COMPOSITIONS AND METHODS FOR THE MODULATION OF ADAPTIVE IMMUNITY

Abstract

Disclosed are compositions and methods for simultaneously providing a gene therapy and preventing an adaptive immune response to a cell modified by the gene therapy by the immune system of a subject. In some embodiments, compositions of the disclosure modify a level of expression of an RNA molecule associated with a disease or disorder as well as inhibit expression or activity of a component of an adaptive immune response to mask the modified cell from a subject's immune system.

Description

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 involved in an adaptive immune response.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “LOCN_003_001 US_SeqList_ST25”, which was created on Jun. 6, 2019 and is 2.93 MB in size, are hereby incorporated by reference in their entirety.

BACKGROUND

There has been a long-felt but unmet need in the art for simultaneously providing a gene therapy and suppressing the adaptive immune response that may arise when the gene therapy is delivered by, for example, a viral vector. The disclosure provides compositions and methods for specifically targeting RNA molecules in a sequence-specific manner that provides a gene therapy in vivo while masking the modified cells from the immune system of a subject, thereby preventing an adaptive immune response to the modified cell.

SUMMARY

The disclosure provides a composition comprising a nucleic acid sequence comprising a guide RNA (gRNA) sequence that specifically binds a target RNA sequence, wherein the target RNA sequence encodes a protein component of an adaptive immune response, and wherein the gRNA sequence comprises a spacer sequence comprising a portion of a nucleic acid sequence encoding the protein component, and wherein the protein component is selected from the group consisting of Beta-2-microglobulin (β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL 12), and CC Chemokine Receptor 7 (CCR7).

The disclosure also provides a composition comprising (a) a first sequence comprising a guide RNA (gRNA) that specifically binds a target sequence within an RNA molecule, wherein the target sequence comprises a sequence encoding a component of an adaptive immune response 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.

The disclosure provides a composition comprising: (a) a first sequence comprising a guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and (b) a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule and (c) 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.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the first target sequence or the second target sequence comprises at least one repeated sequence.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the first sequence comprising a first promoter capable of expressing the gRNA in a eukaryotic cell and/or the second sequence comprising a second promoter capable of expressing the gRNA in a eukaryotic cell. In some embodiments, the first promoter and the second promoter are identical. In some embodiments, the first promoter and the second promoter are not identical.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response, and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the first sequence and second sequence comprising a promoter capable of expressing the first gRNA and the second gRNA in a eukaryotic cell.

In some embodiments of the compositions of the disclosure, including those wherein a gRNA sequence comprises a promoter capable of expressing the gRNA in a eukaryotic cell, 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, including those wherein a gRNA sequence comprises a promoter capable of expressing the gRNA in a eukaryotic cell, the promoter is a constitutively active promoter.

In some embodiments of the compositions of the disclosure, including those wherein a gRNA sequence comprises a promoter capable of expressing the gRNA in a eukaryotic cell, the gRNA sequence comprises a sequence 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 of the compositions of the disclosure, including those wherein a gRNA sequence comprises a promoter capable of expressing the gRNA in a eukaryotic cell, the promoter comprises a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the promoter sequence 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 sequence is isolated or derived from a valine tRNA promoter.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the sequence comprising the first gRNA further comprises a first spacer sequence that specifically binds to the first target RNA sequence. In some embodiments, the first 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 first target RNA sequence. In some embodiments, the first spacer sequence has 100% complementarity to the target RNA sequence. In some embodiments, the first spacer sequence comprises or consists of 20 nucleotides. In some embodiments, the first spacer sequence comprises or consists of 21 nucleotides. In some embodiments, the first spacer sequence comprises or consists of 20 nucleotides of an amino acid sequence encoding a Beta-2-microglobulin (β2M) protein. In some embodiments, the first spacer sequence comprises or consists of 20 nucleotides of an amino acid sequence of

(SEQ ID NO: 88)
MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPADIEVD
LLKNGERIEK VEHSDLSFSK DWSFYLLYYT EFTPTEKDEY
ACRVNHVTLS QPKIVKWDRD M.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the sequence comprising the first gRNA further comprises a first scaffold sequence that specifically binds to the first RNA binding protein. In some embodiments, the first 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 the sequence

(SEQ ID NO: 12)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC
CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
or
(SEQ ID NO: 13)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the sequence comprising the second gRNA further comprises a second spacer sequence that specifically binds to the second target RNA sequence. In some embodiments, the second 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 first target RNA sequence. In some embodiments, the second spacer sequence has 100% complementarity to the target RNA sequence. In some embodiments, the second spacer sequence comprises or consists of 20 nucleotides. In some embodiments, the second spacer sequence comprises or consists of 21 nucleotides. In some embodiments, the second spacer sequence comprises or further comprises 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the sequence comprising the second gRNA further comprises a second scaffold sequence that specifically binds to the first RNA binding protein. In some embodiments, the second scaffold sequence comprises a stem-loop structure. In some embodiments, the scaffold sequence comprises or consists of 85 nucleotides. In some embodiments, the scaffold sequence comprises the sequence

(SEQ ID NO: 12)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
or
(SEQ ID NO: 13)
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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the first gRNA does not bind or does not selectively bind to a second sequence within the first RNA molecule.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second gRNA does not bind or does not selectively bind to a second sequence within the second 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, an RNA genome or an RNA transcriptome comprises the first RNA molecule or the second RNA molecule.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 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 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 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 wherein 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, an RNA genome or an RNA transcriptome comprises the RNA molecule.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the sequence encoding the first RNA binding protein further comprises a sequence encoding a nuclear localization signal (NLS). 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a nuclease domain. In some embodiments, 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. 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. 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. 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. 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 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a NOB1 polypeptide. In some embodiments, the NOB1 polypeptide comprises or consists of SEQ ID NO: 31.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 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 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 comprises or consists of SEQ ID NO: 34.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a Human flap endonuclease-1 (hFEN1). In some embodiments, the hFEN1 comprises or consists of SEQ ID NO: 35.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a human Schlafen 14 (hSLFN14) polypeptide. In some embodiments, the hSLFN14 comprises or consists of SEQ ID NO: 36.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a human beta-lactamase-like protein 2 (hLACTB2) polypeptide. In some embodiments, the hLACTB2 comprises or consists of SEQ ID NO: 37.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of an apurinic/apyrimidinic (AP) endodeoxyribonuclease (APEX2) polypeptide. In some embodiments, the APEX2 comprises or consists of SEQ ID NO: 38. In some embodiments, the APEX2 comprises or consists of SEQ ID NO: 39.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of an angiogenin (ANG) polypeptide. In some embodiments, the ANG comprises or consists of SEQ ID NO: 40.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a heat responsive protein 12 (HRSP12) polypeptide. In some embodiments, the HRSP12 comprises or consists of SEQ ID NO: 41.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a Zinc Finger CCCH-Type Containing 12A (ZC3H12A). In some embodiments, the ZC3H12A comprises or consists of SEQ ID NO: 42. In some embodiments, the ZC3H12A comprises or consists of SEQ ID NO: 43.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of an argonaute 2 (AGO2) polypeptide. In some embodiments, encoding the AGO2 polypeptide comprises or consists of SEQ ID NO: 128.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of an hCG_2002731polypeptide. In some embodiments, the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 135. In some embodiments, the sequence encoding the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 136.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule 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.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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).

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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 of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a Teneurin Transmembrane Protein 1 (TENM1) polypeptide. In some embodiments, the TENM1 polypeptide comprises or consists of SEQ ID NO: 144.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a Teneurin Transmembrane Protein 1 (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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of a Ribonuclease Kappa (RNAseK) polypeptide. In some embodiments, the RNAseK protein comprises or consists of SEQ ID NO: 204.

In some embodiments of the compositions of the disclosure, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, 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, including those wherein the composition comprises a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule, the second RNA binding protein comprises or consists of 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 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, the sequence encoding 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, the sequence encoding a nuclease comprises a sequence isolated or derived from a TALEN or a nuclease domain thereof. In some embodiments, the sequence encoding a nuclease comprises a sequence isolated or derived from a zinc finger nuclease or a nuclease domain thereof. In some embodiments, the target sequence comprises a sequence encoding a component of an adaptive immune response.

The disclosure provides a vector comprising a composition of the disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector comprises a sequence isolated or derived from a lentivirus, an adenovirus, an adeno-associated virus (AAV) vector, or a retrovirus. In some embodiments, the vector is replication incompetent.

The disclosure provides a vector comprising a composition of the disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector comprises a sequence isolated or derived from an adeno-associated vector (AAV). In some embodiments, the adeno-associated virus (AAV) is an isolated AAV. In some embodiments, the adeno-associated virus (AAV) is a self-complementary adeno-associated virus (scAAV). In some embodiments, the adeno-associated virus (AAV) is a recombinant adeno-associated virus (rAAV). In some embodiments, the adeno-associated virus (AAV) comprises a sequence 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 adeno-associated virus (AAV) comprises a sequence isolated or derived from an AAV of serotype AAV9. In some embodiments, the adeno-associated virus (AAV) comprise a sequence isolated or derived from Anc80.

The disclosure provides a vector comprising a composition of the disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retrovirus.

The disclosure provides a vector comprising a composition of the disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentivirus.

The disclosure provides a vector comprising a composition of the disclosure. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector comprises a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.

The disclosure provides a composition comprising a vector of the disclosure.

The disclosure provides a cell comprising a vector of the disclosure.

The disclosure provides a cell comprising a cell of the disclosure.

In some embodiments of cells of the disclosure, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

In some embodiments of cells of the disclosure, the cell is an immune cell. In some embodiments, the immune cell is a T lymphocyte (T-cell). In some embodiments, the T-cell is an effector T-cell, a helper T-cell, a memory T-cell, a regulatory T-cell, a natural Killer T-cell, a mucosal-associated invariant T-cell, or a gamma delta T cell.

In some embodiments of cells of the disclosure, the cell is an immune cell. In some embodiments, the immune cell is an antigen-presenting cell. In some embodiments, the antigen-presenting cell is a dendritic cell, a macrophage, or a B cell. In some embodiments, the antigen-presenting cell is a somatic cell.

In some embodiments of cells of the disclosure, the cell is an immune cell. In some embodiments, the cell is a healthy cell. In some embodiments, the cell is not a healthy cell. In some embodiments, the cell is isolated or derived from a subject having a disease or disorder.

The disclosure provides a composition comprising a cell of the disclosure.

The disclosure provides a composition comprising a plurality of cells of the disclosure.

The disclosure provides a method of masking a cell from an adaptive immune response comprising contacting a composition of the disclosure to the cell to produce a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the RNA molecule encodes a component of an adaptive immune response. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the cell is in vitro or ex vivo. In some embodiments, a plurality of cells comprises the cell. In some embodiments, each cell of the plurality of cells contacts the composition, thereby producing a plurality of modified cells. In some embodiments, the method further comprises administering the modified cell to a subject. In some embodiments, the method further comprises administering the plurality of modified cells to a subject. In some embodiments, the cell is autologous. In some embodiments, the cell is allogeneic. In some embodiments, the plurality of modified cells is autologous. In some embodiments, the plurality of modified cells is allogeneic. In some embodiments, the component of an adaptive immune response comprises or consists of a component of a type I major histocompatibility complex (MHC I), a type II major histocompatibility complex (MEW II), a T-cell receptor (TCR), a costimulatory molecule or a combination thereof. In some embodiments, the MHC I component comprises an α1 chain, an α2 chain, an α3 chain, or a β2M protein. In some embodiments, the component of an adaptive immune response comprises or consists of an MHC I β2M protein. In some embodiments, the MEW II component comprises an α1 chain, an α2 chain, a β1 chain, or a β2 chain. In some embodiments, the TCR component comprises an α-chain and a β-chain. In some embodiments, the costimulatory molecule comprises a Cluster of Differentiation 28 (CD28), a Cluster of Differentiation 80 (CD80), a Cluster of Differentiation 86 (CD86), an Inducible T-cell COStimulator (ICOS), or an ICOS Ligand (ICOSLG) protein. In some embodiments, a protein component of an adaptive immune response is, without limitation, Beta-2-microglobulin (β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), or CC Chemokine Receptor 7 (CCR7).

The disclosure provides a method of preventing or reducing an adaptive immune response in a subject comprising administering a therapeutically effective amount of a composition of the disclosure to the subject, wherein the composition contacts at least one cell in the subject producing a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the RNA molecule encodes a component of an adaptive immune response.

The disclosure provides a method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of a composition of the disclosure to the subject, wherein the composition contacts at least one cell in the subject producing a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the composition prevents or reduces an adaptive immune response to the modified cell.

In some embodiments of the methods of the disclosure, the component of an adaptive immune response comprises or consists of a component of a type I major histocompatibility complex (MHC I), a type II major histocompatibility complex (MHC II), a T-cell receptor (TCR), a costimulatory molecule or a combination thereof. In some embodiments, the MHC I component comprises an α1 chain, an α2 chain, an α3 chain, or a β2M protein. In some embodiments, the component of an adaptive immune response comprises or consists of an MHC I β2M protein. In some embodiments, the MHC II component comprises an al chain, an α2 chain, a β1 chain, or a β2 chain. In some embodiments, the TCR component comprises an α-chain and a β-chain. In some embodiments, the costimulatory molecule comprises a Cluster of Differentiation 28 (CD28), a Cluster of Differentiation 80 (CD80), a Cluster of Differentiation 86 (CD86), an Inducible T-cell COStimulator (ICOS), or an ICOS Ligand (ICOSLG) protein.

In some embodiments of the methods of treating a disease or disorder of the disclosure, the disease or disorder is a genetic disease or disorder. In some embodiments, the disease or disorder is a single gene genetic disease or disorder. In some embodiments, the disease or disorder results from microsatellite instability. In some embodiments, the microsatellite instability occurs in a DNA sequence at least 1, 2, 3, 4, 5 or 6 repeated motifs. In some embodiments, an RNA molecule comprises a transcript of the DNA sequence and wherein the composition binds to a target sequence of the RNA molecule comprising at least 1, 2, 3, 4, 5, or 6 repeated motifs.

In some embodiments of the methods of the disclosure, the composition is administered systemically. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered by an injection or an infusion.

In some embodiments of the methods of the disclosure, the composition is administered locally. In some embodiments, the composition is administered by an intraosseous, intraocular, intracerebral, or intraspinal route. In some embodiments, the composition is administered by an injection or an infusion.

In some embodiments of the methods of the disclosure, a therapeutically effective amount of the composition is a single dose.

In some embodiments of the methods of the disclosure, the composition is non-genome integrating.

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.

FIG. 1A is a schematic diagram depicting an exemplary RNA Endonuclease-C. jejuni Cas9 fusion protein.

FIG. 1B 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 8. Zika NS5 expression is displayed as fold change relative to the endonuclease loaded with an sgRNA containing a control (Lambda) spacer sequence.

FIG. 2A is a fluorescence microscopy image of cells transfected with CjeCas9-endonuclease fusions loaded with an sgRNA containing a Zika NS5-targeting spacer sequence.

FIG. 2B 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 CjeCas9-endonuclease fusions loaded with a non-Zika NS5 targeting sgRNA.

FIG. 3 is a list of exemplary endonucleases for use in the compositions of the disclosure.

FIG. 4 is a schematic diagram depicting a construct encoding an exemplary RNA Endonuclease-C. jejuni Cas9 fusion protein and two gRNA molecules for modulating immune response in the context of a gene therapy. The present invention describes a means to address human disease using a CRISPR-based gene therapy or other non-self protein encoded in AAV while simultaneously altering host gene expression to prevent adaptive immune response to the non-self protein. In one embodiment, the AAV particle (left) carries a pair of guide RNAs and a CRISPR-associated (Cas) protein. The guides target a gene associated with adaptive immune response and a gene (or gene product) to promote therapeutic benefit, respectively. Upon delivery to target tissue, the immune response-targeted guide reduces expression of genes associated with antigen presentation (beta-2-microglobulin, B2M) or co-stimulation of T cells (ICOSLG, CD80, CD86, OX40L, IL12, CCR7). Antigen presentation inhibition prevents formation of T helper (Th) cells specific to the therapeutic transgenes such as Cas proteins while co-stimulation inhibition prevents the activation of Th cells that are specific to the transgene.

DETAILED DESCRIPTION

The disclosure provides compositions and methods for the simultaneous treatment of disease by targeting RNA molecules of a modified cell while masking the modified cell from an adaptive immune response. By inhibiting or reducing expression of a component of an adaptive immune response in the modified cell, the modified cell is invisible to a host immune system. For example, compositions of the disclosure may simultaneously target an RNA molecule associated with a genetic disease or disorder and an RNA molecule that encodes the β2M subunit of the MHC I. By selectively targeting an RNA molecule that encodes the β2M subunit of the MHC I, the composition prevents the modified cell from displaying one or more antigen peptides derived from an RNA targeting construct, vector, or combination thereof on the surface of the modified cell. Consequently, a subject's immune system does not identify the modified cell as containing foreign sequences and does not attempt to mount an immune response directed at the modified cell. This method increases the therapeutic efficacy of the treatment of the genetic disease or disorder while avoiding a common side effect of gene therapy.

RNA-Targeting Fusion Protein Compositions

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.

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 gRNA sequence comprises 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 sequence is isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the promoter sequence 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 sequence 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: 84)
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 AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC U (SEQ ID NO: 83). 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)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC
CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
or
(SEQ ID NO: 13)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

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 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. 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: 208)
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

(SEQ ID NO: 209)
1   MEMNMDMDMD MELASIVSSL SALSHSNNNG GQAAAAGIVN GGAAGSQQIG GFRRSSFTTA
61  NEVDSEILLL HGSSESSPIF KKTALSVGTA PPFSTNSKKF FGNGGNYYQY RSTDTASLSS
121 ASYNNYHTHH TAANLGKNNK VNHLLGQYSA SIAGPVYYNG NDNNNSGGEG FFEKFGKSLI
181 DGTRELESQD RPDAVNTQSQ FISKSVSNAS 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 PYIYYPTPNP IPVKMPKDEK TFKKRNNKNH PANNSNNANK QANPYLENSI PTKNTSKKNA
481 SSKSNESTAN NHKSHSHSHP HSQSLQQQQQ TYHRSPLLEQ LRNSSSDKNS NSNMSLKDIF
541 GHSLEFCKDQ HGSRFIQREL ATSPASEKEV IFNEIRDDAI ELSNDVFGNY VIQKFFEFGS
601 KIQKNTLVDQ FKGNMKQLSL QMYACRVIQK ALEYIDSNQR IELVLELSDS VLQMIKDQNG
661 NHVIQKAIET IPIEKLPFIL SSLTGHIYHL STHSYGCRVI QRLLEFGSSE DQESILNELK
721 DFIPYLIQDQ YGNYVIQYVL QQDQFTNKEM VDIKQEIIET VANNVVEYSK HKFASNVVEK
781 SILYGSKNQK DLIISKILPR DKNHALNLED DSPMILMIKD QFANYVIQKL VNVSEGEGKK
841 LIVIAIRAYL DKLNKSNSLG NRHLASVEKL AALVENAEV.

In some embodiments, a PUF4 protein of the disclosure comprises or consists of the amino acid sequence of

(SEQ ID NO: 210)
1   MSTKGLKEEI DDVPSVDPVV SETVNSALFQ 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

(SEQ ID NO: 211)
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 SGKKIIESLQ KLNVPMTMTN EPMPYWAMPT PLMDISAHFM
541 NKLNFQKNSV FDE.

In some embodiments, a PUF6 protein of the disclosure comprises or consists of the amino acid sequence of

(SEQ ID NO: 212)
1   MTPNRRSTDS YNMLGASFDF DPDFSLLSNK THKNKNPKPP VKLLPYRHGS NTTSSDLDNY
61  IFNSGSGSSD DETPPPAAPI FISLEEVLLN GLLIDFAIDP SGVKFLEANY PLDSEDQIRK
121 AVFEKLTEST TLFVGLCHSR NGNFIVQKLV ELATPAEQRE LLRQMIDGGL LVMCKDKFAC
181 RVVQLALQKF DHSNVFQLIQ ELSTFDLAAM CTDQISIHVI QRVVKQLPVD MWTFFVHFLS
241 SGDSLMAVCQ DKYGCRLVQQ VIDRLAENTK LPCFKFRIQL LHSLMTCIVR NCYRLSSNEF
301 ANYVIQYVIK SSGIMEMYRD TIIDKCLLRN LLSMSQDKYA SHVIEGAFLF APPALLHEMM
361 EEIFSGYVKD VELNRDALDI LLFHQYGNYV VQQMISICTA ALIGKEERQL PPAILLLYSG
421 WYEKMKQRVL QHASRLERFS SGKKIIDSVM RHGVPTAAAI NAQAAPSLME LTAQFDAMFP
481 SFLAR.

In some embodiments, a PUF7 protein of the disclosure comprises or consists of the amino acid sequence of

(SEQ ID NO: 213)
1   MTPNRRSTDS YNMLGASFDF DPDFSLLSNK THKNKNPKPP VKLLPYRHGS 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 LPCFKFRIQL 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.

In some embodiments, a PUF8 protein of the disclosure comprises or consists of the amino acid sequence of

(SEQ ID NO: 214)
1   MSRPISIGNT CTFDPSASPI ESLGRSIGAQ KIVDSVCGSP IRSYGRHIST NPKNERLPDT
61  PEFQFATYMH QGGKVIGQNT LHMFGTPPSC YCAQENIPIS SNVGHVLSTI NNNYMNHQYN
121 GSNMFSNQMT QMLQAQAYND LQMHQAHSQS IRVPVQPSAT GIFSNPYREP TTTDDLLTRY
181 RANPAMMKNL KLSDIRGALL KFAKDQVGSR FIQQELASSK DRFEKDSIFD EVVSNADELV
241 DDIFGNYVVQ KFFEYGEERH WARLVDAIID RVPEYAFQMY ACRVLQKALE KINEPLQIKI
301 LSQIRHVIHR CMKDQNGNHV VQKAIEKVSP QYVQFIVDTL LESSNTIYEM SVDPYGCRVV
361 QRCLEHCSPS QTKPVIGQIH KRFDEIANNQ YGNYVVQHVI EHGSEEDRMV IVTRVSNNLF
421 EFATHKYSSN VIEKCLEQGA VYHKSMIVGA ACHHQEGSVP IVVQMMKDQY ANYVVQKMFD
481 QVTSEQRREL ILTVRPHIPV LRQFPHGKHI LAKLEKYFQK PAVMSYPYQD MQGSH.

