWQHCPYGKKCTYGIKCKFYHPERPHHAQLAVADELRAKTGARPGA

GAEEQRPPRAPGGSAGARAAPREPFAHSLPPARGSPDLAALRGSFSRLAF

SDDLGPLGPPLPVPACSLTPRLGGPDWVSAGGRVPGPLSLPSPESQFSPG

DLPPPPGLQLQPRGEHRPRDLHGDLLSPRRPPDDPWARPPRSDRFPGRSV

WAEPAWGDGATGGLSVYATEDDEGDARARARIALYSVFPRDQVDRVMAAF

PELSDLARLILLVQRCQSAGAPLGKP.

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)

RQQQPQVVEKQQETPLAPADFAHISQDAQSLHSGASRRSQKRLQSPSKQA

QPLDDPEAEQLTVVGKISFNPKDVLGRGAGGTFVFRGQFEGRAVAVKRLL

RECFGLVRREVQLLQESDRHPNVLRYFCTERGPQFHYIALELCRASLQEY

VENPDLDRGGLEPEVVLQQLMSGLAHLHSLHIVHRDLKPGNILITGPDSQ

GLGRVVLSDFGLCKKLPAGRCSFSLHSGIPGTEGWMAPELLQLLPPDSPT

SAVDIFSAGCVFYYVLSGGSHPFGDSLYRQANILTGAPCLAHLEEEVHDK

VVARDLVGAMLSPLPQPRPSAPQVLAHPFFWSRAKQLQFFQDVSDWLEKE

SEQEPLVRALEAGGCAVVRDNWHEHISMPLQTDLRKFRSYKGTSVRDLLR

AVRNKKHHYRELPVEVRQALGQVPDGFVQYFTNRFPRLLLHTHRAMRSCA

SESLFLPYYPPDSEARRPCPGATGR.

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)

KLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTES

STGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHT

IELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVAAVVMQEGLAHICL

VTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDV

VKCILVASPGFVREQFCDYLFQQAVKTDNKLLLENRSKFLQVHASSGHKY

SLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEK

ANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTVRIFSSLHV

SGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED.

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)

SLVIRNLQRVIPIRRAPLRSKIEIVRRILGVQKFDLGIICVDNKNIQHIN

RIYRDRNVPTDVLSFPFHEHLKAGEFPQPDFPDDYNLGDIFLGVEYIFHQ

CKENEDYNDVLTVTATHGLCHLLGFTHGTEAEWQQMFQKEKAVLDELGRR

TGTRLQPLTRGLFGGS.

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)

QEVIAGLERFTFAFEKDVEMQKGTGLLPFQGMDKSASAVCNFFTKGLCEK

GKLCPFRHDRGEKMVVCKHWLRGLCKKGDHCKFLHQYDLTRMPECYFYSK

FGDCSNKECSFLHVKPAFKSQDCPWYDQGFCKDGPLCKYRHVPRIMCLNY

LVGFCPEGPKCQFAQKIREFKLLPGSKI.

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:

(SEQ ID NO: 135)

KLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTES

STGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHT

IELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVAAVVMQEGLAHICL

VTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDV

VKCILVASPGFVREQFCDYMFQQAVKTDNKLLLENRSKFLQVHASSGHKY

SLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEK

ANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTVRIFSSLHV

SGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED.

In some embodiments, the hCG_2002731 polypeptide comprises or consists of:

(SEQ ID NO: 136)

DPAWSADVAAVVMQEGLAHICLVTPSMTLTRAKVEVNIPRKRKGNCSQHD

RALERFYEQVVQAIQRHIHFDVVKCILVASPGFVREQFCDYMFQQAVKTD

NKLLLENRSKFLQVHASSGHKYSLKEALCDPTVASRLSDTKAAGEVKALD

DFYKMLQHEPDRAFYGLKQVEKANEAMAIDTLLISDELFRHQDVATRSRY

VRLVDSVKENAGTVRIFSSLHVSGEQLSQLTGVAAILRFPVPELSDQEGD

SSSEED.

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)

MDPGKDKEGVPQPSGPPARKKFVIPLDEDEVPPGVRGNPVLKFVRNVPWE

FGDVIPDYVLGQSTCALFLSLRYHNLHPDYIHGRLQSLGKNFALRVLLVQ

VDVKDPQQALKELAKMCILADCTLILAWSPEEAGRYLETYKAYEQKPADL

LMEKLEQDFVSRVTECLTTVKSVNKTDSQTLLTTFGSLEQLIAASREDLA

LCPGLGPQK.

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)

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEP

LVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS

PKERHIIVACEGSPYVPVHFDASVEDST.

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)

QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNK

RSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGF

RNVVVACENGLPVHLDQSIFRRP.

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)

GLGLVQPSYGQDGMYQRFLRQHVHPEETGGSDRYCNLMMQRRKMTLYHCK

RFNTFIHEDIWNIRSICSTTNIQCKNGKMNCHEGVVKVTDCRDTGSSRAP

NCRYRAIASTRRVVIACEGNPQVPVHFDG.

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)

XSAVDNILLKLAKFKIGFLRLGQIQKVHPAIQQFTEQEICRSKSIKSLAL

LEELYNSQLIVATTCMGINHPIFSRKIFDFCIVDEASQISQPICLGPLFF

SRRFVLVGDHQQLPPLVLNREARALGMSESLFKRLEQNKSAVVQLTVQYR

MNSKIMSLSNKLTYEGKLECGSDKVANAVINLRHFKDVKLELEFYADYSD

NPWLMGVFEPNNPVCFLNTDKVPAPEQVEKGGVSNVTEAKLIVFLTSIFV

KAGCSPSDIGIIAPYRQQLKIINDLLARSIGMVEVNTVDKYQGRDKSIVL

VSFVRSNKDGTVGELLKDWRRLNVAITRAKHKLILLGCVPSLNCYPPLEK

LLNHLNSEKLISFFFCIWSHLIALL.

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)

MALRSHDRSTRPLYISVGHRMSLEAAVRLTCCCCRFRIPEPVRQADICSR

EHIRKSLGLPGPPTPRSPKAQRPVACPKGDSGESSALC.

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)

CYTNHALSYDQAKRVPRWVLEHISKSKIMGDADRKHCKFKPDPNIPPTFS

AFNEDYVGSGWSRGHMAPAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYW

NRIEMYCRELTERFEDVWVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVP

SHLYKVILARRSSVSTEPLALGAFVVPNEAIGFQPQLTEFQVSLQDLEKL

SGLVFFPHLDRT.

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)

VTVSQMTSVLNGKTRRFADIQLQHGALCFNIRYGTTVEEEKNHVLEIARQ

RAVAQAWTKEQRRLQEGEEGIRAWTEGEKQQLLSTGRVQGYDGYFVLSVE

QYLELSDSANNIHFMRQSEIGRR.

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)

TVSQPTLLVNGKTRRFTNIEFQYSTLLLSIRYGLTPDTLDEEKARVLDQA

RQRALGTAWAKEQQKARDGREGSRLWTEGEKQQLLSTGRVQGYEGYYVLP

VEQYPELADSSSNIQFLRQNEMGKR.

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)

MGWLRPGPRPLCPPARASWAFSHRFPSPLAPRRSPTPFFMASLLCCGPKL

AACGIVLSAWGVIMLIMLGIFFNVHSAVLIEDVPFTEKDFENGPQNIYNL

YEQVSYNCFIAAGLYLLLGGFSFCQVRLNKRKEYMVR.

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:

(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.

In some embodiments, the TALEN polypeptide comprises or consists of:

(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.

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:

(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.

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.

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

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.

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.

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.

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.

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

(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

In some embodiments, a PUF3 protein of the disclosure comprises or consists of the amino acid sequence of