In some embodiments, a PUF9 protein of the disclosure comprises or consists of the amino acid sequence of

(SEQ ID NO: 215)
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, 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 nuclear localization signal (NLS). 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 polypeptide. In some embodiments, the RNAse1 polypeptide comprises or consists of: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGLCKPVNTFVHEPLVDVQNV CFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYV PVHFDASVEDST (SEQ ID NO: 20). In some embodiments, the second RNA binding protein comprises or consists of an RNAse4 polypeptide. In some embodiments, the RNAse4 polypeptide comprises or consists of: QDGMYQRFLRQHVHPEETGGSDRYCDLMMQRRKMTLYHCKRFNTFIHEDIWNIRSIC S TTNIQCKNGKMNCHEGVVKVTDCRDTGS SRAPNCRYRAIASTRRVVIACEGNPQVPVH FDG (SEQ ID NO: 21). In some embodiments, the second RNA binding protein comprises or consists of an RNAse6 polypeptide. In some embodiments, the RNAse6 polypeptide comprises or consists of: WPKRLTKAHWFEIQHIQPSPLQCNRAMSGINNYTQHCKHQNTFLHDSFQNVAAVCDLL SIVCKNRRHNCHQSSKPVNMTDCRLTSGKYPQCRYSAAAQYKFFIVACDPPQKSDPPYK LVPVHLDSIL (SEQ ID NO: 22). In some embodiments, the second RNA binding protein comprises or consists of an RNAse7 polypeptide. In some embodiments, the RNAse7 polypeptide comprises or consists of: APARAGFCPLLLLLLLGLWVAEIPVSAKPKGMTSSQWFKIQHMQPSPQACNSAMKNINK HTKRCKDLNTFLHEPFSSVAATCQTPKIACKNGDKNCHQSHGPVSLTMCKLTSGKYPNC RYKEKRQNKSYVVACKPPQKKDSQQFHLVPVHLDRVL (SEQ ID NO: 23). In some embodiments, the second RNA binding protein comprises or consists of an RNAse8 polypeptide. In some embodiments, the RNAse8 polypeptide comprises or consists of: TSSQWFKTQHVQPSPQACNSAMSIINKYTERCKDLNTFLHEPFSSVAITCQTPNIACKNSC KNCHQSHGPMSLTMGELTSGKYPNCRYKEKHLNTPYIVACDPPQQGDPGYPLVPVHLD KVV (SEQ ID NO: 24). In some embodiments, the second RNA binding protein comprises or consists of an RNAse2 polypeptide. In some embodiments, the RNAse2 polypeptide comprises or consists of: KPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGN PNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQ RRDPPQYPVVPVHLDRII (SEQ ID NO: 25). In some embodiments, the second RNA binding protein comprises or consists of an RNAse6PL polypeptide. In some embodiments, the RNAse6PL polypeptide comprises or consists of: DKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPF NLEEIKKNWMEITDSSLPSPSMGPAPPRWMRSTPRRSTLAEAWNSTGSWTSTGGCALPP AALPSGDLCCRPSLTAGSRGVGVDLTALHQLLHVHYSATGIIPEECSEPTKPFQIILHHDH TEWVQSIGMPIWGTISSSESAIGKNEESQPACAVLSHDS (SEQ ID NO: 26). In some embodiments, the second RNA binding protein comprises or consists of an RNAseL polypeptide. In some embodiments, the RNAseL polypeptide comprises or consists of: AAVEDNHLLIKAVQNEDVDLVQQLLEGGANVNFQEEEGGWTPLHNAVQMSREDIVEL LLRHGADPVLRKKNGATPFILAAIAGSVKdLLKLFLSKGADVNECDFYGFTAFMEAAVY GKVKALKFLYKRGANVNLRRKTKEDQERLRKGGATALMDAAEKGHVEVLKILLDEM GADVNACDNMGRNALIHALLSSDDSDVEAITHLLLDHGADVNVRGERGKTPLILAVEK KHLGLVQRLLEQEHIEINDTDSDGKTALLLAVELKLKKIAELLCKRGASTDCGDLVMTA RRNYDHSLVKVLLSHGAKEDFHPPAEDWKPQSSHWGAALKDLHRIYRPMIGKLKFFID EKYKIADTSEGGIYLGEYEKQEVAVKTFCEGSPRAQREVSCLQSSRENSHLVTFYGSESH RGHLEVCVTLCEQTLEACLDVHRGEDVENEEDEFARNVLSSIFKAVQELHLSCGYTHQD LQPQNILIDSKKAAHLADFDKSIKWAGDPQEVKRDLEDLGRLVLYVVKKGSISFEDLKA QSNEEVVQLSPDEETKDLIHRLFHPGEHVRDCLSDLLGHPFFWTWESRYRTLRNVGNES DIKTRKSESEILRLLQPGPSEHSKSFDKWTTKINECVMKKMNKFYEKRGNFYQNTVGDL LKFIRNLGEHIDEEKHKKMKLKIGDPSLYFQKTFPDLVIYVYTKLQNTEYRKHFPQTHSP NKPQCDGAGGASGLASPGC (SEQ ID NO: 27). In some embodiments, the second RNA binding protein comprises or consists of an RNAseT2 polypeptide. In some embodiments, the RNAseT2 polypeptide comprises or consists of: VQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDLLPEMRAYW PDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGI KPSINYYQVADFKDALARVYGVIPKIQCLPPSQDEEVQTIGQIELCLTKQDQQLQNCTEP GEQPSPKQEVWLANGAAESRGLRVCEDGPVFYPPPKKTKH (SEQ ID NO: 28). In some embodiments, the second RNA binding protein comprises or consists of an RNAse11 polypeptide. In some embodiments the RNAse11 polypeptide comprises or consists of: EASESTMKIIKEEFTDEEMQYDMAKSGQEKQTIEILMNPILLVKNTSLSMSKDDMSSTLL TFRSLHYNDPKGNSSGNDKECCNDMTVWRKVSEANGSCKWSNNFIRSSTEVMRRVHR APSCKFVQNPGISCCESLELENTVCQFTTGKQFPRCQYHSVTSLEKILTVLTGHSLMSWL VCGSKL (SEQ ID NO: 29). In some embodiments, the second RNA binding protein comprises or consists of an RNAseT2-like polypeptide. In some embodiments, the RNAseT2-like polypeptidec omprises 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: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEPLVDVQNV CFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYV PVHFDASVEDST (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)) comprises or consists of: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEPLVDVQNV CFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYV PVHFEASVEDST (SEQ ID NO: 117). 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: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCRPVNTFVHEPLVDVQNV CFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYV PVNFEASVEDST (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 of: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNV CFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYV PVNFDASVEDST (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: 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: KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEPLVDVQNV CFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTSPKERHIIVACEGSPYV PVNFEASVEDST (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)
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 comprises or consists of:

(SEQ ID NO: 225)
KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGDCRPVNTFVHEP
LVDVQNVCFQEKVTCKDGQGNCYKSNSSMHITDCRLTADSDYPNCAYRTS
PKERHIIVACEGSPYVPVNFEASVEDST.

In some embodiments of the compositions of the disclosure, the second RNA binding protein comprises or consists of a NOB1 polypeptide. In some embodiments, the NOB1 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 polypeptide comprises or consists of: AFSGLQRVGGVDVSFVKGDSVRACASLVVLSFPELEVVYEESRMVSLTAPYVSGFLAFR EVPFLLELVQQLREKEPGLMPQVLLVDGNGVLHHRGEGVACHLGVLTDLPCVGVAKKL LQVDGLENNALHKEKIRLLQTRGDSFPLLGDSGTVLGMALRSHDRSTRPLYISVGHRMS LEAAVRLTCCCCRFRIPEPVRQADICSREHIRKS (SEQ ID NO: 32). In some embodiments, the second RNA binding protein comprises or consists of an endonuclease G (ENDOG) polypeptide. In some embodiments, the ENDOG polypeptide comprises or consists of: AELPPVPGGPRGPGELAKYGLPGLAQLKSRESYVLCYDPRTRGALWVVEQLRPERLRG DGDRRECDFREDDSVHAYHRATNADYRGSGFDRGHLAAAANHRWSQKAMDDTFYLS NVAPQVPHLNQNAWNNLEKYSRSLTRSYQNVYVCTGPLFLPRTEADGKSYVKYQVIGK NHVAVPTHFEKVLILEAAGGQIELRTYVMPNAPVDEAIPLERFLVPIESIERASGLLEVPNI LARAGSLKAITAGSK (SEQ ID NO: 33). In some embodiments, the second RNA binding protein comprises or consists of an endonuclease D1 (ENDOD1) polypeptide. In some embodiments, the ENDOD1 polypeptide comprises or consists of: RLVGEEEAGFGECDKFFYAGTPPAGLAAD SHVKICQRAEGAERFATLYSTRDRIPVYSA FRAPRPAPGGAEQRWLVEPQIDDPNSNLEEAINEAEAITSVNSLGSKQALNTDYLDSDYQ RGQLYPFSLSSDVQVATFTLTNSAPMTQSFQERWYVNLHSLMDRALTPQCGSGEDLYIL TGTVPSDYRVKDKVAVPEFVWLAACCAVPGGGWAMGFVKHTRDSDIIEDVMVKDLQ KLLPFNPQLFQNNCGETEQDTEKMKKILEVVNQIQDEERMVQSQKSSSPLSSTRSKRSTL LPPEASEGSSSFLGKLMGFIATPFIKLFQLIYYLVVAILKNIVYFLWCVTKQVINGIESCLY RLGSATISYFMAIGEELVSIPWKVLKVVAKVIRALLRILCCLLKAICRVLSIPVRVLVDVA TFPVYTMGAIPIVCKDIALGLGGTVSLLFDTAFGTLGGLFQVVFSVCKRIGYKVTFDNSG EL (SEQ ID NO: 34). In some embodiments, the second RNA binding protein comprises or consists of a Human flap endonuclease-1 (hFEN1) polypeptide. In some embodiments, the hFEN1 polypeptide comprises or consists of: MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGET TSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAG AEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAA TEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCE SIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELK WSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKE PEPKGSTKKKAKTGAAGKFKRGK (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 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: MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETKVTRDALTEP LAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLSGLFATQNGDVGCYGNMD EFTQEELRALDSEGRALLTQHKIRTWEGKEKTLTLINVYCPHADPGRPERLVFKMRFYR LLQIRAEALLAAGSHVIILGDLNTAHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQ SASHVGPFIDSYRCFQPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASF LLPEVMGSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQSPVLEQ STLQHNNQTRVQTCQNKAQVRSTRPQPSQVGSSRGQKNLKSYFQPSPSCPQASPDIELPS LPLMSALMTPKTPEEKAVAKVVKGQAKTSEAKDEKELRTSFWKSVLAGPLRTPLCGGH REPCVMRTVKKPGPNLGRRFYMCARPRGPPTDPSSRCNFFLWSRPS (SEQ ID NO: 38). In some embodiments, the APEX2 polypeptide comprises or consists of: MLRVVSWNINGIRRPLQGVANQEPSNCAAVAVGRILDELDADIVCLQETKVTRDALTEP LAIVEGYNSYFSFSRNRSGYSGVATFCKDNATPVAAEEGLSGLFATQNGDVGCYGNMD EFTQEELRALDSEGRALLTQHKIRTWEGKEKTLTLINVYCPHADPGRPERLVFKMRFYR LLQIRAEALLAAGSHVIILGDLNTAHRPIDHWDAVNLECFEEDPGRKWMDSLLSNLGCQ SASHVGPFIDSYRCFQPKQEGAFTCWSAVTGARHLNYGSRLDYVLGDRTLVIDTFQASF LLPEVMGSDHCPVGAVLSVSSVPAKQCPPLCTRFLPEFAGTQLKILRFLVPLEQSP (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)
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: GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVF SCRGILLAVNWFLER GHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIV KLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLD NFLRKKPLTLE (SEQ ID NO: 42). 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 polypeptide comprises 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
RWVLEHISKSKIMGDADRKHCKFKPDPMPPTFSAFNEDYVGSGWSRGHMA
PAGNNKFSSKAMAETFYLSNIVPQDFDNNSGYWNRIEMYCRELTERFEDV
WVVSGPLTLPQTRGDGKKIVSYQVIGEDNVAVPSHLYKVILARRSSVSTE
PLALGAFVVPNEAIGFQPQLTEFQVSLQDLEKLSGLVFFPHLDRTSDIRN
ICSVDTCKLLDFQEFTLYLSTRKIEGARSVLRLEKIMENLKNAEIEPDDY
FMSRYEKKLEELKAKEQSGTQIRKPS.

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 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: KLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTESSTGSVGSN RVRTTLTLCVEAIDFD SQACQLRVKGTNIQENEYVKMGAYHTIELEPNRQFTLAKKQW DSVVLERIEQACDPAWSADVAAVVMQEGLAHICLVTP SMTLTRAKVEVNIPRKRKGNC SQHDRALEREYEQVVQAIQRHIHFDVVKCILVASPGFVREQFCDYMFQQAVKTDNKLLL ENRSKFLQVHASSGHKYSLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRA FYGLKQVEKANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTVRIFSSLHV SGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED (SEQ ID NO: 135). 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: VTVSQMTSVLNGKTRRFADIQLQHGALCFNIRYGTTVEEEKNHVLEIARQRAVAQAWT KEQRRLQEGEEGIRAWTEGEKQQLLSTGRVQGYDGYFVLSVEQYLELSDSANNIHFMR QSEIGRR (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)
TVSQPTLLVNGKTRRFTNIEFQYSTLLLSIRYGLTPDTLDEEKARVLDQA
RQRALGTAWAKEQQKARDGREGSRLWTEGEKQQLLSTGRVQGYEGYYVLP
VEQYPELADSSSNIQFLRQNEMGKR.

In some embodiments of the compositions of the disclosure, the second RNA binding protein comprises or consists of 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 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 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.

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 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, the spacer sequence and the scaffolding sequence are not contiguous. In some embodiments, a sequence encoding a 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 scaffold 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 not comprise a nuclear localization sequence (NLS).

In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof 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, a guide RNA sequence of the disclosure comprises a promoter to drive expression of the guide RNA. In some embodiments, a vector comprising a guide RNA sequence of the disclosure comprises a promoter to drive expression of the guide RNA. In some embodiments, the promoter is a constitutive promoter. In some embodiments, a promoter is a tissue-specific and/or cell-type specific promoter. In some embodiments, a promoter is an inducible promoter. In some embodiments, a promoter is a hybrid or a recombinant promoter. In some embodiments, a promoter is a promoter capable of driving expression in a mammalian cell. In some embodiments, a promoter is a promoter capable of expression in a human cell. In some embodiments, a promoter is a promoter capable of expressing the guide RNA sequence and restricting the expression to the nucleus of the cell. In some embodiments, a promoter is a human RNA polymerase promoter or a promoter sequence isolated or derived from a a human RNA polymerase promoter. In some embodiments, a promoter is a U6 promoter or a sequence isolated or derived from a sequence encoding a U6 promoter. In some embodiments, a promoter is a human tRNA promoter or a promoter sequence isolated or derived from a sequence a human tRNA promoter. In some embodiments, a promoter is a human valine tRNA promoter or a promoter sequence isolated or derived from a human valine tRNA promoter.

In some embodiments of the compositions of the disclosure, a promoter further comprises a regulatory element. In some embodiments, a vector comprising a promoter which 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 guide RNA sequence, a promoter to drive expression of the guide RNA and a regulatory element to enhance expression of the guide RNA. In some embodiments of the compositions of the disclosure, the vector further comprises a nucleic acid 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 a 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 T denticola Cas 9 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
KGGVKLDLQRAHCAVPKNKTHECNWREVDLISLLKKYQMKRYPTSYTGT
PR

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
QSAPDITFPLPVSAENHREYYVITNEQNEVIRLFPKQGETPRIEKGELLL
TGEVERKVFRCKGMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAPKPISAD
GQIVLKGRIEKGVFVCNQLKQKLKTGLPDGSYWISLPVISQTFKEGESVN
NSKLTSQQVQLFGRVREGIFRCHNYQCPASGADGNFWCTLDTDTAQPAFT
PIKNAPPGVGGGQIILTGDVDDKGIFHADDDLHYELPASLPKGKYYGIFT
VESCDPTLIPIELSAPKTSKGENLIEGNIWVDEHTGEVRFDPKKNREDQR
HHAIDAIVIALSSQSLFQRLSTYNARRENKKRGLDSTEHFPSPWPGFAQD
VRQSVVPLLVSYKQNPKTLCKISKTLYKDGKKIHSCGNAVRGQLHKETVY
GQRTAPGATEKSYHIRKDIRELKTSKHIGKVVDITIRQMLLKHLQENYHI
DITQEFNIPSNAFFKEGVYRIFLPNKHGEPVPIKKIRMKEELGNAERLKD
NINQYVNPRNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPRE
GRNIVSILQINDTFLIGLKEEEPEVYRNDLSTLSKHLYRVQKLSGMYYTF
RHHLASTLNNEREEFRIQSLEAWKRANPVKVQIDEIGRITFLNGPLC.

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 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: 179)
		NK4A179
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: 181)
		4847
Cas13a8	Cga2Cas13a	Carnobacterium	WP_034563842.1	AATATAAACTACCTCTAAATGTAAG
		gallinarum DSM		AGGACTATAAC (SEQ ID NO: 182)
		4847
Cas13a9	Pprcas13a	Paludibacter	WP_013443710.1	CTTGTGGATTATCCCAAAATTGAAG
		propionicigenes		GGAACTACAAC (SEQ ID NO: 183)
		WB4
Cas13a10	LweCas13a	Listeria	WP_036059185.1	GATTTAGAGTACCTCAAAATAGAAG
		weihenstephanensis		AGGTCTAAAAC (SEQ ID NO: 184)
		FSL R9-0317
Cas13a11	LbfCas13a	Listeriaceae	WP_036091002.1	GATTTAGAGTACCTCAAAACAAAAG
		bacterium FSL		AGGACTAAAAC (SEQ ID NO: 185)
		M6-0635
		(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: 189)
		DE442

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:

Species	Cas13b Accession	Cas13b 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	EHO06562.1	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 = DSM	WP_004919755.1	1096
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 = DSM 20706	WP_036860899.1	1127
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 15124	WP_036884929.1	1134
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	EOA10535.1	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 iekektyaey faevfkkeke alik.

In some embodiments of the compositions of the disclosure, the sequence encoding the first RNA binding protein, or RNA-guided target 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.

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_contig13552000311:
(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_metagenome_contig2643000492:
(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|OBXZ01000094.1|and
emb|OBJF01000033.1|):
(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|DJXD01000002.1| (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|DJXD01000002.1| (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|OGDF01008514.1|(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|OBLI01038679 (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. 30

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.molce1.2018.02.2018) 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. W02018/183703 (CasM) and W02019/006471 (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):
(SEQ ID NO: 47)
GTGAGAAGTCTCCTTATGGGGAGATGCTAC.

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 |DJXDO1000002.1|; 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):
(SEQ ID NO: 51).
CAACTACAACCCCGTAAAAATACGGGGTTCTGAAAC

In some embodiments of the disclosure, a CjeCas9-endonuclease fusions and gRNA molecule may comprise or consist of the nucleic acid sequence of:

E43-CjeCas9 and sgRNA plasmid
(U6: N's = sgRNA spacer, E43, CjeCas9)
(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
GAGAGGACTTCAAAAAATCAGGTCCA              CCCAAGAAAAAACGCAAGGTGGAA
GATCCGAAGAAAAAGCGAAAAGTGGATGTGtaaCGTTTTCCGGGACGCCG
GCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCAC
CCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT
CACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT
TGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCG.

In some embodiments of the disclosure, a CjeCas9-endonuclease fusions and gRNA molecule may comprise or consist of the nucleic acid sequence of:

E67-CjeCas9 and sgRNA plasmid
(U6: N's = sgRNA spacer, E67, CjeCas9)
(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
GTCCA              CCCAAGAAAAAACGCAAGGTGGAAGATCCGAAGAAAAAGCGAAAA
GTGGATGTGtaaCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCG
GGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCT
TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGC
ATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTAT
CTTATCATGTCTGTATACCG.

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-nRNA) 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.

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 Pumilio1, 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, 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 sequence encoding 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, the sequence encoding a nuclear localization signal (NLS) is positioned 3′ to the sequence encoding 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 sequence encoding 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 sequence encoding the first NLS or the second NLS is positioned 3′ to the sequence encoding 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 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, 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.

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 (e.g., spacer sequence) that hybridizes to or specifically binds to a target RNA sequence; and on its 3′ end, an RNA sequence (e.g., scaffold sequence) capable of specifically 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 comprised within a single (e.g., unitary) 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, the spacer sequence of the 2-component system targets an RNA sequence involved in an adaptive immune response. In some embodiments, a spacer sequence of the 2-component system comprises a portion of a nucleic acid sequence encoding a protein component of an adaptive immune response, and wherein the protein component is selected from the group consisting of Beta-2-microglobulin β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), and CC Chemokine Receptor 7 (CCR7). In some embodiments, the 2-component system comprises a spacer which is a portion of a nucleic acid sequence encoding a protein component of an adaptive immune response and which is about 20 or 21 nucleotides in length. In some embodiments, the 2-component system comprises a first and second spacer comprised within a singular gRNA. In some embodiments, the 2-component system comprises a first and second spacer sequence comprised within first and second gRNA sequences. In some embodiments, the first spacer targets a repeat sequence and the second spacer targets RNA involved in an adaptive immune response.

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 total of 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, a 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., N.Y.).

“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 lx SSC, 0.1x 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 hematopoetic 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, naive T cells, effector T cells, helper T cells, memory T cells, regulatory T cells (Tregs) and Gamma delta T cells. In some embodiments, an immune cell of the disclosure is a B lymphocyte. In some embodiments, an immune cell of the disclosure is a natural killer cell. In some embodiments, an immune cell of the disclosure is an antigen-presenting cell.

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.

Masking Modified Cells of the Disclosure

Compositions of the disclosure simultaneously deliver a gene therapy and prevent expression of antigens derived from the gene therapy construct or associated delivery vector from display on the surface of a modified cell of the disclosure.

By inhibiting or reducing expression of a component of an adaptive immune response in the modified cell, the modified cell is invisible to a host immune system. For example, compositions of the disclosure may simultaneously target an RNA molecule associated with a genetic disease or disorder and an RNA molecule that encodes the β2M subunit of the MEW I. By selectively targeting an RNA molecule that encodes the β2M subunit of the MHC I, the composition prevents the modified cell from displaying one or more antigen peptides derived from an RNA targeting construct, vector, or combination thereof on the surface of the modified cell. Consequently, a subject's immune system does not identify the modified cell as containing foreign sequences and does not attempt to mount an immune response directed at the modified cell. This method increases the therapeutic efficacy of the treatment of the genetic disease or disorder while avoiding a common side effect of gene therapy.

In some embodiments of the compositions and methods of the disclosure, the component of an adaptive immune response comprises or consists of a component of a type I major histocompatibility complex (MEW I), a type II major histocompatibility complex (MHC II), a T-cell receptor (TCR), a costimulatory molecule or a combination thereof. In some embodiments, the MHC I component comprises an α1 chain, an α2 chain, an α3 chain, or a β2M protein. In some embodiments, the component of an adaptive immune response comprises or consists of an MEW I β2M protein. In some embodiments, the MHC II component comprises an α1 chain, an α2 chain, a α1 chain, or a α2 chain. In some embodiments, the TCR component comprises an α-chain and a β-chain. In some embodiments, the costimulatory molecule comprises a Cluster of Differentiation 28 (CD28), a Cluster of Differentiation 80 (CD80), a Cluster of Differentiation 86 (CD86), an Inducible T-cell COStimulator (ICOS), or an ICOS Ligand (ICOSLG) protein.

An α-chain of an MHC I may be encoded by an HLA gene, including but not limited to, HLA-A, HLA-B and HLA-C.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding an α-chain derived from an HLA-A gene comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 216)
1    atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggctct ggccctgacc
61   cagacctggg cgggctctca ctccatgagg tatttcttca catccgtgtc ccggcccggc
121  cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc
181  gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggt
241  ccggagtatt gggacgggga gacacggaaa gtgaaggccc actcacagac tcaccgagtg
301  gacctgggga ccctgcgcgg ctactacaac cagagcgagg ccggttctca caccgtccag
361  aggatgtgtg gctgcgacgt ggggtcggac tggcgcttcc tccgcgggta ccaccagtac
421  gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg
481  gacatggcag ctcagaccac caagcacaag tgggaggcgg cccatgtggc ggagcagttg
541  agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag
601  gagacgctgc agcgcacgga cgcccccaaa acgcatatga ctcaccacgc tgtctctgac
661  catgaagcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc
721  tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca
781  ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaca ggagcagaga
841  taaacctgcc atgtgcagca tgagggtttg cccaagcccc tcaccctgag atgggagccg
901  tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct
961  gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa
1021 ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc
1081 acagcttgta aagtgtga.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding an α-chain derived from an HLA-B gene comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 217)
1    tggtgtagga gaagagggat caggacgaag tcccaggccc cgggcggggc tctcagggtc
61   tcaggctccg agggccgcgt ctgcaatggg gaggcgcagc gttggggatt ccccactccc
121  acgagtttca cttcttctcc caacctatgt cgggtccttc ttccaggata ctcgtgacgc
181  gtccccattt cccactccca ttgggtgtcg ggtgtctaga gaagccaatc agcgtcgccg
241  tggtcccagt tctaaagtcc ccacgcaccc acccggactc agaatctcct cagacgccga
301  gatgcgggtc acggcacccc gaaccgtcct cctgctgctc tcggcggccc tggccctgac
361  cgagacctgg gccggtgagt gcgggtcggc agggaaatgg cctctgtggg gaggagcgag
421  gggaccgcag gcgggggcgc aggacccggg gagccgcgcc gggaggaggg tcgggcgggt
481  ctcagcccct cctcgccccc aggctcccac tccatgaggt atttccacac cgccatgtcc
541  cggcccggcc gcggggagcc ccgcttcatc accgtgggct acgtggacga cacgctgttc
601  gtgaggttcg acagcgacgc cacgagtccg aggaaggagc cgcgggcgcc atggatagag
661  caggaggggc cggagtattg ggaccgggag acacagatct ccaagaccaa cacacagact
721  taccgagaga gcctgcggaa cctgcgcggc tactacaacc agagcgaggc cggtgagtga
781  ccccggcccg gggcgcaggt cacgactccc catcccccac gtacggcccg ggtcgccccg
841  agtctccggg tccgagatcc gcccccctga ggccgcggga cccgcccaga ccctcgaccg
901  gcgagagccc caggcgcgtt tacccggttt cattttcagt tgaggccaaa atccccgcgg
961  gttggtcggg gcggggcggg gcggggctcg ggggacgggg ctgaccgcgg ggcctgggcc
1021 agggtctcac acttggcaga ggatgtatgg ctgcgacctg gggcccgacg ggcgcctcct
1081 ccgcgggtat aaccagttag cctacgacgg caaggattac atcgccctga acgaggacct
1141 gagctcctgg accgcggcgg acaccgcggc tcagatcacc cagcgcaagt gggaggcggc
1201 ccgtgtggcg gagcaggaca gagcctacct ggagggcctg tgcgtggagt cgctccgcag
1261 atacctggag aacgggaagg agacgctgca gcgcgcgggt accaggggca gtggggagcc
1321 ttccccatct cctataggtc gccggggatg gcctcccacg agaagaggag gaaaatggga
1381 tcagcgctag aatgtcgccc tcccttgaat ggagaatggc atgagttttc ctgagtttcc
1441 tctgagggcc ccctcttctc tctaggacaa taaggaatga cgtctctgag gaaatggagg
1501 ggaagacagt ccctagaata ctgatcaggg gtcccctttg acccctgcag cagccttggg
1561 aaccgtgact ttcctctcag gccttgttct ctgcctcaca ctcagtgtgt ttggggctct
1621 gattccagca cttctgagtc actttacctc cactcagatc gggagcagaa gtccctgttc
1681 cccgctcaga gactcgaact ttccaatgaa taggagatta tcccaggtgc ctgcgtccag
1741 gctggtgtct gggttctgtg ccccttcccc accccaggtg tcctgtccat tctcaggctg
1801 gtcacatggg tggtcctagg gtgtcccatg agagatgcaa agcgcctgaa ttttctgact
1861 cttcccatca gaccccccaa agacacatgt gacccaccac cccatctctg accatgaggc
1921 caccctgagg tgctgggccc tgggcttcta ccctgcggag atcacactga cctggcagcg
1981 ggatggcgag gaccaaactc aggacaccga gcttgtggag accagaccag caggagatag
2041 aaccttccag aagtgggcag ctgtggtggt gccttctgga gaagagcaga gatacacatg
2101 ccatgtacag catgaggggc tgccgaagcc cctcaccctg agatggggta aggaggggga
2161 tgaggggtca tatctgttct cagggaaagc aggagccctt ctggagccct tcagcagggt
2221 cagggcccct catcttcccc tcctttccca gagccatctt cccagtccac catccccatc
2281 gtgggcattg ttgctggcct ggctgtccta gcagttgtgg tcatcggagc tgtggtcgct
2341 actgtgatgt gtaggaggaa gagctcaggt agggaagggg tgaggggtgg ggtctgggtt
2401 ttcttgtccc actgggggtt tcaagcccca ggtagaagtg ttccctgcct cattactggg
2461 aagcagcatc cacacagggg ctaacgcagc ctgggaccct gtgtgccagc acttactctt
2521 ttgtgcagca catgtgacaa tgaaggacgg atgtatcgcc ttgatggttg tggtgttggg
2581 gtcctgattc cagcattcat gagtcagggg aaggtccctg ctaaggacag accttaggag
2641 ggcagttggt ccaggaccca cacttgcttt cctcgtgttt cctgatcctg ccttgggtct
2701 gtagtcatac ttctggaaat tccttttggt tccaagacga ggaggttcct ctaagatctc
2761 atggccctgc ttcctcccag tcccctcaca ggacattttc ttcccacagg tggaaaagga
2821 gggagctact ctcaggctgc gtgtaagtgg tgggggtggg agtgtggagg agctcaccca
2881 ccccataatt cctcctgtcc cacgtctcct gagggctctg accaggtcct gtttttgttc
2941 tactccagcc agcgacagtg cccagggctc tgatgtgtct ctcacagctt gaaaaggtga
3001 gattcttggg gtctagagtg ggtggggtgg cgggtctggg ggtgggtggg gcagtgggga
3061 aaggcctggg taatggagat tctttgattg ggatgtttcg cgtgtgtggt gggctgttca
3121 gagtgtcatc acttaccatg actaaccaga atttgttcat gactgttgtt ttctgtagcc
3181 tgagacagct gtcttgtgag ggactgagat gcaggatttc ttcacgcctc ccctttgtga
3241 cttcaagagc ctctggcatc tctttctgca aaggcacctg aatgtgtctg cgtccctgtt
3301 agcataatgt gaggaggtgg agagacagcc cacccttgtg tccactgtga cccctgttcg
3361 catgctgacc tgtgtttcct cccca.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding an α-chain derived from an HLA-C gene comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 218)
1    tccgcagtcc cggttctaaa gtccccagtc acccacccgg actcacattc tccccagagg
61   ccgagatgcg ggtcatggcg ccccgagccc tcctcctgct gctctcggga ggcctggccc
121  tgaccgagac ctgggcctgc tcccactcca tgaggtattt cgacaccgcc gtgtcccggc
181  ccggccgcgg agagccccgc ttcatctcag tgggctacgt ggacgacacg cagttcgtgc
241  ggttcgacag cgacgccgcg agtccgagag gggagccgcg ggcgccgtgg gtggagcagg
301  aggggccgga gtattgggac cgggagacac agaagtacaa gcgccaggca caggctgacc
361  gagtgagcct gcggaacctg cgcggctact acaaccagag cgaggacggg tctcacaccc
421  tccagaggat gtctggctgc gacctggggc ccgacgggcg cctcctccgc gggtatgacc
481  agtccgccta cgacggcaag gattacatcg ccctgaacga ggacctgcgc tcctggaccg
541  ccgcggacac cgcggctcag atcacccagc gcaagttgga ggcggcccgt gcggcggagc
601  agctgagagc ctacctggag ggcacgtgcg tggagtggct ccgcagatac ctggagaacg
661  ggaaggagac gctgcagcgc gcagaacccc caaagacaca cgtgacccac caccccctct
721  ctgaccatga ggccaccctg aggtgctggg ccctgggctt ctaccctgcg gagatcacac
781  tgacctggca gcgggatggg gaggaccaga cccaggacac cgagcttgtg gagaccaggc
841  cagcaggaga tggaaccttc cagaagtggg cagctgtggt ggtgccttct ggacaagagc
901  agagatacac gtgccatatg cagcacgagg ggctgcaaga gcccctcacc ctgagctggg
961  agccatcttc ccagcccacc atccccatca tgggcatcgt tgctggcctg gctgtcctgg
1021 ttgtcctagc tgtccttgga gctgtggtca ccgctatgat gtgtaggagg aagagctcag
1081 gtggaaaagg agggagctgc tctcaggctg cgtgcagcaa cagtgcccag ggctctgatg
1141 agtctctcat cacttgtaaa gcctgagaca gctgcctgtg tgggactgag atgcaggatt
1201 tcttcacacc tctcctttgt gacttcaaga gcctctggca tctctttctg caaaggcacc
1261 tgaatgtgtc tgcgttcctg ttagcataat gtgaggaggt ggagagacag cccacccccg
1321 tgtccaccgt gacccctgtc cccacactga cctgtgttcc ctccccgatc atctttcctg
1381 ttccagagag gtggggctgg atgtctccat ctctgtctca aattcatggt gcactgagct
1441 gcaacttctt acttccctaa tgaagttaag aacctgaata taaatttgtg ttctcaaata
1501 tttgctatga agcgttgatg gattaattaa ataagtcaat tcctagaagt tgagagagca
1561 aataaagacc tgagaacctt ccagaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding an α-chain derived from an HLA-C gene comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 219)
1    tccgcagtcc cggttctaaa gtccccagtc acccacccgg actcacattc tccccagagg
61   ccgagatgcg ggtcatggcg ccccgagccc tcctcctgct gctctcggga ggcctggccc
121  tgaccgagac ctgggcctgc tcccactcca tgaggtattt cgacaccgcc gtgtcccggc
181  ccggccgcgg agagccccgc ttcatctcag tgggctacgt ggacgacacg cagttcgtgc
241  ggttcgacag cgacgccgcg agtccgagag gggagccgcg ggcgccgtgg gtggagcagg
301  aggggccgga gtattgggac cgggagacac agaactacaa gcgccaggca caggctgacc
361  gagtgagcct gcggaacctg cgcggctact acaaccagag cgaggacggg tctcacaccc
421  tccagaggat gtatggctgc gacctggggc ccgacgggcg cctcctccgc gggtatgacc
481  agtccgccta cgacggcaag gattacatcg ccctgaacga ggacctgcgc tcctggaccg
541  ccgcggacac cgcggctcag atcacccagc gcaagttgga ggcggcccgt gcggcggagc
601  agctgagagc ctacctggag ggcacgtgcg tggagtggct ccgcagatac ctggagaacg
661  ggaaggagac gctgcagcgc gcagaacccc caaagacaca cgtgacccac caccccctct
721  ctgaccatga ggccaccctg aggtgctggg ccctgggctt ctaccctgcg gagatcacac
781  tgacctggca gcgggatggg gaggaccaga cccaggacac cgagcttgtg gagaccaggc
841  cagcaggaga tggaaccttc cagaagtggg cagctgtggt ggtgccttct ggacaagagc
901  agagatacac gtgccatatg cagcacgagg ggctgcaaga gcccctcacc ctgagctggg
961  agccatcttc ccagcccacc atccccatca tgggcatcgt tgctggcctg gctgtcctgg
1021 ttgtcctagc tgtccttgga gctgtggtca ccgctatgat gtgtaggagg aagagctcag
1081 gtggaaaagg agggagctgc tctcaggctg cgtgcagcaa cagtgcccag ggctctgatg
1141 agtctctcat cacttgtaaa gcctgagaca gctgcctgtg tgggactgag atgcaggatt
1201 tcttcacacc tctcctttgt gacttcaaga gcctctggca tctctttctg caaaggcgtc
1261 tgaatgtgtc tgcgttcctg ttagcataat gtgaggaggt ggagagacag cccacccccg
1321 tgtccaccgt gacccctgtc cccacactga cctgtgttcc ctccccgatc atctttcctg
1381 ttccagagag gtggggctgg atgtctccat ctctgtctca aattcatggt gcactgagct
1441 gcaacttctt acttccctaa tgaagttaag aacctgaata taaatttgtg ttctcaaata
1501 tttgctatga agcgttgatg gattaattaa ataagtcaat tcctagaagt tgagagagca
1561 aataaagacc tgagaacctt ccagaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding an β2M protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 220)
1    attcctgaag ctgacagcat tcgggccgag atgtctcgct ccgtggcctt agctgtgctc
61   gcgctactct ctctttctgg cctggaggct atccagcgta ctccaaagat tcaggtttac
121  tcacgtcatc cagcagagaa tggaaagtca aatttcctga attgctatgt gtctgggttt
181  catccatccg acattgaagt tgacttactg aagaatggag agagaattga aaaagtggag
241  cattcagact tgtctttcag caaggactgg tctttctatc tcttgtacta cactgaattc
301  acccccactg aaaaagatga gtatgcctgc cgtgtgaacc atgtgacttt gtcacagccc
361  aagatagtta agtgggatcg agacatgtaa gcagcatcat ggaggtttga agatgccgca
421  tttggattgg atgaattcca aattctgctt gcttgctttt taatattgat atgcttatac
481  acttacactt tatgcacaaa atgtagggtt ataataatgt taacatggac atgatcttct
541  ttataattct actttgagtg ctgtctccat gtttgatgta tctgagcagg ttgctccaca
601  ggtagctcta ggagggctgg caacttagag gtggggagca gagaattctc ttatccaaca
661  tcaacatctt ggtcagattt gaactcttca atctcttgca ctcaaagctt gttaagatag
721  ttaagcgtgc ataagttaac ttccaattta catactctgc ttagaatttg ggggaaaatt
781  tagaaatata attgacagga ttattggaaa tttgttataa tgaatgaaac attttgtcat
841  ataagattca tatttacttc ttatacattt gataaagtaa ggcatggttg tggttaatct
901  ggtttatttt tgttccacaa gttaaataaa tcataaaact tgatgtgtta tctcttatat
961  ctcactccca ctattacccc tttattttca aacagggaaa cagtcttcaa gttccacttg
1021 gtaaaaaatg tgaacccctt gtatatagag tttggctcac agtgtaaagg gcctcagtga
1081 ttcacatttt ccagattagg aatctgatgc tcaaagaagt taaatggcat agttggggtg
1141 acacagctgt ctagtgggag gccagccttc tatattttag ccagcgttct ttcctgcggg
1201 ccaggtcatg aggagtatgc agactctaag agggagcaaa agtatctgaa ggatttaata
1261 ttttagcaag gaatagatat acaatcatcc cttggtctcc ctgggggatt ggtttcagga
1321 ccccttcttg gacaccaaat ctatggatat ttaagtccct tctataaaat ggtatagtat
1381 ttgcatataa cctatccaca tcctcctgta tactttaaat catttctaga ttacttgtaa
1441 tacctaatac aatgtaaatg ctatgcaaat agttgttatt gtttaaggaa taatgacaag
1501 aaaaaaaagt ctgtacatgc tcagtaaaga cacaaccatc cctttttttc cccagtgttt
1561 ttgatccatg gtttgctgaa tccacagatg tggagcccct ggatacggaa ggcccgctgt
1621 actttgaatg acaaataaca gatttaaaat tttcaaggca tagttttata cctga.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD28 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 221)
1    taaagtcatc aaaacaacgt tatatcctgt gtgaaatgct gcagtcagga tgccttgtgg
61   tttgagtgcc ttgatcatgt gccctaaggg gatggtggcg gtggtggtgg ccgtggatga
121  cggagactct caggccttgg caggtgcgtc tttcagttcc cctcacactt cgggttcctc
181  ggggaggagg ggctggaacc ctagcccatc gtcaggacaa agatgctcag gctgctcttg
241  gctctcaact tattcccttc aattcaagta acaggaaaca agattttggt gaagcagtcg
301  cccatgcttg tagcgtacga caatgcggtc aaccttagct gcaagtattc ctacaatctc
361  ttctcaaggg agttccgggc atcccttcac aaaggactgg atagtgctgt ggaagtctgt
421  gttgtatatg ggaattactc ccagcagctt caggtttact caaaaacggg gttcaactgt
481  gatgggaaat tgggcaatga atcagtgaca ttctacctcc agaatttgta tgttaaccaa
541  acagatattt acttctgcaa aattgaagtt atgtatcctc ctccttacct agacaatgag
601  aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt
661  cccggacctt ctaagccctt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat
721  agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc
781  ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac
841  cagccctatg ccccaccacg cgacttcgca gcctatcgct cctgacacgg acgcctatcc
901  agaagccagc cggctggcag cccccatctg ctcaatatca ctgctctgga taggaaatga
961  ccgccatctc cagccggcca cctcaggccc ctgttgggcc accaatgcca atttttctcg
1021 agtgactaga ccaaatatca agatcatttt gagactctga aatgaagtaa aagagatttc
1081 ctgtgacagg ccaagtctta cagtgccatg gcccacattc caacttacca tgtacttagt
1141 gacttgactg agaagttagg gtagaaaaca aaaagggagt ggattctggg agcctcttcc
1201 ctttctcact cacctgcaca tctcagtcaa gcaaagtgtg gtatccacag acattttagt
1261 tgcagaagaa aggctaggaa atcattcctt ttggttaaat gggtgtttaa tcttttggtt
1321 agtgggttaa acggggtaag ttagagtagg gggagggata ggaagacata tttaaaaacc
1381 attaaaacac tgtctcccac tcatgaaatg agccacgtag ttcctattta atgctgtttt
1441 cctttagttt agaaatacat agacattgtc ttttatgaat tctgatcata tttagtcatt
1501 ttgaccaaat gagggatttg gtcaaatgag ggattccctc aaagcaatat caggtaaacc
1561 aagttgcttt cctcactccc tgtcatgaga cttcagtgtt aatgttcaca atatactttc
1621 gaaagaataa aatagttctc ctacatgaag aaagaatatg tcaggaaata aggtcacttt
1681 atgtcaaaat tatttgagta ctatgggacc tggcgcagtg gctcatgctt gtaatcccag
1741 cactttggga ggccgaggtg ggcagatcac ttgagatcag gaccagcctg gtcaagatgg
1801 tgaaactccg tctgtactaa aaatacaaaa tttagcttgg cctggtggca ggcacctgta
1861 atcccagctg cccaagaggc tgaggcatga gaatcgcttg aacctggcag gcggaggttg
1921 cagtgagccg agatagtgcc acagctctcc agcctgggcg acagagtgag actccatctc
1981 aaacaacaac aacaacaaca acaacaacaa caaaccacaa aattatttga gtactgtgaa
2041 ggattatttg tctaacagtt cattccaatc agaccaggta ggagctttcc tgtttcatat
2101 gtttcagggt tgcacagttg gtctctttaa tgtcggtgtg gagatccaaa gtgggttgtg
2161 gaaagagcgt ccataggaga agtgagaata ctgtgaaaaa gggatgttag cattcattag
2221 agtatgagga tgagtcccaa gaaggttctt tggaaggagg acgaatagaa tggagtaatg
2281 aaattcttgc catgtgctga ggagatagcc agcattaggt gacaatcttc cagaagtggt
2341 caggcagaag gtgccctggt gagagctcct ttacagggac tttatgtggt ttagggctca
2401 gagctccaaa actctgggct cagctgctcc tgtaccttgg aggtccattc acatgggaaa
2461 gtattttgga atgtgtcttt tgaagagagc atcagagttc ttaagggact gggtaaggcc
2521 tgaccctgaa atgaccatgg atatttttct acctacagtt tgagtcaact agaatatgcc
2581 tggggacctt gaagaatggc ccttcagtgg ccctcaccat ttgttcatgc ttcagttaat
2641 tcaggtgttg aaggagctta ggttttagag gcacgtagac ttggttcaag tctcgttagt
2701 agttgaatag cctcaggcaa gtcactgccc acctaagatg atggttcttc aactataaaa
2761 tggagataat ggttacaaat gtctcttcct atagtataat ctccataagg gcatggccca
2821 agtctgtctt tgactctgcc tatccctgac atttagtagc atgcccgaca tacaatgtta
2881 gctattggta ttattgccat atagataaat tatgtataaa aattaaactg ggcaatagcc
2941 taagaagggg ggaatattgt aacacaaatt taaacccact acgcagggat gaggtgctat
3001 aatatgagga ccttttaact tccatcattt tcctgtttct tgaaatagtt tatcttgtaa