• • 1 MEMNMDMDMD MELASIVSSL SALSHSNNNG GQAAAAGIVi GGAAGSQQIG GFRRSSFTTA
  • 61 NEVDSEILLL HGSSESSPIF KKTALSVGTA PPFSTNSKKF FGNGGNYYQY RSTDTASLSS
  • 121 ASYNNYHTHH TAANLGKNNK VNHLLGQYSA SIAGPVYYNG NDNNNSGGEG FFEKFGKSLI
  • 181 DGTRELESQD RPDANTQSQ FITKSVSNAS LDTQNTFEQN VESDKNFNKL NRNTTNSGSL
  • 241 YHSSSNSGSS ASLESENAHY PKRNIWNVAN TPVFRPSNNP AAVGATNVAL PNQQDGPANN
  • 301 NFPPYMNGFP PNQFHQGPHY QNFPNYLIGS PSNFISQMIS VQIPANEDTE DSNGKKKKKA
  • 361 NRPSSVSSPS SPPNNSPFPF AYPNPMMFMP PPPLSAPQQQ QQQQQQQQQE DQQQQQQQEN
  • 421 PYTYYPTPNP IPVKMPKDEK TFKKRNNKNH PANNNNANK QANPIYLENSI PTKNTSKKNA
  • 481 SSKSNESTAN NHKSHSHSHP HSQSLQQQQQ TYHRSPLLEQ LRNSSSDKNS NSNMSLKDIF
  • 541 GHSLEFCKDQ HGSRFIQREL ATSPASEKEV I FNEIRDDAI ELSNDVFGNY VIQKFFEFGS
  • 601 KIQKNTLVDQ FKGNMKIQLSL QMYACRVQK ALEYIDSNQR IELVLELSDS VLQMIKDQNG
  • 661 NHVIQKAIE IPiEKLPFIL SSLTGHIYHL STHSYGCRVI QRLLEFGSSE DQESILNELK
  • 721 DFIPYLIQDQ YGNYVIQYVL QQDQFTNKEM VDIKQEIIET VANNVVEYSK HKEASNVVEK
  • 781 SILYGSKNQK DLIISKILPR DKNHALNLED DSPMILMIKD QFANYVIQKL VNVSEGEGKK
  • 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

(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.

In some embodiments, a PUF5 protein of the disclosure comprises or consists of the amino acid sequence of

• • 1 MSDSTGRINS KASDSSSISD HQTADLSIFN GSFDGGAFSS SNIPLFNFMG TGNQRFQYSP
  • 61 HPFAKSSDPC RLAALTPSTP KGPLNLTPAD FGLADFSVGN ESFADFTANN TSFVGNVQSN
  • 121 VRSTRLLPAW AVDNSGNIRD DLTLQDVVSN GSLIDFAMDR TGVKFLERHF PEDHDNEMHF
  • 181 VLFDKLTEQG AVFTSLCRSA AGNFIIQKFV EHATLDEQER LVRKMCDNGL IEMCLDKFAC
  • 241 RVVQMSIQKF DVSIAMKLVE KISSLDFLPL CTDQCAIHVL QKVVKLLPIS AWSFFVKFLC
  • 301 RDDNLMTVCQ DKYGCRLVQQ TIDKLSDNPK LHCFNTRLQL LHGLMTSVAR NCFRLSSNEF
  • 361 ANYVVQYVIK SSGVMEMYRD TIIEKCLLRN ILSMSQDKYA SHVVEGAFLF APPLLLSEMM
  • 421 DEIFDGYVKD QETNRDALDI LLFHQYGNYV VQQMISICIS ALLGKEERKM VASEMRLYAK
  • 481 WFDRIKNRVN RHSGRLERFS SGKKITIESLQ KLNVPMTMTN EPMPYWAMPT PLMDISAHFM
  • 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
  • 1 MTPNRRSTDS YNMLGASFDF DPDFSLLSNK THKNKNPKPP VKLLPYRHGS NTTSSDLDNY
  • 61 IFNSGSGSSD DETPPPAAPI FISLEEVLLN GLLIDFAIDP SGVKFLEANY PLDSEDQIRPK
  • 121 AVFEKLTEST TLFVGLCHSR NGNFIVQKLV ELATPAEQRE LLRQMIDGGL LVMCKDKFAC
  • 181 RVVQLALQKF DHSNVFQLIQ ELSTFDLAAM CTDQISIHVI QRVVKQLPVD MWTFFVHFLS
  • 241 SGDSLMAVCQ DKYGC:LVQQ VIDRLAENPK LPCFKFRIQL LHSLMTCIVR NCYRLSSNEF
  • 301 ANYVIQYVIK SSGIMEMYRD TIIDKCLLRN LLSMSQDKYA SHVIEGAFLF APPALLHEMM
  • 361 EEIFSGYVKD VELNRDALDI LLFHQYGNYV VQQMISICTA ALIGKEEPQL PPAILLLYSG
  • 421 WYEKMKQRVL QHASRLERFS SGKKIIDSVM RHGVPTAAAI NAQAAPSLME LTAQFDAFP
  • 4181 SFLAR (SEQ ID NO: 213). In some embodiments, a PUF7 protein of the disclosure comprises or consists of the amino acid sequence of
  • 1 MTPNRRSTDS YNMLGASFDF DPDFSLLSNK THKNKNPKPP VKLLPYRIGS NTTSSDSDSY
  • 61 IFNSGSGSSD AETPAPVAPI FISLEDVLLN GQLIDFAIDP SGVKFLEANY PLDSEDQIRK
  • 121 AVFEKFTEST TLFVGLCHSR NGNFIVQKLV ELATPAEQRE LLRQMIDGGL LAMCKDKFAC
  • 181 RVVQLALQKF DHSNVFQLIQ ELSTFDLAAM CTDQISIHVI QRVVKQLPVD MWTFFVHFLS
  • 241 SGDSLMAVCQ DKYGCRLVQQ VIDRLAENPK LPCFKFEPRIQL LHSLMTCIVR NCYRLSSNEF
  • 301 ANYVIQYVIK SSGIMEMYRD TIIDKCLLRN LLSMSQDKYA SHVIEGAFLF APPALLHEMM
  • 361 EEIFSGYVKD VESNRDALDI LLFHQYGNYV VQQMISICTA ALIGKEEREL PPAILLLYSG
  • 421 WYEKMKQRVL QHASRLERFS SGKKIIDSVM RHGVPTAAAV NAQAAPSLME LTAQFDAMFP
  • 481 SFLAR (SEQ ID NO: 214). In some embodiments, a PUF8 protein of the disclosure comprises or consists of the amino acid sequence of
  • 1 MSRPISIGNT CTFDPSASPI ESLGRSIGAQ KIVDSVCGSP IRSYGRHIST NPKNERLPDT
  • 61 PEFQFATYMH QGGKVIGQNT LHMFGTPPSC YCAQENIPIS SNVGHVLSTI NNNYMNHQYN
  • 121 GSNMFSNQMT QMLQAQAYND LQMHQAIHSQS IRVPVQPSAT GIFSNPYPREP TTTDDLLTRY
  • 181 RANPAMMKNL KLSDIRGALL KFAKDQVGSR FIQQELASSK DRFEKDSIFD EVVSNADELV
  • 241 DDIFGNYVVQ KFFEYGEERH WARLVDAIID RVPEYAFQMY ACRVLQKALE KINEPLQIKI
  • 301 LSQIRHVIHR CMKDCNGNHV VOKAIEKVSP QYVQFIVDTL LESSNTIYEM SVDPYGCRVV
  • 361 QRCLEHCSPS QTKPVIGQIH KRFDEIANNQ YGNYVQIIHVI EHGSEEDRMV IVTRVSNNLF
  • 421 EFATHKYSSN VIEKCLEQGA VYHKSMIVGA ACHHQEGSVP IVVQMMKDQY ANYVVQKMFD
  • 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

(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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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).

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.

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, nβ2, 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.

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.

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_SM) vector, a modified sooty mangabey simian immunodeficiency virus (SIV_SM) vector, a African green monkey simian immunodeficiency virus (SIV_AGM) vector, a modified African green monkey simian immunodeficiency virus (SIV_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

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.

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).

“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.

Examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C.; hybridization buffer concentrations of about 6×SSC to about 10×SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4×SSC to about 8×SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C.; buffer concentrations of about 9×SSC to about 2×SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×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.

“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

In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a prokaryotic cell.

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.

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.

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).

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, naïve 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.

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.

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.

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.

In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a primary cell.

In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a cultured cell.

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.

In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is autologous or allogeneic.

Methods of Use

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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), Baló 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, Sjögren'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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In some embodiments of the methods of the disclosure, a subject of the disclosure is a human.

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.

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.