3061 tgaaatataa ggcacctccc acttttatgt atagaaagag gtcttttaat ttttttttaa
3121 tgtgagaagg aagggaggag taggaatctt gagattccag atcgaaaata ctgtactttg
3181 gttgattttt aagtgggctt ccattccatg gatttaatca gtcccaagaa gatcaaactc
3241 agcagtactt gggtgctgaa gaactgttgg atttaccctg gcacgtgtgc cacttgccag
3301 cttcttgggc acacagagtt cttcaatcca agttatcaga ttgtatttga aaatgacaga
3361 gctggagagt tttttgaaat ggcagtggca aataaataaa tacttttttt taaatggaaa
3421 gacttgatct atggtaataa atgattttgt tttctgactg gaaaaatagg cctactaaag
3481 atgaatcaca cttgagatgt ttcttactca ctctgcacag aaacaaagaa gaaatgttat
3541 acagggaagt ccgttttcac tattagtatg aaccaagaaa tggttcaaaa acagtggtag
3601 gagcaatgct ttcatagttt cagatatggt agttatgaag aaaacaatgt catttgctgc
3661 tattattgta agagtcttat aattaatggt actcctataa tttttgattg tgagctcacc
3721 tatttgggtt aagcatgcca atttaaagag accaagtgta tgtacattat gttctacata
3781 ttcagtgata aaattactaa actactatat gtctgcttta aatttgtact ttaatattgt
3841 cttttggtat taagaaagat atgctttcag aatagatatg cttcgctttg gcaaggaatt
3901 tggatagaac ttgctattta aaagaggtgt ggggtaaatc cttgtataaa tctccagttt
3961 agcctttttt gaaaaagcta gactttcaaa tactaatttc acttcaagca gggtacgttt
4021 ctggtttgtt tgcttgactt cagtcacaat ttcttatcag accaatggct gacctctttg
4081 agatgtcagg ctaggcttac ctatgtgttc tgtgtcatgt gaatgctgag aagtttgaca
4141 gagatccaac ttcagccttg accccatcag tccctcgggt taactaactg agccaccggt
4201 cctcatggct attttaatga gggtattgat ggttaaatgc atgtctgatc ccttatccca
4261 gccatttgca ctgccagctg ggaactatac cagacctgga tactgatccc aaagtgttaa
4321 attcaactac atgctggaga ttagagatgg tgccaataaa ggacccagaa ccaggatctt
4381 gattgctata gacttattaa taatccaggt caaagagagt gacacacact ctctcaagac
4441 ctggggtgag ggagtctgtg ttatctgcaa ggccatttga ggctcagaaa gtctctcttt
4501 cctatagata tatgcatact ttctgacata taggaatgta tcaggaatac tcaaccatca
4561 caggcatgtt cctacctcag ggcctttaca tgtcctgttt actctgtcta gaatgtcctt
4621 ctgtagatga cctggcttgc ctcgtcaccc ttcaggtcct tgctcaagtg tcatcttctc
4681 ccctagttaa actaccccac accctgtctg ctttccttgc ttatttttct ccatagcatt
4741 ttaccatctc ttacattaga catttttctt atttatttgt agtttataag cttcatgagg
4801 caagtaactt tgctttgttt cttgctgtat ctccagtgcc cagagcagtg cctggtatat
4861 aataaatatt tattgactga gtgaaaaaaa aaaaaaaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD28 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 222)
1    taaagtcatc aaaacaacgt tatatcctgt gtgaaatgct gcagtcagga tgccttgtgg
61   tttgagtgcc ttgatcatgt gccctaaggg gatggtggcg gtggtggtgg ccgtggatga
121  cggagactct caggccttgg caggtgcgtc tttcagttcc cctcacactt cgggttcctc
181  ggggaggagg ggctggaacc ctagcccatc gtcaggacaa agatgctcag gctgctcttg
241  gctctcaact tattcccttc aattcaagta acaggaaaca agattttggt gaagcagtcg
301  cccatgcttg tagcgtacga caatgcggtc aaccttagct ggaaacacct ttgtccaagt
361  cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg tggagtcctg
421  gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg
481  agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc
541  aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc ctgacacgga
601  cgcctatcca gaagccagcc ggctggcagc ccccatctgc tcaatatcac tgctctggat
661  aggaaatgac cgccatctcc agccggccac ctcaggcccc tgttgggcca ccaatgccaa
721  tttttctcga gtgactagac caaatatcaa gatcattttg agactctgaa atgaagtaaa
781  agagatttcc tgtgacaggc caagtcttac agtgccatgg cccacattcc aacttaccat
841  gtacttagtg acttgactga gaagttaggg tagaaaacaa aaagggagtg gattctggga
901  gcctcttccc tttctcactc acctgcacat ctcagtcaag caaagtgtgg tatccacaga
961  cattttagtt gcagaagaaa ggctaggaaa tcattccttt tggttaaatg ggtgtttaat
1021 cttttggtta gtgggttaaa cggggtaagt tagagtaggg ggagggatag gaagacatat
1081 ttaaaaacca ttaaaacact gtctcccact catgaaatga gccacgtagt tcctatttaa
1141 tgctgttttc ctttagttta gaaatacata gacattgtct tttatgaatt ctgatcatat
1201 ttagtcattt tgaccaaatg agggatttgg tcaaatgagg gattccctca aagcaatatc
1261 aggtaaacca agttgctttc ctcactccct gtcatgagac ttcagtgtta atgttcacaa
1321 tatactttcg aaagaataaa atagttctcc tacatgaaga aagaatatgt caggaaataa
1381 ggtcacttta tgtcaaaatt atttgagtac tatgggacct ggcgcagtgg ctcatgcttg
1441 taatcccagc actttgggag gccgaggtgg gcagatcact tgagatcagg accagcctgg
1501 tcaagatggt gaaactccgt ctgtactaaa aatacaaaat ttagcttggc ctggtggcag
1561 gcacctgtaa tcccagctgc ccaagaggct gaggcatgag aatcgcttga acctggcagg
1621 cggaggttgc agtgagccga gatagtgcca cagctctcca gcctgggcga cagagtgaga
1681 ctccatctca aacaacaaca acaacaacaa caacaacaac aaaccacaaa attatttgag
1741 tactgtgaag gattatttgt ctaacagttc attccaatca gaccaggtag gagctttcct
1801 gtttcatatg tttcagggtt gcacagttgg tctctttaat gtcggtgtgg agatccaaag
1861 tgggttgtgg aaagagcgtc cataggagaa gtgagaatac tgtgaaaaag ggatgttagc
1921 attcattaga gtatgaggat gagtcccaag aaggttcttt ggaaggagga cgaatagaat
1981 ggagtaatga aattcttgcc atgtgctgag gagatagcca gcattaggtg acaatcttcc
2041 agaagtggtc aggcagaagg tgccctggtg agagctcctt tacagggact ttatgtggtt
2101 tagggctcag agctccaaaa ctctgggctc agctgctcct gtaccttgga ggtccattca
2161 catgggaaag tattttggaa tgtgtctttt gaagagagca tcagagttct taagggactg
2221 ggtaaggcct gaccctgaaa tgaccatgga tatttttcta cctacagttt gagtcaacta
2281 gaatatgcct ggggaccttg aagaatggcc cttcagtggc cctcaccatt tgttcatgct
2341 tcagttaatt caggtgttga aggagcttag gttttagagg cacgtagact tggttcaagt
2401 ctcgttagta gttgaatagc ctcaggcaag tcactgccca cctaagatga tggttcttca
2461 actataaaat ggagataatg gttacaaatg tctcttccta tagtataatc tccataaggg
2521 catggcccaa gtctgtcttt gactctgcct atccctgaca tttagtagca tgcccgacat
2581 acaatgttag ctattggtat tattgccata tagataaatt atgtataaaa attaaactgg
2641 gcaatagcct aagaaggggg gaatattgta acacaaattt aaacccacta cgcagggatg
2701 aggtgctata atatgaggac cttttaactt ccatcatttt cctgtttctt gaaatagttt
2761 atcttgtaat gaaatataag gcacctccca cttttatgta tagaaagagg tcttttaatt
2821 tttttttaat gtgagaagga agggaggagt aggaatcttg agattccaga tcgaaaatac
2881 tgtactttgg ttgattttta agtgggcttc cattccatgg atttaatcag tcccaagaag
2941 atcaaactca gcagtacttg ggtgctgaag aactgttgga tttaccctgg cacgtgtgcc
3001 acttgccagc ttcttgggca cacagagttc ttcaatccaa gttatcagat tgtatttgaa
3061 aatgacagag ctggagagtt ttttgaaatg gcagtggcaa ataaataaat actttttttt
3121 aaatggaaag acttgatcta tggtaataaa tgattttgtt ttctgactgg aaaaataggc
3181 ctactaaaga tgaatcacac ttgagatgtt tcttactcac tctgcacaga aacaaagaag
3241 aaatgttata cagggaagtc cgttttcact attagtatga accaagaaat ggttcaaaaa
3301 cagtggtagg agcaatgctt tcatagtttc agatatggta gttatgaaga aaacaatgtc
3361 atttgctgct attattgtaa gagtcttata attaatggta ctcctataat ttttgattgt
3421 gagctcacct atttgggtta agcatgccaa tttaaagaga ccaagtgtat gtacattatg
3481 ttctacatat tcagtgataa aattactaaa ctactatatg tctgctttaa atttgtactt
3541 taatattgtc ttttggtatt aagaaagata tgctttcaga atagatatgc ttcgctttgg
3601 caaggaattt ggatagaact tgctatttaa aagaggtgtg gggtaaatcc ttgtataaat
3661 ctccagttta gccttttttg aaaaagctag actttcaaat actaatttca cttcaagcag
3721 ggtacgtttc tggtttgttt gcttgacttc agtcacaatt tcttatcaga ccaatggctg
3781 acctctttga gatgtcaggc taggcttacc tatgtgttct gtgtcatgtg aatgctgaga
3841 agtttgacag agatccaact tcagccttga ccccatcagt ccctcgggtt aactaactga
3901 gccaccggtc ctcatggcta ttttaatgag ggtattgatg gttaaatgca tgtctgatcc
3961 cttatcccag ccatttgcac tgccagctgg gaactatacc agacctggat actgatccca
4021 aagtgttaaa ttcaactaca tgctggagat tagagatggt gccaataaag gacccagaac
4081 caggatcttg attgctatag acttattaat aatccaggtc aaagagagtg acacacactc
4141 tctcaagacc tggggtgagg gagtctgtgt tatctgcaag gccatttgag gctcagaaag
4201 tctctctttc ctatagatat atgcatactt tctgacatat aggaatgtat caggaatact
4261 caaccatcac aggcatgttc ctacctcagg gcctttacat gtcctgttta ctctgtctag
4321 aatgtccttc tgtagatgac ctggcttgcc tcgtcaccct tcaggtcctt gctcaagtgt
4381 catcttctcc cctagttaaa ctaccccaca ccctgtctgc tttccttgct tatttttctc
4441 catagcattt taccatctct tacattagac atttttctta tttatttgta gtttataagc
4501 ttcatgaggc aagtaacttt gctttgtttc ttgctgtatc tccagtgccc agagcagtgc
4561 ctggtatata ataaatattt attgactgag tgaaaaaaaa aaaaaaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD28 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 223)
1    taaagtcatc aaaacaacgt tatatcctgt gtgaaatgct gcagtcagga tgccttgtgg
61   tttgagtgcc ttgatcatgt gccctaaggg gatggtggcg gtggtggtgg ccgtggatga
121  cggagactct caggccttgg caggtgcgtc tttcagttcc cctcacactt cgggttcctc
181  ggggaggagg ggctggaacc ctagcccatc gtcaggacaa agatgctcag gctgctcttg
241  gctctcaact tattcccttc aattcaagta acagggaaac acctttgtcc aagtccccta
301  tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttggtggagt cctggcttgc
361  tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg
421  ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat
481  taccagccct atgccccacc acgcgacttc gcagcctatc gctcctgaca cggacgccta
541  tccagaagcc agccggctgg cagcccccat ctgctcaata tcactgctct ggataggaaa
601  tgaccgccat ctccagccgg ccacctcagg cccctgttgg gccaccaatg ccaatttttc
661  tcgagtgact agaccaaata tcaagatcat tttgagactc tgaaatgaag taaaagagat
721  ttcctgtgac aggccaagtc ttacagtgcc atggcccaca ttccaactta ccatgtactt
781  agtgacttga ctgagaagtt agggtagaaa acaaaaaggg agtggattct gggagcctct
841  tccctttctc actcacctgc acatctcagt caagcaaagt gtggtatcca cagacatttt
901  agttgcagaa gaaaggctag gaaatcattc cttttggtta aatgggtgtt taatcttttg
961  gttagtgggt taaacggggt aagttagagt agggggaggg ataggaagac atatttaaaa
1021 accattaaaa cactgtctcc cactcatgaa atgagccacg tagttcctat ttaatgctgt
1081 tttcctttag tttagaaata catagacatt gtcttttatg aattctgatc atatttagtc
1141 attttgacca aatgagggat ttggtcaaat gagggattcc ctcaaagcaa tatcaggtaa
1201 accaagttgc tttcctcact ccctgtcatg agacttcagt gttaatgttc acaatatact
1261 ttcgaaagaa taaaatagtt ctcctacatg aagaaagaat atgtcaggaa ataaggtcac
1321 tttatgtcaa aattatttga gtactatggg acctggcgca gtggctcatg cttgtaatcc
1381 cagcactttg ggaggccgag gtgggcagat cacttgagat caggaccagc ctggtcaaga
1441 tggtgaaact ccgtctgtac taaaaataca aaatttagct tggcctggtg gcaggcacct
1501 gtaatcccag ctgcccaaga ggctgaggca tgagaatcgc ttgaacctgg caggcggagg
1561 ttgcagtgag ccgagatagt gccacagctc tccagcctgg gcgacagagt gagactccat
1621 ctcaaacaac aacaacaaca acaacaacaa caacaaacca caaaattatt tgagtactgt
1681 gaaggattat ttgtctaaca gttcattcca atcagaccag gtaggagctt tcctgtttca
1741 tatgtttcag ggttgcacag ttggtctctt taatgtcggt gtggagatcc aaagtgggtt
1801 gtggaaagag cgtccatagg agaagtgaga atactgtgaa aaagggatgt tagcattcat
1861 tagagtatga ggatgagtcc caagaaggtt ctttggaagg aggacgaata gaatggagta
1921 atgaaattct tgccatgtgc tgaggagata gccagcatta ggtgacaatc ttccagaagt
1981 ggtcaggcag aaggtgccct ggtgagagct cctttacagg gactttatgt ggtttagggc
2041 tcagagctcc aaaactctgg gctcagctgc tcctgtacct tggaggtcca ttcacatggg
2101 aaagtatttt ggaatgtgtc ttttgaagag agcatcagag ttcttaaggg actgggtaag
2161 gcctgaccct gaaatgacca tggatatttt tctacctaca gtttgagtca actagaatat
2221 gcctggggac cttgaagaat ggcccttcag tggccctcac catttgttca tgcttcagtt
2281 aattcaggtg ttgaaggagc ttaggtttta gaggcacgta gacttggttc aagtctcgtt
2341 agtagttgaa tagcctcagg caagtcactg cccacctaag atgatggttc ttcaactata
2401 aaatggagat aatggttaca aatgtctctt cctatagtat aatctccata agggcatggc
2461 ccaagtctgt ctttgactct gcctatccct gacatttagt agcatgcccg acatacaatg
2521 ttagctattg gtattattgc catatagata aattatgtat aaaaattaaa ctgggcaata
2581 gcctaagaag gggggaatat tgtaacacaa atttaaaccc actacgcagg gatgaggtgc
2641 tataatatga ggacctttta acttccatca ttttcctgtt tcttgaaata gtttatcttg
2701 taatgaaata taaggcacct cccactttta tgtatagaaa gaggtctttt aatttttttt
2761 taatgtgaga aggaagggag gagtaggaat cttgagattc cagatcgaaa atactgtact
2821 ttggttgatt tttaagtggg cttccattcc atggatttaa tcagtcccaa gaagatcaaa
2881 ctcagcagta cttgggtgct gaagaactgt tggatttacc ctggcacgtg tgccacttgc
2941 cagcttcttg ggcacacaga gttcttcaat ccaagttatc agattgtatt tgaaaatgac
3001 agagctggag agttttttga aatggcagtg gcaaataaat aaatactttt ttttaaatgg
3061 aaagacttga tctatggtaa taaatgattt tgttttctga ctggaaaaat aggcctacta
3121 aagatgaatc acacttgaga tgtttcttac tcactctgca cagaaacaaa gaagaaatgt
3181 tatacaggga agtccgtttt cactattagt atgaaccaag aaatggttca aaaacagtgg
3241 taggagcaat gctttcatag tttcagatat ggtagttatg aagaaaacaa tgtcatttgc
3301 tgctattatt gtaagagtct tataattaat ggtactccta taatttttga ttgtgagctc
3361 acctatttgg gttaagcatg ccaatttaaa gagaccaagt gtatgtacat tatgttctac
3421 atattcagtg ataaaattac taaactacta tatgtctgct ttaaatttgt actttaatat
3481 tgtcttttgg tattaagaaa gatatgcttt cagaatagat atgcttcgct ttggcaagga
3541 atttggatag aacttgctat ttaaaagagg tgtggggtaa atccttgtat aaatctccag
3601 tttagccttt tttgaaaaag ctagactttc aaatactaat ttcacttcaa gcagggtacg
3661 tttctggttt gtttgcttga cttcagtcac aatttcttat cagaccaatg gctgacctct
3721 ttgagatgtc aggctaggct tacctatgtg ttctgtgtca tgtgaatgct gagaagtttg
3781 acagagatcc aacttcagcc ttgaccccat cagtccctcg ggttaactaa ctgagccacc
3841 ggtcctcatg gctattttaa tgagggtatt gatggttaaa tgcatgtctg atcccttatc
3901 ccagccattt gcactgccag ctgggaacta taccagacct ggatactgat cccaaagtgt
3961 taaattcaac tacatgctgg agattagaga tggtgccaat aaaggaccca gaaccaggat
4021 cttgattgct atagacttat taataatcca ggtcaaagag agtgacacac actctctcaa
4081 gacctggggt gagggagtct gtgttatctg caaggccatt tgaggctcag aaagtctctc
4141 tttcctatag atatatgcat actttctgac atataggaat gtatcaggaa tactcaacca
4201 tcacaggcat gttcctacct cagggccttt acatgtcctg tttactctgt ctagaatgtc
4261 cttctgtaga tgacctggct tgcctcgtca cccttcaggt ccttgctcaa gtgtcatctt
4321 ctcccctagt taaactaccc cacaccctgt ctgctttcct tgcttatttt tctccatagc
4381 attttaccat ctcttacatt agacattttt cttatttatt tgtagtttat aagcttcatg
4441 aggcaagtaa ctttgctttg tttcttgctg tatctccagt gcccagagca gtgcctggta
4501 tataataaat atttattgac tgagtgaaaa aaaaaaaaaa aaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD80 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 224)
1    gacaagtact gagtgaactc aaaccctctg taaagtaaca gaagttagaa ggggaaatgt
61   cgcctctctg aagattaccc aaagaaaaag tgatttgtca ttgctttata gactgtaaga
121  agagaacatc tcagaagtgg agtcttaccc tgaaatcaaa ggatttaaag aaaaagtgga
181  atttttcttc agcaagctgt gaaactaaat ccacaacctt tggagaccca ggaacaccct
241  ccaatctctg tgtgttttgt aaacatcact ggagggtctt ctacgtgagc aattggattg
301  tcatcagccc tgcctgtttt gcacctggga agtgccctgg tcttacttgg gtccaaattg
361  ttggctttca cttttgaccc taagcatctg aagccatggg ccacacacgg aggcagggaa
421  catcaccatc caagtgtcca tacctcaatt tctttcagct cttggtgctg gctggtcttt
481  ctcacttctg ttcaggtgtt atccacgtga ccaaggaagt gaaagaagtg gcaacgctgt
541  cctgtggtca caatgtttct gttgaagagc tggcacaaac tcgcatctac tggcaaaagg
601  agaagaaaat ggtgctgact atgatgtctg gggacatgaa tatatggccc gagtacaaga
661  accggaccat ctttgatatc actaataacc tctccattgt gatcctggct ctgcgcccat
721  ctgacgaggg cacatacgag tgtgttgttc tgaagtatga aaaagacgct ttcaagcggg
781  aacacctggc tgaagtgacg ttatcagtca aagctgactt ccctacacct agtatatctg
841  actttgaaat tccaacttct aatattagaa ggataatttg ctcaacctct ggaggttttc
901  cagagcctca cctctcctgg ttggaaaatg gagaagaatt aaatgccatc aacacaacag
961  tttcccaaga tcctgaaact gagctctatg ctgttagcag caaactggat ttcaatatga
1021 caaccaacca cagcttcatg tgtctcatca agtatggaca tttaagagtg aatcagacct
1081 tcaactggaa tacaaccaag caagagcatt ttcctgataa cctgctccca tcctgggcca
1141 ttaccttaat ctcagtaaat ggaatttttg tgatatgctg cctgacctac tgctttgccc
1201 caagatgcag agagagaagg aggaatgaga gattgagaag ggaaagtgta cgccctgtat
1261 aacagtgtcc gcagaagcaa ggggctgaaa agatctgaag gtcccacctc catttgcaat
1321 tgacctcttc tgggaacttc ctcagatgga caagattacc ccaccttgcc ctttacgtat
1381 ctgctcttag gtgcttcttc acttcagttg ctttgcagga agtgtctaga ggaatatggt
1441 gggcacagaa gtagctctgg tgaccttgat caaggtgttt tgaaatgcag aattcttgag
1501 ttctggaagg gactttagag aataccagtg ttattaatga caaaggcact gaggcccagg
1561 gaggtgaccc gaattataaa ggccagcgcc agaacccaga tttcctaact ctggtgctct
1621 ttccctttat cagtttgact gtggcctgtt aactggtata tacatatata tgtcaggcaa
1681 agtgctgctg gaagtagaat ttgtccaata acaggtcaac ttcagagact atctgatttc
1741 ctaatgtcag agtagaagat tttatgctgc tgtttacaaa agcccaatgt aatgcatagg
1801 aagtatggca tgaacatctt taggagacta atggaaatat tattggtgtt tacccagtat
1861 tccatttttt tcattgtgtt ctctattgct gctctctcac tcccccatga ggtacagcag
1921 aaaggagaac tatccaaaac taatttcctc tgacatgtaa gacgaatgat ttaggtacgt
1981 caaagcagta gtcaaggagg aaagggatag tccaaagact taactggttc atattggact
2041 gataatctct ttaaatggct ttatgctagt ttgacctcat ttgtaaaata tttatgagaa
2101 agttctcatt taaaatgaga tcgttgttta cagtgtatgt actaagcagt aagctatctt
2161 caaatgtcta aggtagtaac tttccatagg gcctccttag atccctaaga tggctttttc
2221 tccttggtat ttctgggtct ttctgacatc agcagagaac tggaaagaca tagccaactg
2281 ctgttcatgt tactcatgac tcctttctct aaaactgcct tccacaattc actagaccag
2341 aagtggacgc aacttaagct gggataatca cattatcatc tgaaaatctg gagttgaaca
2401 gcaaaagaag acaacatttc tcaaatgcac atctcatggc agctaagcca catggctggg
2461 atttaaagcc tttagagcca gcccatggct ttagctacct cactatgctg cttcacaaac
2521 cttgctcctg tgtaaaacta tattctcagt gtagggcaga gaggtctaac accaacataa
2581 ggtactagca gtgtttcccg tattgacagg aatacttaac tcaataattc ttttcttttc
2641 catttagtaa cagttgtgat gactatgttt ctattctaag taattcctgt attctacagc
2701 agatactttg tcagcaatac taagggaaga aacaaagttg aaccgtttct ttaataa