In some embodiments of the methods of the disclosure, a therapeutically effective amount eliminates the disease or disorder.

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.

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.

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.

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

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; or

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

The composition of embodiment 1, wherein the target sequence comprises at least one repeated sequence.

Embodiment 3

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

The composition of embodiment 3, wherein the eukaryotic cell is an animal cell.

Embodiment 5

The composition of embodiment 4, wherein the animal cell is a mammalian cell.

Embodiment 6

The composition of embodiment 5, wherein the animal cell is a human cell.

Embodiment 7

The composition of any one of embodiments 1-6, wherein the promoter is a constitutively active promoter.

Embodiment 8

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

The composition of embodiment 8, wherein the promoter is isolated or derived from a U6 promoter.

Embodiment 10

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

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

The composition of embodiment 10, wherein the promoter is isolated or derived from a valine tRNA promoter.

Embodiment 13

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

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

The composition of embodiment 13, wherein the spacer sequence has 100% complementarity to the target RNA sequence.

Embodiment 16

The composition of any one of embodiments 13-15, wherein the spacer sequence comprises or consists of 20 nucleotides.

Embodiment 17

The composition of any one of embodiments 13-15, wherein the spacer sequence comprises or consists of 21 nucleotides.

Embodiment 18

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

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

The composition of embodiment 19, wherein the scaffold sequence comprises a stem-loop structure.

Embodiment 21

The composition of embodiment 19 or 20, wherein the scaffold sequence comprises or consists of 90 nucleotides.

Embodiment 22

The composition of embodiment 19 or 20, wherein the scaffold sequence comprises or consists of 93 nucleotides.

Embodiment 23

The composition of embodiment 22, wherein the scaffold sequence comprises the sequence

(SEQ ID NO: 13)

GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

Embodiment 24

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

The composition of embodiment 19 or 24, wherein the scaffold sequence comprises a step-loop structure.

Embodiment 26

The composition of embodiment 25, wherein the scaffold sequence comprises or consists of 85 nucleotides.

Embodiment 27

The composition of embodiment 26, wherein the scaffold sequence comprises the sequence

(SEQ ID NO: 17)

GGACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA

GUGGCACCGAGUCGGUGCUUUUU.

Embodiment 28

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

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

The composition of embodiment 29, wherein the scaffold sequence comprises a stem-loop structure.

Embodiment 31

The composition of embodiment 29 or 30, wherein the scaffold sequence comprises or consists of 90 nucleotides.

Embodiment 32

The composition of embodiment 30 or 31, wherein the scaffold sequence comprises or consists of 93 nucleotides.

Embodiment 33

The composition of embodiment 32, wherein the scaffold sequence comprises the sequence

(SEQ ID NO: 82)

GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU

or

(SEQ ID NO: 83)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

Embodiment 34

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

The composition of embodiment 34, wherein an RNA genome or an RNA transcriptome comprises the RNA molecule.

Embodiment 36

The composition of any one of embodiments 1-35, wherein the first RNA binding protein comprises a CRISPR-Cas protein.

Embodiment 37

The composition of embodiment 36, wherein the CRISPR-Cas protein is a Type II CRISPR-Cas protein.

Embodiment 38

The composition of embodiment 37, wherein the first RNA binding protein comprises a Cas9 polypeptide or an RNA-binding portion thereof.

Embodiment 39

The composition of embodiment 36, wherein the CRISPR-Cas protein is a Type V CRISPR-Cas protein.

Embodiment 40

The composition of embodiment 39, wherein the first RNA binding protein comprises a Cpf1 polypeptide or an RNA-binding portion thereof.

Embodiment 41

The composition of embodiment 36, wherein the CRISPR-Cas protein is a Type VI CRISPR-Cas protein.

Embodiment 42

The composition of embodiment 41, wherein the first RNA binding protein comprises a Cas13 polypeptide or an RNA-binding portion thereof.

Embodiment 43

The composition of any one of embodiments 36-42, wherein the CRISPR-Cas protein comprises a native RNA nuclease activity.

Embodiment 44

The composition of embodiment 43, wherein the native RNA nuclease activity is reduced or inhibited.

Embodiment 45

The composition of embodiment 43, wherein the native RNA nuclease activity is increased or induced.