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding a CD80 protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of comprising SEQ ID NO: 330 to SEQ ID NO: 3067.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD86 protein comprising or consisting of 20 nucleotides of the sequence of:

(SEQ ID NO: 226)
1    agtcattgcc gaggaaggct tgcacagggt gaaagctttg cttctctgct gctgtaacag
61   ggactagcac agacacacgg atgagtgggg tcatttccag atattaggtc acagcagaag
121  cagccaaaat ggatccccag tgcactatgg gactgagtaa cattctcttt gtgatggcct
181  tcctgctctc tggtgctgct cctctgaaga ttcaagctta tttcaatgag actgcagacc
241  tgccatgcca atttgcaaac tctcaaaacc aaagcctgag tgagctagta gtattttggc
301  aggaccagga aaacttggtt ctgaatgagg tatacttagg caaagagaaa tttgacagtg
361  ttcattccaa gtatatgggc cgcacaagtt ttgattcgga cagttggacc ctgagacttc
421  acaatcttca gatcaaggac aagggcttgt atcaatgtat catccatcac aaaaagccca
481  caggaatgat tcgcatccac cagatgaatt ctgaactgtc agtgcttgct aacttcagtc
541  aacctgaaat agtaccaatt tctaatataa cagaaaatgt gtacataaat ttgacctgct
601  catctataca cggttaccca gaacctaaga agatgagtgt tttgctaaga accaagaatt
661  caactatcga gtatgatggt attatgcaga aatctcaaga taatgtcaca gaactgtacg
721  acgtttccat cagcttgtct gtttcattcc ctgatgttac gagcaatatg accatcttct
781  gtattctgga aactgacaag acgcggcttt tatcttcacc tttctctata gagcttgagg
841  accctcagcc tcccccagac cacattcctt ggattacagc tgtacttcca acagttatta
901  tatgtgtgat ggttttctgt ctaattctat ggaaatggaa gaagaagaag cggcctcgca
961  actcttataa atgtggaacc aacacaatgg agagggaaga gagtgaacag accaagaaaa
1021 gagaaaaaat ccatatacct gaaagatctg atgaagccca gcgtgttttt aaaagttcga
1081 agacatcttc atgcgacaaa agtgatacat gtttttaatt aaagagtaaa gcccatacaa
1141 gtattcattt tttctaccct ttcctttgta agttcctggg caaccttttt gatttcttcc
1201 agaaggcaaa aagacattac catgagtaat aagggggctc caggactccc tctaagtgga
1261 atagcctccc tgtaactcca gctctgctcc gtatgccaag aggagacttt aattctctta
1321 ctgcttcttt tcacttcaga gcacacttat gggccaagcc cagcttaatg gctcatgacc
1381 tggaaataaa atttaggacc aatacctcct ccagatcaga ttcttctctt aatttcatag
1441 attgtgtttt ttttttaaat agacctctca atttctggaa aactgccttt tatctgccca
1501 gaattctaag ctggtgcccc actgaatttt gtgtacctgt gactaaacaa ctacctcctc
1561 agtctgggtg ggacttatgt atttatgacc ttatagtgtt aatatcttga aacatagaga
1621 tctatgtact gtaatagtgt gattactatg ctctagagaa aagtctaccc ctgctaagga
1681 gttctcatcc ctctgtcagg gtcagtaagg aaaacggtgg cctagggtac aggcaacaat
1741 gagcagacca acctaaattt ggggaaatta ggagaggcag agatagaacc tggagccact
1801 tctatctggg ctgttgctaa tattgaggag gcttgcccca cccaacaagc catagtggag
1861 agaactgaat aaacaggaaa atgccagagc ttgtgaaccc tgtttctctt gaagaactga
1921 ctagtgagat ggcctgggga agctgtgaaa gaaccaaaag agatcacaat actcaaaaga
1981 gagagagaga gaaaaaagag agatcttgat ccacagaaat acatgaaatg tctggtctgt
2041 ccaccccatc aacaagtctt gaaacaagca acagatggat agtctgtcca aatggacata
2101 agacagacag cagtttccct ggtggtcagg gaggggtttt ggtgataccc aagttattgg
2161 gatgtcatct tcctggaagc agagctgggg agggagagcc atcaccttga taatgggatg
2221 aatggaagga ggcttaggac tttccactcc tggctgagag aggaagagct gcaacggaat
2281 taggaagacc aagacacaga tcacccgggg cttacttagc ctacagatgt cctacgggaa
2341 cgtgggctgg cccagcatag ggctagcaaa tttgagttgg atgattgttt ttgctcaagg
2401 caaccagagg aaacttgcat acagagacag atatactggg agaaatgact ttgaaaacct
2461 ggctctaagg tgggatcact aagggatggg gcagtctctg cccaaacata aagagaactc
2521 tggggagcct gagccacaaa aatgttcctt tattttatgt aaaccctcaa gggttataga
2581 ctgccatgct agacaagctt gtccatgtaa tattcccatg tttttaccct gcccctgcct
2641 tgattagact cctagcacct ggctagtttc taacatgttt tgtgcagcac agtttttaat
2701 aaatgcttgt tacattcatt taaaaaaaaa aaaaa.

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding a CD86 protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of SEQ ID NO: 3068 to SEQ ID NO: 5783.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD86 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 227)
1    ccctttctgt atttgagttc taccgtcagt cctggcatta tttctctctc tacaaggagc
61   cttaggaggt acggggagct cgcaaatact ccttttggtt tattcttacc accttgcttc
121  tgtgttcctt gggaatgctg ctgtgcttat gcatctggtc tctttttgga gctacagtgg
181  acaggcattt gtgacagcac tatgggactg agtaacattc tctttgtgat ggccttcctg
241  ctctctggtg ctgctcctct gaagattcaa gcttatttca atgagactgc agacctgcca
301  tgccaatttg caaactctca aaaccaaagc ctgagtgagc tagtagtatt ttggcaggac
361  caggaaaact tggttctgaa tgaggtatac ttaggcaaag agaaatttga cagtgttcat
421  tccaagtata tgggccgcac aagttttgat tcggacagtt ggaccctgag acttcacaat
481  cttcagatca aggacaaggg cttgtatcaa tgtatcatcc atcacaaaaa gcccacagga
541  atgattcgca tccaccagat gaattctgaa ctgtcagtgc ttgctaactt cagtcaacct
601  gaaatagtac caatttctaa tataacagaa aatgtgtaca taaatttgac ctgctcatct
661  atacacggtt acccagaacc taagaagatg agtgttttgc taagaaccaa gaattcaact
721  atcgagtatg atggtattat gcagaaatct caagataatg tcacagaact gtacgacgtt
781  tccatcagct tgtctgtttc attccctgat gttacgagca atatgaccat cttctgtatt
841  ctggaaactg acaagacgcg gcttttatct tcacctttct ctatagagct tgaggaccct
901  cagcctcccc cagaccacat tccttggatt acagctgtac ttccaacagt tattatatgt
961  gtgatggttt tctgtctaat tctatggaaa tggaagaaga agaagcggcc tcgcaactct
1021 tataaatgtg gaaccaacac aatggagagg gaagagagtg aacagaccaa gaaaagagaa
1081 aaaatccata tacctgaaag atctgatgaa gcccagcgtg tttttaaaag ttcgaagaca
1141 tcttcatgcg acaaaagtga tacatgtttt taattaaaga gtaaagccca tacaagtatt
1201 cattttttct accctttcct ttgtaagttc ctgggcaacc tttttgattt cttccagaag
1261 gcaaaaagac attaccatga gtaataaggg ggctccagga ctccctctaa gtggaatagc
1321 ctccctgtaa ctccagctct gctccgtatg ccaagaggag actttaattc tcttactgct
1381 tcttttcact tcagagcaca cttatgggcc aagcccagct taatggctca tgacctggaa
1441 ataaaattta ggaccaatac ctcctccaga tcagattctt ctcttaattt catagattgt
1501 gttttttttt taaatagacc tctcaatttc tggaaaactg ccttttatct gcccagaatt
1561 ctaagctggt gccccactga attttgtgta cctgtgacta aacaactacc tcctcagtct
1621 gggtgggact tatgtattta tgaccttata gtgttaatat cttgaaacat agagatctat
1681 gtactgtaat agtgtgatta ctatgctcta gagaaaagtc tacccctgct aaggagttct
1741 catccctctg tcagggtcag taaggaaaac ggtggcctag ggtacaggca acaatgagca
1801 gaccaaccta aatttgggga aattaggaga ggcagagata gaacctggag ccacttctat
1861 ctgggctgtt gctaatattg aggaggcttg ccccacccaa caagccatag tggagagaac
1921 tgaataaaca ggaaaatgcc agagcttgtg aaccctgttt ctcttgaaga actgactagt
1981 gagatggcct ggggaagctg tgaaagaacc aaaagagatc acaatactca aaagagagag
2041 agagagaaaa aagagagatc ttgatccaca gaaatacatg aaatgtctgg tctgtccacc
2101 ccatcaacaa gtcttgaaac aagcaacaga tggatagtct gtccaaatgg acataagaca
2161 gacagcagtt tccctggtgg tcagggaggg gttttggtga tacccaagtt attgggatgt
2221 catcttcctg gaagcagagc tggggaggga gagccatcac cttgataatg ggatgaatgg
2281 aaggaggctt aggactttcc actcctggct gagagaggaa gagctgcaac ggaattagga
2341 agaccaagac acagatcacc cggggcttac ttagcctaca gatgtcctac gggaacgtgg
2401 gctggcccag catagggcta gcaaatttga gttggatgat tgtttttgct caaggcaacc
2461 agaggaaact tgcatacaga gacagatata ctgggagaaa tgactttgaa aacctggctc
2521 taaggtggga tcactaaggg atggggcagt ctctgcccaa acataaagag aactctgggg
2581 agcctgagcc acaaaaatgt tcctttattt tatgtaaacc ctcaagggtt atagactgcc
2641 atgctagaca agcttgtcca tgtaatattc ccatgttttt accctgcccc tgccttgatt
2701 agactcctag cacctggcta gtttctaaca tgttttgtgc agcacagttt ttaataaatg
2761 cttgttacat tcatttaaaa aaaaaaaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD86 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 228)
1    ccctttctgt atttgagttc taccgtcagt cctggcatta tttctctctc tacaaggagc
61   cttaggaggt acggggagct cgcaaatact ccttttggtt tattcttacc accttgcttc
121  tgtgttcctt gggaatgctg ctgtgcttat gcatctggtc tctttttgga gctacagtgg
181  acaggcattt gtgacagcac tatgggactg agtaacattc tctttgtgat ggccttcctg
241  ctctctggtg ctgctcctct gaagattcaa gcttatttca atgagactgc agacctgcca
301  tgccaatttg caaactctca aaaccaaagc ctgagtgagc tagtagtatt ttggcaggac
361  caggaaaact tggttctgaa tgaggtatac ttaggcaaag agaaatttga cagtgttcat
421  tccaagtata tgggccgcac aagttttgat tcggacagtt ggaccctgag acttcacaat
481  cttcagatca aggacaaggg cttgtatcaa tgtatcatcc atcacaaaaa gcccacagga
541  atgattcgca tccaccagat gaattctgaa ctgtcagtgc ttgctaactt cagtcaacct
601  gaaatagtac caatttctaa tataacagaa aatgtgtaca taaatttgac ctgctcatct
661  atacacggtt acccagaacc taagaagatg agtgttttgc taagaaccaa gaattcaact
721  atcgagtatg atggtattat gcagaaatct caagataatg tcacagaact gtacgacgtt
781  tccatcagct tgtctgtttc attccctgat gttacgagca atatgaccat cttctgtatt
841  ctggaaactg acaagacgcg gcttttatct tcacctttct ctataggaac caacacaatg
901  gagagggaag agagtgaaca gaccaagaaa agagaaaaaa tccatatacc tgaaagatct
961  gatgaagccc agcgtgtttt taaaagttcg aagacatctt catgcgacaa aagtgataca
1021 tgtttttaat taaagagtaa agcccataca agtattcatt ttttctaccc tttcctttgt
1081 aagttcctgg gcaacctttt tgatttcttc cagaaggcaa aaagacatta ccatgagtaa
1141 taagggggct ccaggactcc ctctaagtgg aatagcctcc ctgtaactcc agctctgctc
1201 cgtatgccaa gaggagactt taattctctt actgcttctt ttcacttcag agcacactta
1261 tgggccaagc ccagcttaat ggctcatgac ctggaaataa aatttaggac caatacctcc
1321 tccagatcag attcttctct taatttcata gattgtgttt tttttttaaa tagacctctc
1381 aatttctgga aaactgcctt ttatctgccc agaattctaa gctggtgccc cactgaattt
1441 tgtgtacctg tgactaaaca actacctcct cagtctgggt gggacttatg tatttatgac
1501 cttatagtgt taatatcttg aaacatagag atctatgtac tgtaatagtg tgattactat
1561 gctctagaga aaagtctacc cctgctaagg agttctcatc cctctgtcag ggtcagtaag
1621 gaaaacggtg gcctagggta caggcaacaa tgagcagacc aacctaaatt tggggaaatt
1681 aggagaggca gagatagaac ctggagccac ttctatctgg gctgttgcta atattgagga
1741 ggcttgcccc acccaacaag ccatagtgga gagaactgaa taaacaggaa aatgccagag
1801 cttgtgaacc ctgtttctct tgaagaactg actagtgaga tggcctgggg aagctgtgaa
1861 agaaccaaaa gagatcacaa tactcaaaag agagagagag agaaaaaaga gagatcttga
1921 tccacagaaa tacatgaaat gtctggtctg tccaccccat caacaagtct tgaaacaagc
1981 aacagatgga tagtctgtcc aaatggacat aagacagaca gcagtttccc tggtggtcag
2041 ggaggggttt tggtgatacc caagttattg ggatgtcatc ttcctggaag cagagctggg
2101 gagggagagc catcaccttg ataatgggat gaatggaagg aggcttagga ctttccactc
2161 ctggctgaga gaggaagagc tgcaacggaa ttaggaagac caagacacag atcacccggg
2221 gcttacttag cctacagatg tcctacggga acgtgggctg gcccagcata gggctagcaa
2281 atttgagttg gatgattgtt tttgctcaag gcaaccagag gaaacttgca tacagagaca
2341 gatatactgg gagaaatgac tttgaaaacc tggctctaag gtgggatcac taagggatgg
2401 ggcagtctct gcccaaacat aaagagaact ctggggagcc tgagccacaa aaatgttcct
2461 ttattttatg taaaccctca agggttatag actgccatgc tagacaagct tgtccatgta
2521 atattcccat gtttttaccc tgcccctgcc ttgattagac tcctagcacc tggctagttt
2581 ctaacatgtt ttgtgcagca cagtttttaa taaatgcttg ttacattcat ttaaaaaaaa
2641 aaaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD86 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 229)
1    agtcattgcc gaggaaggct tgcacagggt gaaagctttg cttctctgct gctgtaacag
61   ggactagcac agacacacgg atgagtgggg tcatttccag atattaggtc acagcagaag
121  cagccaaaat ggatccccag tgcactatgg gactgagtaa cattctcttt gtgatggcct
181  tcctgctctc tgctaacttc agtcaacctg aaatagtacc aatttctaat ataacagaaa
241  atgtgtacat aaatttgacc tgctcatcta tacacggtta cccagaacct aagaagatga
301  gtgttttgct aagaaccaag aattcaacta tcgagtatga tggtattatg cagaaatctc
361  aagataatgt cacagaactg tacgacgttt ccatcagctt gtctgtttca ttccctgatg
421  ttacgagcaa tatgaccatc ttctgtattc tggaaactga caagacgcgg cttttatctt
481  cacctttctc tatagagctt gaggaccctc agcctccccc agaccacatt ccttggatta
541  cagctgtact tccaacagtt attatatgtg tgatggtttt ctgtctaatt ctatggaaat
601  ggaagaagaa gaagcggcct cgcaactctt ataaatgtgg aaccaacaca atggagaggg
661  aagagagtga acagaccaag aaaagagaaa aaatccatat acctgaaaga tctgatgaag
721  cccagcgtgt ttttaaaagt tcgaagacat cttcatgcga caaaagtgat acatgttttt
781  aattaaagag taaagcccat acaagtattc attttttcta ccctttcctt tgtaagttcc
841  tgggcaacct ttttgatttc ttccagaagg caaaaagaca ttaccatgag taataagggg
901  gctccaggac tccctctaag tggaatagcc tccctgtaac tccagctctg ctccgtatgc
961  caagaggaga ctttaattct cttactgctt cttttcactt cagagcacac ttatgggcca
1021 agcccagctt aatggctcat gacctggaaa taaaatttag gaccaatacc tcctccagat
1081 cagattcttc tcttaatttc atagattgtg tttttttttt aaatagacct ctcaatttct
1141 ggaaaactgc cttttatctg cccagaattc taagctggtg ccccactgaa ttttgtgtac
1201 ctgtgactaa acaactacct cctcagtctg ggtgggactt atgtatttat gaccttatag
1261 tgttaatatc ttgaaacata gagatctatg tactgtaata gtgtgattac tatgctctag
1321 agaaaagtct acccctgcta aggagttctc atccctctgt cagggtcagt aaggaaaacg
1381 gtggcctagg gtacaggcaa caatgagcag accaacctaa atttggggaa attaggagag
1441 gcagagatag aacctggagc cacttctatc tgggctgttg ctaatattga ggaggcttgc
1501 cccacccaac aagccatagt ggagagaact gaataaacag gaaaatgcca gagcttgtga
1561 accctgtttc tcttgaagaa ctgactagtg agatggcctg gggaagctgt gaaagaacca
1621 aaagagatca caatactcaa aagagagaga gagagaaaaa agagagatct tgatccacag
1681 aaatacatga aatgtctggt ctgtccaccc catcaacaag tcttgaaaca agcaacagat
1741 ggatagtctg tccaaatgga cataagacag acagcagttt ccctggtggt cagggagggg
1801 ttttggtgat acccaagtta ttgggatgtc atcttcctgg aagcagagct ggggagggag
1861 agccatcacc ttgataatgg gatgaatgga aggaggctta ggactttcca ctcctggctg
1921 agagaggaag agctgcaacg gaattaggaa gaccaagaca cagatcaccc ggggcttact
1981 tagcctacag atgtcctacg ggaacgtggg ctggcccagc atagggctag caaatttgag
2041 ttggatgatt gtttttgctc aaggcaacca gaggaaactt gcatacagag acagatatac
2101 tgggagaaat gactttgaaa acctggctct aaggtgggat cactaaggga tggggcagtc
2161 tctgcccaaa cataaagaga actctgggga gcctgagcca caaaaatgtt cctttatttt
2221 atgtaaaccc tcaagggtta tagactgcca tgctagacaa gcttgtccat gtaatattcc
2281 catgttttta ccctgcccct gccttgatta gactcctagc acctggctag tttctaacat
2341 gttttgtgca gcacagtttt taataaatgc ttgttacatt catttaaaaa aaaaaaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CD86 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 230)
1    agtcattgcc gaggaaggct tgcacagggt gaaagctttg cttctctgct gctgtaacag
61   ggactagcac agacacacgg atgagtgggg tcatttccag atattaggtc acagcagaag
121  cagccaaaat ggatccccag tggtgctgct cctctgaaga ttcaagctta tttcaatgag
181  actgcagacc tgccatgcca atttgcaaac tctcaaaacc aaagcctgag tgagctagta
241  gtattttggc aggaccagga aaacttggtt ctgaatgagg tatacttagg caaagagaaa
301  tttgacagtg ttcattccaa gtatatgggc cgcacaagtt ttgattcgga cagttggacc
361  ctgagacttc acaatcttca gatcaaggac aagggcttgt atcaatgtat catccatcac
421  aaaaagccca caggaatgat tcgcatccac cagatgaatt ctgaactgtc agtgcttgct
481  aacttcagtc aacctgaaat agtaccaatt tctaatataa cagaaaatgt gtacataaat
541  ttgacctgct catctataca cggttaccca gaacctaaga agatgagtgt tttgctaaga
601  accaagaatt caactatcga gtatgatggt attatgcaga aatctcaaga taatgtcaca
661  gaactgtacg acgtttccat cagcttgtct gtttcattcc ctgatgttac gagcaatatg
721  accatcttct gtattctgga aactgacaag acgcggcttt tatcttcacc tttctctata
781  gagcttgagg accctcagcc tcccccagac cacattcctt ggattacagc tgtacttcca
841  acagttatta tatgtgtgat ggttttctgt ctaattctat ggaaatggaa gaagaagaag
901  cggcctcgca actcttataa atgtggaacc aacacaatgg agagggaaga gagtgaacag
961  accaagaaaa gagaaaaaat ccatatacct gaaagatctg atgaagccca gcgtgttttt
1021 aaaagttcga agacatcttc atgcgacaaa agtgatacat gtttttaatt aaagagtaaa
1081 gcccatacaa gtattcattt tttctaccct ttcctttgta agttcctggg caaccttttt
1141 gatttcttcc agaaggcaaa aagacattac catgagtaat aagggggctc caggactccc
1201 tctaagtgga atagcctccc tgtaactcca gctctgctcc gtatgccaag aggagacttt
1261 aattctctta ctgcttcttt tcacttcaga gcacacttat gggccaagcc cagcttaatg
1321 gctcatgacc tggaaataaa atttaggacc aatacctcct ccagatcaga ttcttctctt
1381 aatttcatag attgtgtttt ttttttaaat agacctctca atttctggaa aactgccttt
1441 tatctgccca gaattctaag ctggtgcccc actgaatttt gtgtacctgt gactaaacaa
1501 ctacctcctc agtctgggtg ggacttatgt atttatgacc ttatagtgtt aatatcttga
1561 aacatagaga tctatgtact gtaatagtgt gattactatg ctctagagaa aagtctaccc
1621 ctgctaagga gttctcatcc ctctgtcagg gtcagtaagg aaaacggtgg cctagggtac
1681 aggcaacaat gagcagacca acctaaattt ggggaaatta ggagaggcag agatagaacc
1741 tggagccact tctatctggg ctgttgctaa tattgaggag gcttgcccca cccaacaagc
1801 catagtggag agaactgaat aaacaggaaa atgccagagc ttgtgaaccc tgtttctctt
1861 gaagaactga ctagtgagat ggcctgggga agctgtgaaa gaaccaaaag agatcacaat
1921 actcaaaaga gagagagaga gaaaaaagag agatcttgat ccacagaaat acatgaaatg
1981 tctggtctgt ccaccccatc aacaagtctt gaaacaagca acagatggat agtctgtcca
2041 aatggacata agacagacag cagtttccct ggtggtcagg gaggggtttt ggtgataccc
2101 aagttattgg gatgtcatct tcctggaagc agagctgggg agggagagcc atcaccttga
2161 taatgggatg aatggaagga ggcttaggac tttccactcc tggctgagag aggaagagct
2221 gcaacggaat taggaagacc aagacacaga tcacccgggg cttacttagc ctacagatgt
2281 cctacgggaa cgtgggctgg cccagcatag ggctagcaaa tttgagttgg atgattgttt
2341 ttgctcaagg caaccagagg aaacttgcat acagagacag atatactggg agaaatgact
2401 ttgaaaacct ggctctaagg tgggatcact aagggatggg gcagtctctg cccaaacata
2461 aagagaactc tggggagcct gagccacaaa aatgttcctt tattttatgt aaaccctcaa
2521 gggttataga ctgccatgct agacaagctt gtccatgtaa tattcccatg tttttaccct
2581 gcccctgcct tgattagact cctagcacct ggctagtttc taacatgttt tgtgcagcac
2641 agtttttaat aaatgcttgt tacattcatt taaaaaaaaa aaaaa.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding ICOSLG protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 231)
1    AGTTAGAGCC GATCTCCCGC GCCCCGAGGT TGCTCCTCTC CGAGGTCTCC CGCGGCCCAA
61   GTTCTCCGCG CCCCGAGGTC TCCGCGCCCC GAGGTCTCCG CGGCCCGAGG TCTCCGCCCG
121  CACCATGCGG CTGGGCAGTC CTGGACTGCT CTTCCTGCTC TTCAGCAGCC TTCGAGCTGA
181  TACTCAGGAG AAGGAAGTCA GAGCGATGGT AGGCAGCGAC GTGGAGCTCA GCTGCGCTTG
241  CCCTGAAGGA AGCCGTTTTG ATTTAAATGA TGTTTACGTA TATTGGCAAA CCAGTGAGTC
301  GAAAACCGTG GTGACCTACC ACATCCCACA GAACAGCTCC TTGGAAAACG TGGACAGCCG
361  CTACCGGAAC CGAGCCCTGA TGTCACCGGC CGGCATGCTG CGGGGCGACT TCTCCCTGCG
421  CTTGTTCAAC GTCACCCCCC AGGACGAGCA GAAGTTTCAC TGCCTGGTGT TGAGCCAATC
481  CCTGGGATTC CAGGAGGTTT TGAGCGTTGA GGTTACACTG CATGTGGCAG CAAACTTCAG
541  CGTGCCCGTC GTCAGCGCCC CCCACAGCCC CTCCCAGGAT GAGCTCACCT TCACGTGTAC
601  ATCCATAAAC GGCTACCCCA GGCCCAACGT GTACTGGATC AATAAGACGG ACAACAGCCT
661  GCTGGACCAG GCTCTGCAGA ATGACACCGT CTTCTTGAAC ATGCGGGGCT TGTATGACGT
721  GGTCAGCGTG CTGAGGATCG CACGGACCCC CAGCGTGAAC ATTGGCTGCT GCATAGAGAA
781  CGTGCTTCTG CAGCAGAACC TGACTGTCGG CAGCCAGACA GGAAATGACA TCGGAGAGAG
841  AGACAAGATC ACAGAGAATC CAGTCAGTAC CGGCGAGAAA AACGCGGCCA CGTGGAGCAT
901  CCTGGCTGTC CTGTGCCTGC TTGTGGTCGT GGCGGTGGCC ATAGGCTGGG TGTGCAGGGA
961  CCGATGCCTC CAACACAGCT ATGCAGGTGC CTGGGCTGTG AGTCCGGAGA CAGAGCTCAC
1021 TGGTGAGTTT GCCGTGGGAA GCAGCAGGTT CTGGGGGGCC CAGGGGAGGC TTGGCTGCCA
1081 GCTGTCTTTC AGAGTTTCAA AAAACTTTCA AAAGGCAAAA GTCCCTTGCC TTGAACAACT
1141 GTTGTTCCTG GAGACGCAGC GAAGCCCTCG ATGGTGCGCA TGGCATTTCC TGCAGCCTCC
1201 CCTTGGCATG GGATGGCATC CTGGTGTGCA CTTTGTCACA CTGCGATGGG ATTTTCCCAA
1261 CATGCACAGA AGCAGAGAGA CGAGTGCTAG ACCCCCGCGC TCCCCAGTGC CCAGCCCCGA
1321 CCAGGGTGTC CAGGGCGGGT CCAGGCACCG GCGCCCAGCC CCCATGGGGT GTCCGGAGTG
1381 GGTCCAGGCA CCGGCGCCCA GCCCCCGTGG GGTGTCCAGG GCGGGTCCAG GCACCGGCGC
1441 CCAGCCCCTG TGGGGTGTCC GGAGTGGGTC CGGGCACCGC CAGCTTCTCT CTGTGGCAGC
1501 CACTCCTGCA GCTCTCGTTT GCCCCTCAGT TCCAGGAGCA ACATAGATGT GGATTCCTGT
1561 CCAATTTGGG AAAAATGTCC ACACACGGTC ACCCACCTGG CAGGTGCCTC TGGCTGCAAG
1621 GGGCGCTGGG CTTCGCAGGC AGGCCAGCCG GGCTCCCCGC CATGGGCCAG GATCCCCTCC
1681 GAGCCCTGTT TGCCGCCCAG GAGAAGGGGT TCCCCGGGGA CAGTGGGCTC AGGGTGTGCG
1741 CAGCCACCAT GCTGTGGTGT CACCTGTGGA CCCAGGCGAG CTGATGGCCG ACCGCAGAAA
1801 CGCACTTCCA AGGCCAGGTC GGCCCATCCA GATGATGCAG GAACACAGCT TGCTAAAAAC
1861 ACGGCCGGCC TGTTCCCGTC GGAGCCAGTC GAAGTTCCCT GAACAGGCCG CTGTTTCCGA
1921 AGCTTTAAAC CCTGTGTTTC CACCAAGCTG AGTCCTGAGA AAACCGACGT CTGCCTGCAG
1981 AAGGGAAAGG GGTGCTTCAT GTTCCTCTCT CTCCTTCATC TCCCT.