Embodiment 46

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

The composition of embodiment 46, wherein the CRISPR-Cas protein comprises a mutation.

Embodiment 48

The composition of embodiment 47, wherein a nuclease domain of the CRISPR-Cas protein comprises the mutation.

Embodiment 49

The composition of embodiment 47, wherein the mutation occurs in a nucleic acid encoding the CRISPR-Cas protein.

Embodiment 50

The composition of embodiment 47, wherein the mutation occurs in an amino acid encoding the CRISPR-Cas protein.

Embodiment 51

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

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

The composition of any one of embodiments 1-35, wherein the first RNA binding protein comprises a Pumilio and FBF (PUF) protein.

Embodiment 54

The composition of embodiment 53, wherein the first RNA binding protein comprises a Pumilio-based assembly (PUMBY) protein.

Embodiment 55

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

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

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

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

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

The composition of embodiment 60, wherein an RNA genome or an RNA transcriptome comprises the RNA molecule.

Embodiment 62

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

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

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

The composition of embodiment 64, wherein the first RNA binding protein comprises an NLS at a C-terminus of the protein.

Embodiment 66

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

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

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

The composition of any one of embodiments 1-68, wherein the second RNA binding protein comprises or consists of a nuclease domain.

Embodiment 70

The composition of embodiment 69, wherein the sequence encoding the second RNA binding protein comprises or consists of an RNAse.

Embodiment 71

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse1.

Embodiment 72

The composition of embodiment 71, wherein the RNAse1 protein comprises or consists of SEQ ID NO: 20.

Embodiment 73

The composition of embodiment 72, wherein the second RNA binding protein comprises or consists of an RNAse4.

Embodiment 74

The composition of embodiment 73, wherein the RNAse4 protein comprises or consists of: (SEQ ID NO: 21.

Embodiment 75

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse6.

Embodiment 76

The composition of embodiment 75, wherein the RNAse6 protein comprises or consists of SEQ ID NO: 22.

Embodiment 77

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse7.

Embodiment 78

The composition of embodiment 77, wherein the RNAse7 protein comprises or consists of SEQ ID NO: 23.

Embodiment 79

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse8.

Embodiment 80

The composition of embodiment 79, wherein the RNAse8 protein comprises or consists of SEQ ID NO: 24.

Embodiment 81

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse2.

Embodiment 82

The composition of embodiment 81, wherein the RNAse2 protein comprises or consists of SEQ ID NO: 25.

Embodiment 83

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse6PL.

Embodiment 84

The composition of embodiment 83, wherein the RNAse6PL protein comprises or consists of SEQ ID NO: 26.

Embodiment 85

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAseL.

Embodiment 86

The composition of embodiment 85, wherein the RNAseL protein comprises or consists of SEQ ID NO: 27.

Embodiment 87

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAseT2.

Embodiment 88

The composition of embodiment 87, wherein the RNAseT2 protein comprises or consists of SEQ ID NO: 28.

Embodiment 89

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAse11.

Embodiment 90

The composition of embodiment 89, wherein the RNAse11 comprises or consists of SEQ ID NO: 29.

Embodiment 91

The composition of embodiment 70, wherein the second RNA binding protein comprises or consists of an RNAseT2-like.

Embodiment 92

The composition of embodiment 91, wherein the RNAseT2-like protein comprises or consists of SEQ ID NO: 30.

Embodiment 93

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a NOB 1 polypeptide.

Embodiment 94

The composition of embodiment 93, wherein the NOB 1 polypeptide comprises or consists of SEQ ID NO: 31.

Embodiment 95

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of an endonuclease.

Embodiment 96

The composition of embodiment 95, wherein the second RNA binding protein comprises or consists of an endonuclease V (ENDOV).

Embodiment 97

The composition of embodiment 96, wherein the ENDOV protein comprises or consists of SEQ ID NO: 32.

Embodiment 98

The composition of embodiment 95, wherein the second RNA binding protein comprises or consists of an endonuclease G (ENDOG).

Embodiment 99

The composition of embodiment 98, wherein the ENDOG protein comprises or consists of SEQ ID NO: 33.

Embodiment 100

The composition of embodiment 95, wherein the second RNA binding protein comprises or consists of an endonuclease D1 (ENDOD1).