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding a IOSLG protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of any one of SEQ ID NO: 5784 to SEQ ID NO: 7789.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding OX40L protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 232)
1    GGCCCTGGGA CCTTTGCCTA TTTTCTGATT GATAGGCTTT GTTTTGTCTT TACCTCCTTC
61   TTTCTGGGGA AAACTTCAGT TTTATCGCAC GTTCCCCTTT TCCATATCTT CATCTTCCCT
121  CTACCCAGAT TGTGAAGATG GAAAGGGTCC AACCCCTGGA AGAGAATGTG GGAAATGCAG
181  CCAGGCCAAG ATTCGAGAGG AACAAGCTAT TGCTGGTGGC CTCTGTAATT CAGGGACTGG
241  GGCTGCTCCT GTGCTTCACC TACATCTGCC TGCACTTCTC TGCTCTTCAG GTATCACATC
301  GGTATCCTCG AATTCAAAGT ATCAAAGTAC AATTTACCGA ATATAAGAAG GAGAAAGGTT
361  TCATCCTCAC TTCCCAAAAG GAGGATGAAA TCATGAAGGT GCAGAACAAC TCAGTCATCA
421  TCAACTGTGA TGGGTTTTAT CTCATCTCCC TGAAGGGCTA CTTCTCCCAG GAAGTCAACA
481  TTAGCCTTCA TTACCAGAAG GATGAGGAGC CCCTCTTCCA ACTGAAGAAG GTCAGGTCTG
541  TCAACTCCTT GATGGTGGCC TCTCTGACTT ACAAAGACAA AGTCTACTTG AATGTGACCA
601  CTGACAATAC CTCCCTGGAT GACTTCCATG TGAATGGCGG AGAACTGATT CTTATCCATC
661  AAAATCCTGG TGAATTCTGT GTCCTTTGAG GGGCTGATGG CAATATCTAA AACCAGGCAC
721  CAGCATGAAC ACCAAGCTGG GGGTGGACAG GGCATGGATT CTTCATTGCA AGTGAAGGAG
781  CCTCCCAGCT CAGCCACGTG GGATGTGACA AGAAGCAGAT CCTGGCCCTC CCGCCCCCAC
841  CCCTCAGGGA TATTTAAAAC TTATTTTATA TACCAGTTAA TCTTATTTAT CCTTATATTT
901  TCTAAATTGC CTAGCCGTCA CACCCCAAGA TTGCCTTGAG CCTACTAGGC ACCTTTGTGA
961  GAAAGAAAAA ATAGATGCCT CTTCTTCAAG ATGCATTGTT TCTATTGGTC AGGCAATTGT
1021 CATAATAAAC TTATGTCATT GAAAACGGTA CCTGACTACC ATTTGCTGGA AATTTGACAT
1081 GTGTGTGGCA TTATCAAAAT GAAGAGGAGC AAGGAGTGAA GGAGTGGGGT TATGAATCTG
1141 CCAAAGGTGG TATGAACCAA CCCCTGGAAG CCAAAGCGGC CTCTCCAAGG TTAAATTGAT
1201 TGCAGTTTGC ATATTGCCTA AATTTAAACT TTCTCATTTG GTGGGGGTTC AAAAGAAGAA
1261 TCAGCTTGTG AAAAATCAGG ACTTGAAGAG AGCCGTCTAA GAAATACCAC GTGCTTTTTT
1321 TCTTTACCAT TTTGCTTTCC CAGCCTCCAA ACATAGTTAA TAGAAATTTC CCTTCAAAGA
1381 ACTGTCTGGG GATGTGATGC TTTGAAAAAT CTAATCAGTG ACTTAAGAGA GATTTTCTTG
1441 TATACAGGGA GAGTGAGATA ACTTATTGTG AAGGGTTAGC TTTACTGTAC AGGATAGCAG
1501 GGAACTGGAC ATCTCAGGGT AAAAGTCAGT ACGGATTTTA ATAGCCTGGG GAGGAAAACA
1561 CATTCTTTGC CACAGACAGG CAAAGCAACA CATGCTCATC CTCCTGCCTA TGCTGAGATA
1621 CGCACTCAGC TCCATGTCTT GTACACACAG AAACATTGCT GGTTTCAAGA AATGAGGTGA
1681 TCCTATTATC AAATTCAATC TGATGTCAAA TAGCACTAAG AAGTTATTGT GCCTTATGAA
1741 AAATAATGAT CTCTGTCTAG AAATACCATA GACCATATAT AGTCTCACAT TGATAATTGA
1801 AACTAGAAGG GTCTATAATC AGCCTATGCC AGGGCTTCAA TGGAATAGTA TCCCCTTATG
1861 TTTAGTTGAA ATGTCCCCTT AACTTGATAT AATGTGTTAT GCTTATGGCG CTGTGGACAA
1921 TCTGATTTTT CATGTCAACT TTCCAGATGA TTTGTAACTT CTCTGTGCCA AACCTTTTAT
1981 AAACATAAAT TTTTGAGATA TGTATTTTAA AATTGTAGCA CATGTTTCCC TGACATTTTC
2041 AATAGAGGAT ACAACATCAC AGAATCTTTC TGGATGATTC TGTGTTATCA AGGAATTGTA
2101 CTGTGCTACA ATTATCTCTA GAATCTCCAG AAAGGTGGAG GGCTGTTCGC CCTTACACTA
2161 AATGGTCTCA GTTGGATTTT TTTTTCCTGT TTTCTATTTC CTCTTAAGTA CACCTTCAAC
2221 TATATTCCCA TCCCTCTATT TTAATCTGTT ATGAAGGAAG GTAAATAAAA ATGCTAAATA
2281 GAAGAAATTG TAGGTAAGGT AAGAGGAATC AAGTTCTGAG TGGCTGCCAA GGCACTCACA
2341 GAATCATAAT CATGGCTAAA TATTTATGGA GGGCCTACTG TGGACCAGGC ACTGGGCTAA
2401 ATACTTACAT TTACAAGAAT CATTCTGAGA CAGATATTCA ATGATATCTG GCTTCACTAC
2461 TCAGAAGATT GTGTGTGTGT TTGTGTGTGT GTGTGTGTGT GTATTTCACT TTTTGTTATT
2521 GACCATGTTC TGCAAAATTG CAGTTACTCA GTGAGTGATA TCCGAAAAAG TAAACGTTTA
2581 TGACTATAGG TAATATTTAA GAAAATGCAT GGTTCATTTT TAAGTTTGGA ATTTTTATCT
2641 ATATTTCTCA CAGATGTGCA GTGCACATGC AGGCCTAAGT ATATGTTGTG TGTGTTGTTT
2701 GTCTTTGATG TCATGGTCCC CTCTCTTAGG TGCTCACTCG CTTTGGGTGC ACCTGGCCTG
2761 CTCTTCCCAT GTTGGCCTCT GCAACCACAC AGGGATATTT CTGCTATGCA CCAGCCTCAC
2821 TCCACCTTCC TTCCATCAAA AATATGTGTG TGTGTCTCAG TCCCTGTAAG TCATGTCCTT
2881 CACAGGGAGA ATTAACCCTT CGATATACAT GGCAGAGTTT TGTGGGAAAA GAATTGAATG
2941 AAAAGTCAGG AGATCAGAAT TTTAAATTTG ACTTAGCCAC TAACTAGCCA TGTAACCTTG
3001 GGAAAGTCAT TTCCCATTTC TGGGTCTTGC TTTTCTTTCT GTTAAATGAG AGGAATGTTA
3061 AATATCTAAC AGTTTAGAAT CTTATGCTTA CAGTGTTATC TGTGAATGCA CATATTAAAT
3121 GTCTATGTTC TTGTTGCTAT GAGTCAAGGA GTGTAACCTT CTCCTTTACT ATGTTGAATG
3181 TATTTTTTTC TGGACAAGCT TACATCTTCC TCAGCCATCT TTGTGAGTCC TTCAAGAGCA
3241 GTTATCAATT GTTAGTTAGA TATTTTCTAT TTAGAGAATG CTTAAGGGAT TCCAATCCCG
3301 ATCCAAATCA TAATTTGTTC TTAAGTATAC TGGGCAGGTC CCCTATTTTA AGTCATAATT
3361 TTGTATTTAG TGCTTTCCTG GCTCTCAGAG AGTATTAATA TTGATATTAA TAATATAGTT
3421 AATAGTAATA TTGCTATTTA CATGGAAACA AATAAAAGAT CTCAGAATTC ACTAAAAAAA
3481 AAAA.

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding a OX40L protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of any one of SEQ ID NO: 7790 to SEQ ID NO: 11254.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding IL12 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 233)
1    TTTCGCTTTC ATTTTGGGCC GAGCTGGAGG CGGCGGGGCC GTCCCGGAAC GGCTGCGGCC
61   GGGCACCCCG GGAGTTAATC CGAAAGCGCC GCAAGCCCCG CGGGCCGGCC GCACCGCACG
121  TGTCACCGAG AAGCTGATGT AGAGAGAGAC ACAGAAGGAG ACAGAAAGCA AGAGACCAGA
181  GTCCCGGGAA AGTCCTGCCG CGCCTCGGGA CAATTATAAA AATGTGGCCC CCTGGGTCAG
241  CCTCCCAGCC ACCGCCCTCA CCTGCCGCGG CCACAGGTCT GCATCCAGCG GCTCGCCCTG
301  TGTCCCTGCA GTGCCGGCTC AGCATGTGTC CAGCGCGCAG CCTCCTCCTT GTGGCTACCC
361  TGGTCCTCCT GGACCACCTC AGTTTGGCCA GAAACCTCCC CGTGGCCACT CCAGACCCAG
421  GAATGTTCCC ATGCCTTCAC CACTCCCAAA ACCTGCTGAG GGCCGTCAGC AACATGCTCC
481  AGAAGGCCAG ACAAACTCTA GAATTTTACC CTTGCACTTC TGAAGAGATT GATCATGAAG
541  ATATCACAAA AGATAAAACC AGCACAGTGG AGGCCTGTTT ACCATTGGAA TTAACCAAGA
601  ATGAGAGTTG CCTAAATTCC AGAGAGACCT CTTTCATAAC TAATGGGAGT TGCCTGGCCT
661  CCAGAAAGAC CTCTTTTATG ATGGCCCTGT GCCTTAGTAG TATTTATGAA GACTTGAAGA
721  TGTACCAGGT GGAGTTCAAG ACCATGAATG CAAAGCTTCT GATGGATCCT AAGAGGCAGA
781  TCTTTCTAGA TCAAAACATG CTGGCAGTTA TTGATGAGCT GATGCAGGCC CTGAATTTCA
841  ACAGTGAGAC TGTGCCACAA AAATCCTCCC TTGAAGAACC GGATTTTTAT AAAACTAAAA
901  TCAAGCTCTG CATACTTCTT CATGCTTTCA GAATTCGGGC AGTGACTATT GATAGAGTGA
961  TGAGCTATCT GAATGCTTCC TAAAAAGCGA GGTCCCTCCA AACCGTTGTC ATTTTTATAA
1021 AACTTTGAAA TGAGGAAACT TTGATAGGAT GTGGATTAAG AACTAGGGAG GGGGAAAGAA
1081 GGATGGGACT ATTACATCCA CATGATACCT CTGATCAAGT ATTTTTGACA TTTACTGTGG
1141 ATAAATTGTT TTTAAGTTTT CATGAATGAA TTGCTAAGAA GGGAAAATAT CCATCCTGAA
1201 GGTGTTTTTC ATTCACTTTA ATAGAAGGGC AAATATTTAT AAGCTATTTC TGTACCAAAG
1261 TGTTTGTGGA AACAAACATG TAAGCATAAC TTATTTTAAA ATATTTATTT ATATAACTTG
1321 GTAATCATGA AAGCATCTGA GCTAACTTAT ATTTATTTAT GTTATATTTA TTAAATTATT
1381 TATCAAGTGT ATTTGAAAAA TATTTTTAAG TGTTCTAAAA ATAAAAGTAT TGAATTAAAG
1441 TGAAAAAAAA.

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding an IL12 protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of any one of SEQ ID NO: 11255 to SEQ ID NO: 12685.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule encoding CCR7 protein comprising or consisting of 20 nucleotides of the sequence of

(SEQ ID NO: 234)
1    CACTTCCTCC CCAGACAGGG GTAGTGCGAG GCCGGGCACA GCCTTCCTGT GTGGTTTTAC
61   CGCCCAGAGA GCGTCATGGA CCTGGGGAAA CCAATGAAAA GCGTGCTGGT GGTGGCTCTC
121  CTTGTCATTT TCCAGGTATG CCTGTGTCAA GATGAGGTCA CGGACGATTA CATCGGAGAC
181  AACACCACAG TGGACTACAC TTTGTTCGAG TCTTTGTGCT CCAAGAAGGA CGTGCGGAAC
241  TTTAAAGCCT GGTTCCTCCC TATCATGTAC TCCATCATTT GTTTCGTGGG CCTACTGGGC
301  AATGGGCTGG TCGTGTTGAC CTATATCTAT TTCAAGAGGC TCAAGACCAT GACCGATACC
361  TACCTGCTCA ACCTGGCGGT GGCAGACATC CTCTTCCTCC TGACCCTTCC CTTCTGGGCC
421  TACAGCGCGG CCAAGTCCTG GGTCTTCGGT GTCCACTTTT GCAAGCTCAT CTTTGCCATC
481  TACAAGATGA GCTTCTTCAG TGGCATGCTC CTACTTCTTT GCATCAGCAT TGACCGCTAC
541  GTGGCCATCG TCCAGGCTGT CTCAGCTCAC CGCCACCGTG CCCGCGTCCT TCTCATCAGC
601  AAGCTGTCCT GTGTGGGCAT CTGGATACTA GCCACAGTGC TCTCCATCCC AGAGCTCCTG
661  TACAGTGACC TCCAGAGGAG CAGCAGTGAG CAAGCGATGC GATGCTCTCT CATCACAGAG
721  CATGTGGAGG CCTTTATCAC CATCCAGGTG GCCCAGATGG TGATCGGCTT TCTGGTCCCC
781  CTGCTGGCCA TGAGCTTCTG TTACCTTGTC ATCATCCGCA CCCTGCTCCA GGCACGCAAC
841  TTTGAGCGCA ACAAGGCCAT CAAGGTGATC ATCGCTGTGG TCGTGGTCTT CATAGTCTTC
901  CAGCTGCCCT ACAATGGGGT GGTCCTGGCC CAGACGGTGG CCAACTTCAA CATCACCAGT
961  AGCACCTGTG AGCTCAGTAA GCAACTCAAC ATCGCCTACG ACGTCACCTA CAGCCTGGCC
1021 TGCGTCCGCT GCTGCGTCAA CCCTTTCTTG TACGCCTTCA TCGGCGTCAA GTTCCGCAAC
1081 GATCTCTTCA AGCTCTTCAA GGACCTGGGC TGCCTCAGCC AGGAGCAGCT CCGGCAGTGG
1141 TCTTCCTGTC GGCACATCCG GCGCTCCTCC ATGAGTGTGG AGGCCGAGAC CACCACCACC
1201 TTCTCCCCAT AGGCGACTCT TCTGCCTGGA CTAGAGGGAC CTCTCCCAGG GTCCCTGGGG
1261 TGGGGATAGG GAGCAGATGC AATGACTCAG GACATCCCCC CGCCAAAAGC TGCTCAGGGA
1321 AAAGCAGCTC TCCCCTCAGA GTGCAAGCCC CTGCTCCAGA AGATAGCTTC ACCCCAATCC
1381 CAGCTACCTC AACCAATGCC AAAAAAAGAC AGGGCTGATA AGCTAACACC AGACAGACAA
1441 CACTGGGAAA CAGAGGCTAT TGTCCCCTAA ACCAAAAACT GAAAGTGAAA GTCCAGAAAC
1501 TGTTCCCACC TGCTGGAGTG AAGGGGCCAA GGAGGGTGAG TGCAAGGGGC GTGGGAGTGG
1561 CCTGAAGAGT CCTCTGAATG AACCTTCTGG CCTCCCACAG ACTCAAATGC TCAGACCAGC
1621 TCTTCCGAAA ACCAGGCCTT ATCTCCAAGA CCAGAGATAG TGGGGAGACT TCTTGGCTTG
1681 GTGAGGAAAA GCGGACATCA GCTGGTCAAA CAAACTCTCT GAACCCCTCC CTCCATCGTT
1741 TTCTTCACTG TCCTCCAAGC CAGCGGGAAT GGCAGCTGCC ACGCCGCCCT AAAAGCACAC
1801 TCATCCCCTC ACTTGCCGCG TCGCCCTCCC AGGCTCTCAA CAGGGGAGAG TGTGGTGTTT
1861 CCTGCAGGCC AGGCCAGCTG CCTCCGCGTG ATCAAAGCCA CACTCTGGGC TCCAGAGTGG
1921 GGATGACATG CACTCAGCTC TTGGCTCCAC TGGGATGGGA GGAGAGGACA AGGGAAATGT
1981 CAGGGGCGGG GAGGGTGACA GTGGCCGCCC AAGGCCCACG AGCTTGTTCT TTGTTCTTTG
2041 TCACAGGGAC TGAAAACCTC TCCTCATGTT CTGCTTTCGA TTCGTTAAGA GAGCAACATT
2101 TTACCCACAC ACAGATAAAG TTTTCCCTTG AGGAAACAAC AGCTTTAAAA GAAAAAGAAA
2161 AAAAAAGTCT TTGGTAAATG GCAAAAAAAA AAAAAAAAAA AAAAAAA.