Embodiment 101

The composition of embodiment 100, wherein the ENDOD1 protein comprises or consists of SEQ ID NO: 34.

Embodiment 102

The composition of embodiment 95, wherein the second RNA binding protein comprises or consists of a Human flap endonuclease-1 (hFEN1).

Embodiment 103

The composition of embodiment 102, wherein the hFEN1 protein comprises or consists of SEQ ID NO: 35.

Embodiment 104

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a human Schlafen 14 (hSLFN14) polypeptide.

Embodiment 105

The composition of embodiment 104, wherein the hSLFN14 polypeptide comprises or consists of SEQ ID NO: 36.

Embodiment 106

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

The composition of embodiment 106, wherein the hLACTB2 polypeptide comprises or consists of SEQ ID NO: 37.

Embodiment 108

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of an apurinic/apyrimidinic (AP) endodeoxyribonuclease (APEX2) polypeptide.

Embodiment 109

The composition of embodiment 108, wherein the APEX2 polypeptide comprises or consists of SEQ ID NO: 38.

Embodiment 110

The composition of embodiment 108, wherein the APEX2 polypeptide comprises or consists of: SEQ ID NO: 39.

Embodiment 111

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of an angiogenin (ANG) polypeptide.

Embodiment 112

The composition of embodiment 111, wherein the ANG polypeptide comprises or consists of SEQ ID NO: 40.

Embodiment 113

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a heat responsive protein 12 (HRSP12) polypeptide.

Embodiment 114

The composition of embodiment 113, wherein the HRSP12 polypeptide comprises or consists of SEQ ID NO: 41.

Embodiment 115

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

The composition of embodiment 115, wherein the ZC3H12A polypeptide comprises or consists of SEQ ID NO: 42.

Embodiment 117

The composition of embodiment 115, wherein the ZC3H12A polypeptide comprises or consists of SEQ ID NO: 43.

Embodiment 118

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

The composition of embodiment 118, wherein the RIDA polypeptide comprises or consists of SEQ ID NO: 44.

Embodiment 120

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

The composition of embodiment 120, wherein the PDL6 polypeptide comprises or consists of: (SEQ ID NO: 126.

Embodiment 122

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

The composition of embodiment 122, wherein the NTHL polypeptide comprises or consists of SEQ ID NO: 123.

Embodiment 124

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

The composition of embodiment 124, wherein the KIAA0391 polypeptide comprises or consists of SEQ ID NO: 127.

Embodiment 126

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

The composition of embodiment 126, wherein the APEX1 polypeptide comprises or consists of SEQ ID NO: 125.

Embodiment 128

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of an argonaute 2 (AGO2) polypeptide.

Embodiment 129

The composition of embodiment 128, wherein the AGO2 polypeptide comprises or consists of SEQ ID NO: 128.

Embodiment 130

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a mitochondrial nuclease EXOG (EXOG) polypeptide.

Embodiment 131

The composition of embodiment 130, wherein the EXOG polypeptide comprises or consists of SEQ ID NO: 129.

Embodiment 132

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

The composition of embodiment 132, wherein the ZC3H12D polypeptide comprises or consists of SEQ ID NO: 130.

Embodiment 134

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

The composition of embodiment 134, wherein the ERN2 polypeptide comprises or consists of SEQ ID NO: 131.

Embodiment 136

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

The composition of embodiment 136, wherein the PELO polypeptide comprises or consists of SEQ ID NO: 132.

Embodiment 138

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a YBEY metallopeptidase (YBEY) polypeptide.

Embodiment 139

The composition of embodiment 138, wherein the YBEY polypeptide comprises or consists of SEQ ID NO: 133.

Embodiment 140

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

The composition of embodiment 140, wherein the CPSF4L comprises or consists of SEQ ID NO: 134.

Embodiment 142

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of an hCG_200273 ipolypeptide.

Embodiment 143

The composition of embodiment 142, wherein the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 135.

Embodiment 144

The composition of embodiment 142, wherein the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 136.

Embodiment 145

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

The composition of embodiment 145, wherein the ERCC1 polypeptide comprises or consists of SEQ ID NO: 137.