Exemplary gRNA spacer sequences of the disclosure that specifically bind to a target sequence of an RNA molecule encoding a CCR7 protein of the disclosure may comprise or consist of a nucleic acid having a sequence selected from any one of any one of SEQ ID NO: 12686 to SEQ ID NO: 14872.

Compositions of the disclosure may comprise a gRNA comprising a spacer sequence that specifically binds to a target sequence of an RNA molecule, wherein the spacer sequence and the target sequence are reverse complements of one another. In some embodiments, compositions of the disclosure may comprise a single (i.e., singular) gRNA comprising a) a first spacer sequence that specifically binds to a first target RNA sequence and b) a second spacer sequence that specifically binds to a second target RNA sequence, wherein the first and second spacer sequences each bind different target RNA sequences. In some embodiments, first and second spacer sequences which bind different target RNA sequences are not comprised within a single (i.e., singular) gRNA but rather a first spacer sequence is comprised within a first gRNA and a second spacer sequence is comprised within a second gRNA sequence. In some embodiments, a spacer sequence disclosed herein comprises a portion of a nucleic acid sequence encoding a protein component of the adaptive immune response, wherein the protein component is selected from the group consisting of Beta-2-microglobulin β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), and CC Chemokine Receptor 7 (CCR7). In some embodiments, a spacer which is a portion of a nucleic acid sequence encoding a protein component of an adaptive immune response is about 20 or 21 nucleotides in length.

All nucleotide sequences of the disclosure may include a uracil (U) or a thymine (T) interchangeably.

Exemplary, non-limiting Zika NS5 targeting spacer sequences of sgRNAs include, but are not limited to: gcaatgatcttcatgttgggagc (SEQ ID NO: 196), gaaccttgttgatgaactcttc (SEQ ID NO: 197), gttggtgattagagcttcattc (SEQ ID NO: 198), and gagtgatcctcgttcaagaatcc (SEQ ID NO: 199).

Exemplary, non-limiting lambda NS5 targeting spacer sequences of sgRNAs include, but are not limited to: GTGATAAGTGGAATGCCATG (SEQ ID NO: 200) and

GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAG UUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU

(SEQ ID NO: 201).

Methods of Simultaneous Treatment of Disease and Prevention of Immune Response

The disclosure provides compositons and methods for the simultaneous treatment of a disease or disorder in a subject by delivering a gene therapy to a cell and prevention of an immune response to the cell receiving the gene therapy. For example, the composition shown in FIG. 4 may be administered to a subject wherein gRNA 1 binds to a target sequence within an RNA molecule that encodes a component of an adapative immune response and gRNA2 binds to a target sequence within an RNA molecule associated with a disease or disorder. By targeting an RNA molecule that encodes a component of an adapative immune response gRNA1 prevents the display of an antigen associated with the composition or a vector comprising the composition on the surface of the cell, thereby masking the cell from the subject's immune system. gRNA2 simultaneously targets a second RNA molecule to treat a disease or disorder of the disclosure.

In alternative embodiments, gRNA1 and gRNA2 of the composition shown in FIG. 4, for example, can each target a distinct RNA molecule encoding a component of the adaptive immune response. For example, while gRNA1 targets an RNA molecule encoding a β2M polypeptide, gRNA2 targets a costimulatory molecule (ICOSLG, CD80, CD86, OX40L, IL12 or CCR7).

In some embodients, compositions of the disclosure may comprise or consist of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs.

In some embodiments, compositions of the disclosure may comprise or consist of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs, the expression of which is under the control of a constitutive promoter (e.g. U6) and a fusion protein comprising a first RNA binding protein and a second RNA binding protein, the expression of which fusion is under the control of a viral promoter, which may be optionally constitutive (e.g. EFS).

In some embodiments, compositions of the disclosure may comprise or consist of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs, the expression of which is under the control of a first promoter and a fusion protein comprising a first RNA binding protein and a second RNA binding protein, the expression of which fusion is under the control of a second promoter, wherein the first promoter drives stronger expression of at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 gRNAs that the second promoter drives expression of the fusion protein. In some embodiments, compositions of the disclosure may comprise or consist of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs, the expression of which is under the control of a first promoter and a fusion protein comprising a first RNA binding protein and a second RNA binding protein, the expression of which fusion is under the control of a second promoter, wherein the first promoter drives weaker expression of at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 gRNAs that the second promoter drives expression of the fusion protein. By varying the relative strength of the promoters driving expression of the gRNA versus fusion protein components of the compositions of the disclosure, the compositions may be provided in ratiometric doses while expressing the gRNA and fusion protein form the same vector. Thus, the compositions of the disclosure may comprise gRNAs that bind RNA molecules associated with two or more diseases as well as two or more components of an adaptive immune response. In some embodiments, the compositions of the disclosure may comprise fusion proteins disclosed herein, wherein at least one of the fusion partner proteins is an endonuclease such as, without limitation, RNAse1, RNAse4, RNAse6, RNAse7, RNAse8, RNAse2, RNAse6PL, RNAseL, RNAseT2, RNAse11, RNAseT2-like, NOB1, ENDOV, ENDOG, ENDOD1, hFEN1, hSLFN14, hLACTB2, APEX2, ANG, HRSP12, ZC3H12A, RIDA, PDL6, NTHL, KIAA0391, APEX1, AGO2, EXOG, ZC3H12D, ERN2, PELO, YBEY, CPSF4L, hCG 2002731, ERCC1, RAC1, RAA1, RAB1, DNA2, F1135220, F1113173, ERCC4, RNAse1(K41R), RNAse1(K41R, D121E), RNAse1(K41R, D121E, H119N), RNAse1(H119N), RNAse1(R39D, N67D, N88A, G89D, R91D, H119N), RNAsel(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E), RNAsel(R39D, N67D, N88A, G89D, R91D), TENM1, TENM2, RNAseK, TALEN, ZNF638, or PIN of hSMG6.

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 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 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, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, or Wegener's granulomatosis.

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, the inflammatory disease or disorder includes, but is not limited to, Alzheimer's disease, ankylosing spondylitis, arthritis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, ulcerative colitis, acute bronchitis, acute appendicitis, tonsillitis, infective meningitis, sinusitis, asthma, chronic peptic ulcer, tuberculosis, rheumatoid arthritis, periodontitis, gout, Scleroderma, vasculitis, and myositis.

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, Sézary 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.

EXAMPLES

Example 1

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 1×10̂5 cells per well of a 24-well plate for RNA isolation or 0.5×10̂5 cells per well. Cells were transfected with plasmids encoding Campylobacter jejuni Cas9 (CjeCas9) fused to the gene NTHL1 (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 8. The sequences of the constructs used in this stud are presented below (SEQ ID NO: 13656 and SEQ ID NO: 13657).

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 7.

FIG. 1 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 8. 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. 2A 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 a CjeCas9-endonuclease fusion loaded with a non-Zika NS5 targeting sgRNA (FIGS. 2A and 2B). FIG. 3 is a list of exemplary endonucleases for use in the compositions of the disclosure.

TABLE 7
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 8
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)

A E43-CjeCas9 and sgRNA plasmid may comprise or consist of the sequence (U6:

N′s=sgRNA spacer, E43, CieCas9):

(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
GAGAGGACTTCAAAAAATCAGGTCCA              CCCAAGAAAAAACGCAAGGTGGAA
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
GTCCA              CCCAAGAAAAAACGCAAGGTGGAAGATCCGAAGAAAAAGCGAAAA
GTGGATGTGtaaCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCG
GGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCT
TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGC
ATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTAT
CTTATCATGTCTGTATACCG.

Example Embodiments

• Embodiment 1. A composition comprising:

(a) a first sequence comprising a first guide RNA (gRNA) that specifically binds a target sequence within an RNA molecule, wherein the target sequence comprises a sequence encoding a component of an adaptive immune response 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.

• Embodiment 2. A composition comprising: (a) a first sequence comprising a first guide RNA (gRNA) that specifically binds a first target sequence within a first RNA molecule, wherein the first target sequence comprises a sequence encoding a component of an adaptive immune response and

(b) a second sequence comprising a second guide RNA (gRNA) that specifically binds a second target sequence within a second RNA molecule and

(c) 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.

• Embodiment 3. The composition of embodiment 2, wherein the first target sequence or the second target sequence comprises at least one repeated sequence.
• Embodiment 4. The composition of embodiment 2, wherein the first sequence comprising the first gRNA further comprises a first promoter capable of expressing the gRNA in a eukaryotic cell and/or the second sequence comprising the second gRNA further comprises a second promoter capable of expressing the gRNA in a eukaryotic cell.
• Embodiment 5. The composition of embodiment 2, wherein a sequence comprising the first sequence comprising the first gRNA and the second sequence comprising the second gRNA comprises a promoter capable of expressing the first gRNA and the second gRNA in a eukaryotic cell.
• Embodiment 6. The composition of embodiment 4, wherein the first promoter and the second promoter are identical.
• Embodiment 7. The composition of embodiment 4, wherein the first promoter and the second promoter are not identical.
• Embodiment 8. The composition of any one of embodiments 4-7, wherein the eukaryotic cell is an animal cell.
• Embodiment 9. The composition of embodiment 8, wherein the animal cell is a mammalian cell.
• Embodiment 10. The composition of embodiment 9, wherein the animal cell is a human cell.
• Embodiment 11. The composition of any one of embodiments 5-10, wherein the promoter is a constitutively active promoter.
• Embodiment 12. The composition of any one of embodiments 5-11, wherein the promoter comprises a sequence isolated or derived from a promoter capable of driving expression of an RNA polymerase.
• Embodiment 13. The composition of embodiment 12, wherein the promoter comprises a sequence isolated or derived from a U6 promoter.
• Embodiment 14. The composition of any one of embodiments 5-12, wherein the promoter comprises a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA).
• Embodiment 15. The composition of embodiment 14, wherein the promoter comprises a sequence 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 16. The composition of embodiment 14, wherein the promoter comprises a sequence isolated or derived from a valine tRNA promoter.
• Embodiment 17. The composition of any one of embodiments 2-16, wherein the sequence comprising the first gRNA further comprises a first spacer sequence that specifically binds to the first target RNA sequence.
• Embodiment 18. The composition of embodiment 17, wherein the first 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 first target RNA sequence.
• Embodiment 19. The composition of embodiment 17, wherein the first spacer sequence has 100% complementarity to the target RNA sequence.
• Embodiment 20. The composition of any one of embodiments 17-19, wherein the first spacer sequence comprises or consists of 20 nucleotides.
• Embodiment 21. The composition of any one of embodiments 17-19, wherein the first spacer sequence comprises or consists of 21 nucleotides.
• Embodiment 22. The composition of embodiment 21, wherein the first spacer sequence comprises or consists of 20 nucleotides of an amino acid sequence encoding a Beta-2-microglobulin β2M) protein.
• Embodiment 23. The composition of embodiment 22, wherein the first spacer sequence comprises or consists of 20 nucleotides of an amino acid sequence of

(SEQ ID NO: 88)
MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPADIEVD
LLKNGERIEK VEHSDLSFSK DWSFYLLYYT EFTPTEKDEY
ACRVNHVTLS QPKIVKWDRD M.

• Embodiment 24. The composition of any one of embodiments 2-23, wherein the sequence comprising the first gRNA further comprises a first scaffold sequence that specifically binds to the first RNA binding protein.
• Embodiment 25. The composition of embodiment 24, wherein the first scaffold sequence comprises a stem-loop structure.
• Embodiment 26. The composition of embodiment 24 or 25, wherein the scaffold sequence comprises or consists of 90 nucleotides.
• Embodiment 27. The composition of embodiment 24 or 25, wherein the scaffold sequence comprises or consists of 93 nucleotides.
• Embodiment 28. The composition of embodiment 27, wherein the scaffold sequence comprises the sequence

(SEQ ID NO: 12)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
or
(SEQ ID NO: 13)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

• Embodiment 29. The composition of any one of embodiments 1-28, wherein the sequence comprising the second gRNA further comprises a second spacer sequence that specifically binds to the second target RNA sequence.
• Embodiment 30. The composition of embodiment 29, wherein the second 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 first target RNA sequence.
• Embodiment 31. The composition of embodiment 29, wherein the second spacer sequence has 100% complementarity to the target RNA sequence.
• Embodiment 32. The composition of any one of embodiments 29-31, wherein the second spacer sequence comprises or consists of 20 nucleotides.
• Embodiment 33. The composition of any one of embodiments 29-31, wherein the second spacer sequence comprises or consists of 21 nucleotides.
• Embodiment 34. The composition of any one of embodiments 2-34, wherein the second spacer sequence comprises or further comprises 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.
• Embodiment 35. The composition of any one of embodiments 2-34, wherein the sequence comprising the second gRNA further comprises a second scaffold sequence that specifically binds to the first RNA binding protein.
• Embodiment 36. The composition of embodiment 35, wherein the second scaffold sequence comprises a stem-loop structure.
• Embodiment 37. The composition of embodiment 35 or 36, wherein the second scaffold sequence comprises or consists of 85 nucleotides.
• Embodiment 38. The composition of embodiment 37, wherein the second scaffold sequence comprises the sequence

(SEQ ID NO: 12)
GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGU
CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
or
(SEQ ID NO: 13)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