Embodiment 147

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

The composition of embodiment 147, wherein the RAC1 polypeptide comprises or consists of SEQ ID NO: 138.

Embodiment 149

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a Ribonuclease A A1 (RAA1) polypeptide.

Embodiment 150

The composition of embodiment 149, wherein the RAA1 polypeptide comprises or consists of SEQ ID NO: 139.

Embodiment 151

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a Ras Related Protein (RAB1) polypeptide.

Embodiment 152

The composition of embodiment 151, wherein the RAB1 polypeptide comprises or consists of SEQ ID NO: 140.

Embodiment 153

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

The composition of embodiment 153, wherein the DNA2 polypeptide comprises or consists of SEQ ID NO: 141.

Embodiment 155

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a FLJ35220 polypeptide.

Embodiment 156

The composition of embodiment 155, wherein the FLJ35220 polypeptide comprises or consists of SEQ ID NO: 142.

Embodiment 157

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a FLJ13173 polypeptide.

Embodiment 158

The composition of embodiment 157, wherein the FLJ13173 polypeptide comprises or consists of: (SEQ ID NO: 143.

Embodiment 159

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a DNA repair endonuclease XPF (ERCC4) polypeptide.

Embodiment 160

The composition of embodiment 159, wherein the ERCC4 polypeptide comprises or consists of SEQ ID NO: 64.

Embodiment 161

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(K41R)) polypeptide.

Embodiment 162

The composition of embodiment 161, wherein the Rnase1(K41R) polypeptide comprises or consists of SEQ ID NO: 116.

Embodiment 163

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E)) polypeptide.

Embodiment 164

The composition of embodiment 163, wherein the Rnase1 (Rnase1(K41R, D121E)) polypeptide comprises or consists of SEQ ID NO: 117.

Embodiment 165

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

The composition of embodiment 165, wherein the Rnase1 (Rnase1(K41R, D121E, H119N)) polypeptide comprises or consists of SEQ ID NO: 118.

Embodiment 167

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(H119N)) polypeptide.

Embodiment 168

The composition of embodiment 167, wherein the Rnase1 (Rnase1(H119N)) polypeptide comprises or consists of SEQ ID NO: 119.

Embodiment 169

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

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

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

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

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

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

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of Teneurin Transmembrane Protein 1 (TENM1) polypeptide.

Embodiment 176

The composition of embodiment 175, wherein the TENM1 polypeptide comprises or consists of SEQ ID NO: 144.

Embodiment 177

The composition of embodiment 69, wherein the second RNA binding protein comprises or consists of Teneurin Transmembrane Protein 2 (TENM2) polypeptide.

Embodiment 178

The composition of embodiment 177, wherein the TENM2 polypeptide comprises or consists of SEQ ID NO: 145.

Embodiment 179

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

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

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

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 1×10{circumflex over ( )}5 cells per well of a 24-well plate for RNA isolation or 0.5×10{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:

	Forward Primer
	TetCTG_DMPK_E15_F

SEQ ID NO: 83

	TCGGAGCGGTTGTGAACT
	Reverse Primer
	TetCTG_DMPK_E15_R

SEQ ID NO: 84

GTTCGCCGTTGTTCTGTC

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.

CTG-targeting spacer

SEQ ID NO: 85

AGCAGCAGCAGCAGCAGCAG

Non-targeting control spacer (λ2)

SEQ ID NO: 86

GTGATAAGTGGAATGCCATG

sgRNA scaffold (N's indicate spacer)

SEQ ID NO: 87

GNNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAUGCUGGAAACAGCAUAG

CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG

UCGGUGCUUUUUUU

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

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

A549 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin (GIBCO) and passaged at 90%-100% confluency. Cells were seeded at 1×10{circumflex over ( )}5 cells per well of a 24-well plate for RNA isolation or 0.5×10{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.

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.

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.

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 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 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 (λ2)
sgRNA scaffold (N's	GNNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAUG
indicate spacer)	CUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
	CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU
	GCUUUUUUU (SEQ ID NO: 201)

(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.

A E67-CjeCas9 and sgRNA plasmid may comprise or consist of the sequence (U6: N's=sgRNA spacer, E67, CieCas9):

(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

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

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.