• Embodiment 39. The composition of embodiment 1, wherein the gRNA does not bind or does not selectively bind to a second sequence within the RNA molecule.
• Embodiment 40. The composition of any one of embodiments 2-38, wherein the first gRNA does not bind or does not selectively bind to a second sequence within the first RNA molecule.
• Embodiment 41. The composition of any one of embodiments 2-38, wherein the second gRNA does not bind or does not selectively bind to a second sequence within the second RNA molecule.
• Embodiment 42. The composition of embodiment 39, wherein an RNA genome or an RNA transcriptome comprises the RNA molecule.
• Embodiment 43. The composition of embodiment 40 or 41, wherein an RNA genome or an RNA transcriptome comprises the first RNA molecule or the second RNA molecule.
• Embodiment 44. The composition of any one of embodiments 1-43, wherein the first RNA binding protein comprises a CRISPR-Cas protein.
• Embodiment 45. The composition of embodiment 44, wherein the CRISPR-Cas protein is a Type II CRISPR-Cas protein.
• Embodiment 46. The composition of embodiment 45, wherein the first RNA binding protein comprises a Cas9 polypeptide or an RNA-binding portion thereof.
• Embodiment 47. The composition of embodiment 44, wherein the CRISPR-Cas protein is a Type V CRISPR-Cas protein.
• Embodiment 48. The composition of embodiment 47, wherein the first RNA binding protein comprises a Cpf1 polypeptide or an RNA-binding portion thereof.
• Embodiment 49. The composition of embodiment 44, wherein the CRISPR-Cas protein is a Type VI CRISPR-Cas protein.
• Embodiment 50. The composition of embodiment 49, wherein the first RNA binding protein comprises a Cas13 polypeptide or an RNA-binding portion thereof.
• Embodiment 51. The composition of any one of embodiments 44-50, wherein the CRISPR-Cas protein comprises a native RNA nuclease activity.
• Embodiment 52. The composition of embodiment 51, wherein the native RNA nuclease activity is reduced or inhibited.
• Embodiment 53. The composition of embodiment 52, wherein the native RNA nuclease activity is increased or induced.
• Embodiment 54. The composition of any one of embodiments 44-53, wherein the CRISPR-Cas protein comprises a native DNA nuclease activity and wherein the native DNA nuclease activity is inhibited.
• Embodiment 55. The composition of embodiment 54, wherein the CRISPR-Cas protein comprises a mutation.
• Embodiment 56. The composition of embodiment 54 or 55, wherein a nuclease domain of the CRISPR-Cas protein comprises the mutation.
• Embodiment 57. The composition of any one of embodiments 54-56, wherein the mutation occurs in a nucleic acid encoding the CRISPR-Cas protein.
• Embodiment 58. The composition of any one of embodiments 54-56, wherein the mutation occurs in an amino acid encoding the CRISPR-Cas protein.
• Embodiment 59. The composition of any one of embodiments 54-58, wherein the mutation comprises a substitution, an insertion, a deletion, a frameshift, an inversion, or a transposition.
• Embodiment 60. The composition of embodiment 59, 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 61. The composition of any one of embodiments 1-43, wherein the first RNA binding protein comprises a Pumilio and FBF (PUF) protein.
• Embodiment 62. The composition of embodiment 61, wherein the first RNA binding protein comprises a Pumilio-based assembly (PUMBY) protein.
• Embodiment 63. The composition of any one of embodiments 1-56, wherein the first RNA binding protein does not require multimerization for RNA-binding activity.
• Embodiment 64. The composition of embodiment 63, wherein the first RNA binding protein is not a monomer of a multimer complex
• Embodiment 65. The composition of embodiment 63, wherein a multimer protein complex does not comprise the first RNA binding protein.
• Embodiment 66. The composition of any one of embodiments 1-65, wherein the first RNA binding protein selectively binds to a target sequence within the RNA molecule.
• Embodiment 67. The composition of embodiment 66, wherein the first RNA binding protein does not comprise an affinity for a second sequence within the RNA molecule.
• Embodiment 68. The composition of embodiment 66 or 67, wherein the first RNA binding protein does not comprise a high affinity for or selectively bind a second sequence within the RNA molecule.
• Embodiment 69. The composition of embodiment 68, wherein an RNA genome or an RNA transcriptome comprises the RNA molecule.
• Embodiment 70. The composition of any one of embodiments 1-69, wherein the first RNA binding protein comprises between 2 and 1300 amino acids, inclusive of the endpoints.
• Embodiment 71. The composition of any one of embodiments 1-70, wherein the sequence encoding the first RNA binding protein further comprises a nuclear localization signal (NLS).
• Embodiment 72. The composition of embodiment 71, wherein the sequence encoding a nuclear localization signal (NLS) is positioned 3′ to the sequence encoding the first RNA binding protein.
• Embodiment 73. The composition of embodiment 72, wherein the first RNA binding protein comprises an NLS at a C-terminus of the protein.
• Embodiment 74. The composition of any one of embodiments 1-70, 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 75. The composition of embodiment 74, wherein the sequence encoding the first NLS or the second NLS is positioned 3′ to the sequence encoding the first RNA binding protein.
• Embodiment 76. The composition of embodiment 75, wherein the first RNA binding protein comprises the first NLS or the second NLS at a C-terminus of the protein.
• Embodiment 77. The composition of any one of embodiments 1-76, wherein the second RNA binding protein comprises or consists of a nuclease domain.
• Embodiment 78. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an RNAse.
• Embodiment 79. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse1.
• Embodiment 80. The composition of embodiment 79, wherein the RNAse1 protein comprises or consists of SEQ ID NO: 20.
• Embodiment 81. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse4.
• Embodiment 82. The composition of embodiment 81, wherein the RNAse4 protein comprises or consists of SEQ ID NO: 21.
• Embodiment 83. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse6.
• Embodiment 84. The composition of embodiment 83, wherein the RNAse6 protein comprises or consists of SEQ ID NO: 22.
• Embodiment 85. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse7.
• Embodiment 86. The composition of embodiment 85, wherein the RNAse7 protein comprises or consists of SEQ ID NO: 23.
• Embodiment 87. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse8.
• Embodiment 88. The composition of embodiment 87, wherein the RNAse8 protein comprises or consists of SEQ ID NO: 24.
• Embodiment 89. The composition of embodiment 88, wherein the second RNA binding protein comprises or consists of an RNAse2.
• Embodiment 90. The composition of embodiment 89, wherein the RNAse2 protein comprises or consists of SEQ ID NO: 25.
• Embodiment 91. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse6PL.
• Embodiment 92. The composition of embodiment 91, wherein the RNAse6PL protein comprises or consists of SEQ ID NO: 26.
• Embodiment 93. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAseL.
• Embodiment 94. The composition of embodiment 93, wherein the RNAseL protein comprises or consists of SEQ ID NO: 27.
• Embodiment 95. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAseT2.
• Embodiment 96. The composition of embodiment 95, wherein the RNAseT2 protein comprises or consists of SEQ ID NO: 28.
• Embodiment 97. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAse11.
• Embodiment 98. The composition of embodiment 97, wherein the RNAse11 protein comprises or consists of SEQ ID NO: 29.
• Embodiment 99. The composition of embodiment 78, wherein the second RNA binding protein comprises or consists of an RNAseT2-like.
• Embodiment 100. The composition of embodiment 99, wherein the RNAseT2-like protein comprises or consists of SEQ ID NO: 30.
• Embodiment 101. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a NOB1 polypeptide.
• Embodiment 102. The composition of embodiment 101, wherein the NOB1 polypeptide comprises or consists of SEQ ID NO: 31.
• Embodiment 103. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an endonuclease.
• Embodiment 104. The composition of embodiment 103, wherein the second RNA binding protein comprises or consists of an endonuclease V (ENDOV) polypeptide.
• Embodiment 105. The composition of embodiment 104, wherein the ENDOV protein comprises or consists of SEQ ID NO: 32.
• Embodiment 106. The composition of embodiment 103, wherein the second RNA binding protein comprises or consists of an endonuclease G (ENDOG).
• Embodiment 107. The composition of embodiment 106, wherein the ENDOG protein comprises or consists of SEQ ID NO: 33.
• Embodiment 108. The composition of embodiment 103, wherein the second RNA binding protein comprises or consists of an endonuclease D1 (ENDOD1) polypeptide.
• Embodiment 109. The composition of embodiment 108, wherein the ENDOD1 comprises or consists of SEQ ID NO: 34.
• Embodiment 110. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Human flap endonuclease-1 (hFEN1) polypeptide.
• Embodiment 111. The composition of embodiment 110, wherein the hFEN1 protein comprises or consists of SEQ ID NO: 35.
• Embodiment 112. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a human Schlafen 14 (hSLFN14) polypeptide.
• Embodiment 113. The composition of embodiment 112, wherein the hSLFN14 polypeptide comprises or consists of SEQ ID NO: 36.
• Embodiment 114. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a human beta-lactamase-like protein 2 (hLACTB2) polypeptide.
• Embodiment 115. The composition of embodiment 114, wherein the hLACTB2 polypeptide comprises or consists of SEQ ID NO: 37.
• Embodiment 116. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an apurinic/apyrimidinic (AP) endodeoxyribonuclease (APEX2) polypeptide.
• Embodiment 117. The composition of embodiment 116, wherein the APEX2 polypeptide comprises or consists of SEQ ID NO: 38.
• Embodiment 118. The composition of embodiment 116, wherein the APEX2 polypeptide comprises or consists of SEQ ID NO: 39.
• Embodiment 119. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an angiogenin (ANG) polypeptide.
• Embodiment 120. The composition of embodiment 119, wherein the ANG polypeptide comprises or consists of SEQ ID NO: 40.
• Embodiment 121. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a heat responsive protein 12 (HRSP12) polypeptide.
• Embodiment 122. The composition of embodiment 121, wherein the HRSP12 polypeptide comprises or consists of SEQ ID NO: 41.
• Embodiment 123. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Zinc Finger CCCH-Type Containing 12A (ZC3H12A) polypeptide.
• Embodiment 124. The composition of embodiment 123, wherein the ZC3H12A polypeptide comprises or consists of SEQ ID NO: 42.
• Embodiment 125. The composition of embodiment 124, wherein the ZC3H12A polypeptide comprises or consists of SEQ ID NO: 43.
• Embodiment 126. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Reactive Intermediate Imine Deaminase A (RIDA) polypeptide.
• Embodiment 127. The composition of embodiment 126, wherein the RIDA polypeptide comprises or consists of SEQ ID NO: 44.
• Embodiment 128. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Phospholipase D Family Member 6 (PDL6) polypeptide.
• Embodiment 129. The composition of embodiment 128, wherein the PDL6 polypeptide comprises or consists of SEQ ID NO: 126.
• Embodiment 130. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Endonuclease III-like protein 1 (NTHL) polypeptide.
• Embodiment 131. The composition of embodiment 130, wherein the NTHL polypeptide comprises or consists of SEQ ID NO: 123.
• Embodiment 132. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Mitochondrial ribonuclease P catalytic subunit (KIAA0391) polypeptide.
• Embodiment 133. The composition of embodiment 132, wherein the KIAA0391 polypeptide comprises or consists of SEQ ID NO: 127.
• Embodiment 134. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an apurinic or apyrimidinic site lyase (APEX1) polypeptide.
• Embodiment 135. The composition of embodiment 134, wherein the APEX1 polypeptide comprises or consists of SEQ ID NO: 125.
• Embodiment 136. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an argonaute 2 (AGO2) polypeptide.
• Embodiment 137. The composition of embodiment 136, wherein the AGO2 polypeptide comprises or consists of SEQ ID NO: 128.
• Embodiment 138. The composition of embodiment 67, wherein the second RNA binding protein comprises or consists of a mitochondrial nuclease EXOG (EXOG) polypeptide.
• Embodiment 139. The composition of embodiment 138, wherein the EXOG polypeptide comprises or consists of SEQ ID NO: 129.
• Embodiment 140. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Zinc Finger CCCH-Type Containing 12D (ZC3H12D) polypeptide.
• Embodiment 141. The composition of embodiment 140, wherein the ZC3H12D polypeptide comprises or consists of SEQ ID NO: 130.
• Embodiment 142. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an endoplasmic reticulum to nucleus signaling 2 (ERN2) polypeptide.
• Embodiment 143. The composition of embodiment 142, wherein the ERN2 polypeptide comprises or consists of SEQ ID NO: 131.
• Embodiment 144. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a pelota mRNA surveillance and ribosome rescue factor (PELO) polypeptide.
• Embodiment 145. The composition of embodiment 144, wherein the PELO polypeptide comprises or consists of SEQ ID NO: 132.
• Embodiment 146. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a YBEY metallopeptidase (YBEY) polypeptide.
• Embodiment 147. The composition of embodiment 146, wherein the YBEY polypeptide comprises or consists of SEQ ID NO: 133.
• Embodiment 148. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a cleavage and polyadenylation specific factor 4 like (CPSF4L) polypeptide.
• Embodiment 149. The composition of embodiment 148, wherein the CPSF4L polypeptide comprises or consists of SEQ ID NO: 134.
• Embodiment 150. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an hCG_2002731polypeptide.
• Embodiment 151. The composition of embodiment 150, wherein the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 135.
• Embodiment 152. The composition of embodiment 150, wherein the hCG_2002731 polypeptide comprises or consists of SEQ ID NO: 136.
• Embodiment 153. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of an Excision Repair Cross-Complementation Group 1 (ERCC1) polypeptide.
• Embodiment 154. The composition of embodiment 153, wherein the ERCC1 polypeptide comprises or consists of SEQ ID NO: 137.
• Embodiment 155. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a ras-related C3 botulinum toxin substrate 1 isoform (RAC1) polypeptide.
• Embodiment 156. The composition of embodiment 155, wherein the RAC1 polypeptide comprises or consists of SEQ ID NO: 138.
• Embodiment 157. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Ribonuclease A A1 (RAA1) polypeptide.
• Embodiment 158. The composition of embodiment 157, wherein the RAA1 polypeptide comprises or consists of SEQ ID NO: 139.
• Embodiment 159. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a Ras Related Protein (RAB1) polypeptide.
• Embodiment 160. The composition of embodiment 159, wherein the RAB1 polypeptide comprises or consists of SEQ ID NO: 140.
• Embodiment 161. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a DNA Replication Helicase/Nuclease 2 (DNA2) polypeptide.
• Embodiment 162. The composition of embodiment 161, wherein the DNA2 polypeptide comprises or consists of SEQ ID NO: 141.
• Embodiment 163. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a FLJ35220 polypeptide.
• Embodiment 164. The composition of embodiment 163, wherein the F1135220 polypeptide comprises or consists o SEQ ID NO: 142.
• Embodiment 165. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a FLJ13173 polypeptide.
• Embodiment 166. The composition of embodiment 165, wherein the FLJ13173 polypeptide comprises or consists of SEQ ID NO: 143.
• Embodiment 167. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a DNA repair endonuclease XPF (ERCC4) polypeptide.
• Embodiment 168. The composition of embodiment 167, wherein the ERCC4 polypeptide comprises or consists of SEQ ID NO: 124.
• Embodiment 169. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(K41R)) polypeptide.
• Embodiment 170. The composition of embodiment 169, wherein the Rnase1(K41R) polypeptide comprises or consists of SEQ ID NO: 116.
• Embodiment 171. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E)) polypeptide.
• Embodiment 172. The composition of embodiment 171, wherein the Rnase1 (Rnase1(K41R, D121E)) polypeptide comprises or consists of SEQ ID NO: 117.
• Embodiment 173. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(K41R, D121E, H119N)) polypeptide.
• Embodiment 174. The composition of embodiment 173, wherein the Rnase1 (Rnase1(K41R, D121E, H119N)) polypeptide comprises or consists of SEQ ID NO: 118.
• Embodiment 175. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(H119N)) polypeptide.
• Embodiment 166. The composition of embodiment 175, wherein the Rnase1 (Rnase1(H119N)) polypeptide comprises or consists of SEQ ID NO: 119.
• Embodiment 177. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
• Embodiment 178. The composition of embodiment 177, wherein the Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide comprises or consists of SEQ ID NO: 120.
• Embodiment 179. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
• Embodiment 180. The composition of embodiment 179, wherein the Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E)) polypeptide comprises or consists of SEQ ID NO: 121.
• Embodiment 181. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of a mutated Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D, H119N)) polypeptide.
• Embodiment 182. The composition of embodiment 181, wherein the Rnase1 (Rnase1(R39D, N67D, N88A, G89D, R91D)) polypeptide comprises or consists of SEQ ID NO: 122.
• Embodiment 183. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of Teneurin Transmembrane Protein 1 (TENM1) polypeptide.
• Embodiment 184. The composition of embodiment 173, wherein the TENM1 polypeptide comprises or consists of SEQ ID NO: 144.
• Embodiment 185. The composition of embodiment 77, wherein the second RNA binding protein comprises or consists of Teneurin Transmembrane Protein 2 (TENM2) polypeptide.
• Embodiment 186. The composition of embodiment 185, wherein the TENM2 polypeptide comprises or consists of SEQ ID NO: 145.
• Embodiment 187. The composition of any one of embodiments 1-77, wherein the second RNA binding protein comprises or consists of a transcription activator-like effector nuclease (TALEN) polypeptide or a nuclease domain thereof.
• Embodiment 188. The composition of embodiment 187, wherein 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.

• Embodiment 189. The composition of embodiment 187, wherein 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.

• Embodiment 190. The composition of any one of embodiments 1-77, wherein the second RNA binding protein comprises or consists of a zinc finger nuclease polypeptide or a nuclease domain thereof.
• Embodiment 191. The composition of embodiment 190, wherein the zinc finger nuclease 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.

• Embodiment 192. The composition of any one of embodiments 1-191, wherein the composition further comprises (a) a sequence comprising a gRNA that specifically binds within an RNA molecule and

(b) a sequence encoding a nuclease.

• Embodiment 193. The composition of embodiment 192, wherein the nuclease comprises a sequence isolated or derived from a CRISPR/Cas protein.
• Embodiment 194. The composition of embodiment 193, wherein 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 ITC, a type V, or a type VI CRISPR/Cas protein.
• Embodiment 195. The composition of embodiment 192, wherein the nuclease comprises a sequence isolated or derived from a TALEN or a nuclease domain thereof.
• Embodiment 196. The composition of embodiment 192, wherein the nuclease comprises a sequence isolated or derived from a zinc finger nuclease or a nuclease domain thereof.
• Embodiment 197. The composition of any one of embodiments 191-196, wherein the target sequence comprises a sequence encoding a component of an adaptive immune response.
• Embodiment 198. A vector comprising the composition of any one of embodiments 1-197.
• Embodiment 199. The vector of embodiment 198, wherein the vector is a viral vector.
• Embodiment 200. The vector of embodiment 199, wherein the vector comprises a sequence isolated or derived from a lentivirus, an adenovirus, an adeno-associated virus (AAV) vector, or a retrovirus.
• Embodiment 201. The vector of embodiment 199 or 200, wherein the vector is replication incompetent.
• Embodiment 202. The vector of embodiment any one of embodiments 100-201, wherein the vector comprises a sequence isolated or derived from an adeno-associated vector (AAV).
• Embodiment 203. The vector of embodiment 202, wherein the adeno-associated virus (AAV) is an isolated AAV.
• Embodiment 204. The vector of embodiment 202 or 203, wherein the adeno-associated virus (AAV) is a self-complementary adeno-associated virus (scAAV).
• Embodiment 205. The vector of any one of embodiments 202-204, wherein the adeno-associated virus (AAV) is a recombinant adeno-associated virus (rAAV).
• Embodiment 206. The vector of any one of embodiments 202-205, wherein the adeno-associated virus (AAV) comprises a sequence isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.
• Embodiment 207. The vector of any one of embodiments 202-206, wherein the adeno-associated virus (AAV) comprises a sequence isolated or derived from an AAV of serotype AAV9.
• Embodiment 208. The vector of any one of embodiments 202-206, wherein the adeno-associated virus (AAV) comprise a sequence isolated or derived from Anc80
• Embodiment 209. The vector of any one of embodiments 100-201, wherein the vector is a retrovirus.
• Embodiment 210. The vector of embodiment any one of claims 100-201, wherein the retrovirus is a lentivirus.
• Embodiment 211. The vector of embodiment 198, wherein the vector is a non-viral vector.
• Embodiment 212. The vector of embodiment 211, wherein the non-viral vector comprises a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
• Embodiment 213. A composition comprising the vector of any one of embodiments 198-212.
• Embodiment 214. A cell comprising the vector of any one of embodiments 198-212.
• Embodiment 215. A cell comprising the composition of embodiment 214.
• Embodiment 216. The cell of embodiment 214 or 215, wherein the cell is a mammalian cell.
• Embodiment 217. The cell of embodiment 216, wherein the cell is a human cell.
• Embodiment 218. The cell of any one of embodiments 215-217, whereinthe cell is an immune cell.
• Embodiment 219. The cell of embodiment 218, wherein the immune cell is a T lymphocyte (T-cell).
• Embodiment 220. The cell of embodiment 219, wherein the T-cell is an effector T-cell, a helper T-cell, a memory T-cell, a regulatory T-cell, a natural Killer T-cell, a mucosal-associated invariant T-cell, or a gamma delta T cell.
• Embodiment 221. The cell of any one of embodiments 215-217, whereinthe immune cell is an antigen presenting cell.
• Embodiment 222. The cell of embodiment 221, wherein the antigen presenting cell is a dendritic cell, a macrophage, or a B cell.
• Embodiment 223. The cell of embodiment 221, wherein the antigen presenting cell is a somatic cell.
• Embodiment 224. The cell of any one of embodiments 215-223, wherein the cell is a healthy cell.
• Embodiment 225. The cell of any one of embodiments 215-223, wherein the cell is not a healthy cell.
• Embodiment 226. The cell of embodiment 225, where the cell is isolated or derived from a subject having a disease or disorder.
• Embodiment 227. A composition comprising the cell of any one of embodiments 215-226.
• Embodiment 228. A method of masking a cell from an adaptive immune response comprising contacting a composition of any one of embodiments 1-197, 213 or 227 to the cell to produce a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the RNA molecule encodes a component of an adaptive immune response.
• Embodiment 229. The method of embodiment 228, wherein the cell is in vivo, in vitro, ex vivo or in situ.
• Embodiment 230. The method of embodiment 228, wherein the cell is in vitro or ex vivo.
• Embodiment 231. The method of any one of embodiments 228-230, wherein a plurality of cells comprises the cell.
• Embodiment 232. The method of embodiment 231, wherein each cell of the plurality of cells contacts the composition, thereby producing a plurality of modified cells.
• Embodiment 233. The method of any one of embodiments 228-230, wherein the method further comprises administering the modified cell to a subject.
• Embodiment 234. The method of any one of embodiments 231-232, wherein the method further comprises administering the plurality of modified cells to a subject.
• Embodiment 235. The method of embodiment 233, wherein the cell is autologous.
• Embodiment 236. The method of embodiment 233, wherein the cell is allogeneic.
• Embodiment 237. The method of embodiment 233, wherein the plurality of modified cells is autologous.
• Embodiment 238. The method of embodiment 233, wherein the plurality of modified cells is allogeneic.
• Embodiment 239. The method of any one of embodiments 228-238, wherein the component of an adaptive immune response comprises or consists of a component of a type I major histocompatibility complex (MHC I), a type II major histocompatibility complex (MHC II), a T-cell receptor (TCR), a costimulatory molecule or a combination thereof.
• Embodiment 240. The method of embodiment 239, wherein the MHC I component comprises an α1 chain, an α2 chain, an α3 chain, or a β2M protein.
• Embodiment 241. The method of any one of embodiments 228-238, wherein the component of an adaptive immune response comprises or consists of an MHC I β2M protein.
• Embodiment 242. The method of embodiment 239, wherein the MHC II component comprises an α1 chain, an α2 chain, a β1 chain, or a β2 chain.
• Embodiment 243. The method of embodiment 239, wherein the TCR component comprises an α-chain and a β-chain.
• Embodiment 244. The method of embodiment 239, wherein the costimulatory molecule comprises a Cluster of Differentiation 28 (CD28), a Cluster of Differentiation 80 (CD80), a Cluster of Differentiation 86 (CD86), an Inducible T-cell COStimulator (ICOS), or an ICOS Ligand (ICOSLG) protein.
• Embodiment 245, A method of preventing or reducing an adaptive immune response in a subject comprising administering a therapeutically effective amount of a composition of any one of embodiments 1-197, 213 or 227 to the subject, wherein the composition contacts at least one cell in the subject producing a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the RNA molecule encodes a component of an adaptive immune response.
• Embodiment 246. A method of treating a disease or disorder in a subject comprising administering a therapeutically effective amount of a composition of any one of embodiments 1-197, 213 or 227 to the subject, wherein the composition contacts at least one cell in the subject producing a modified cell, wherein the composition modifies a level of expression of an RNA molecule of the modified cell and wherein the composition prevents or reduces an adaptive immune response to the modified cell.
• Embodiment 247. The method of embodiment 246, wherein the component of an adaptive immune response comprises or consists of a component of a type I major histocompatibility complex (MHC I), a type II major histocompatibility complex (MHC II), a T-cell receptor (TCR), a costimulatory molecule or a combination thereof.
• Embodiment 248. The method of embodiment 247, wherein the MHC I component comprises an α1 chain, an α2 chain, an α3 chain, or a β2M protein.
• Embodiment 249. The method of embodiment 247 or 248, wherein the component of an adaptive immune response comprises or consists of an MHC I β2M protein.
• Embodiment 250. The method of embodiment 249, wherein the MHC II component comprises an α1 chain, an α2 chain, a β1 chain, or a β2 chain.
• Embodiment 251. The method of embodiment 247, wherein the TCR component comprises an α-chain and a β-chain.
• Embodiment 252. The method of embodiment 247, wherein the costimulatory molecule comprises a Cluster of Differentiation 28 (CD28), a Cluster of Differentiation 80 (CD80), a Cluster of Differentiation 86 (CD86), an Inducible T-cell COStimulator (ICOS), or an ICOS Ligand (ICOSLG) protein.
• Embodiment 253. The method of any one of embodiments 246-252, wherein the disease or disorder is a genetic disease or disorder.
• Embodiment 254. The method of embodiment 253, wherein the disease or disorder is a single gene genetic disease or disorder.
• Embodiment 255. The method of embodiment 254, wherein the disease or disorder results from microsatellite instability.
• Embodiment 256. The method of embodiment 255, wherein the microsatellite instability occurs in a DNA sequence at least 1, 2, 3, 4, 5 or 6 repeated motifs.
• Embodiment 257. The method of embodiment 256, wherein an RNA molecule comprises a transcript of the DNA sequence and wherein the composition binds to a target sequence of the RNA molecule comprising at least 1, 2, 3, 4, 5, or 6 repeated motifs.
• Embodiment 258. The method of any one of embodiments 246-257, wherein the composition is administered systemically.
• Embodiment 259. The method of embodiment 259, wherein the composition is administered intravenously.
• Embodiment 260. The method of embodiment 258 or 259, wherein the composition is administered by an injection or an infusion.
• Embodiment 261. The method of any one of embodiments 246-257, wherein the composition is administered locally.
• Embodiment 262. The method of embodiment 261, wherein the composition is administered by an intraosseous, intraocular, intracerebral, or intraspinal route.
• Embodiment 263. The method of embodiment 261 or 262, wherein the composition is administered by an injection or an infusion.
• Embodiment 264. The method of any one of embodiments 265-263, wherein the therapeutically effective amount is a single dose.
• Embodiment 265. The method of any one of embodiments 265-264, wherein the composition is non-genome integrating.

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 claimed 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.

Claims

1. A composition comprising a nucleic acid sequence comprising a guide RNA (gRNA) sequence that specifically binds a target RNA sequence, wherein the target RNA sequence encodes a protein component of an adaptive immune response, and wherein the gRNA sequence comprises a spacer sequence comprising a portion of a nucleic acid sequence encoding the protein component, and wherein the protein component is selected from the group consisting of Beta-2-microglobulin (β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), and CC Chemokine Receptor 7 (CCR7).

2. The composition of claim 1, wherein the adaptive immune response is selected from the group consisting of type I major histocompatibility complex (MHC I), type II major histocompatibility complex (MHC II), T-cell receptor (TCR), costimulatory molecule and a combination thereof.

3. The composition of claim 1, wherein the spacer sequence is about 20 or 21 nucleotides in length.

4. The composition of claim 1, wherein the spacer sequence and the target RNA sequence are reverse complements of one another.

5. The composition of claim 1, wherein the gRNA sequence comprises a scaffold sequence that specifically binds to a CRISPR/Cas polypeptide or portion thereof.

6. The composition of claim 5, wherein the CRISPR/Cas polypeptide or portion thereof is selected from the group consisting of Cas9, Cpf1 , Cas13a, Cas13b, Cas13c and CasRX/Cas13d, wherein the CRISPR/Cas polypeptide has native, reduced or null activity.

7. The composition of claim 1, wherein the nucleic acid sequence comprises a promoter which drives expression of the gRNA sequence.

8. The composition of claim 7, wherein the promoter is selected from the group consisting of a polymerase III promoter and a tRNA promoter.

9. The composition of claim 8, wherein the polymerase III promoter is a U6 promoter.

10. The composition of claim 1, wherein the spacer sequence is a first spacer sequence that specifically binds a first target RNA sequence, and wherein the composition further comprises a second spacer sequence which specifically binds a second target RNA sequence, wherein the first spacer sequence and the second spacer sequence bind different target RNA sequences.

11. The composition of claim 10, wherein the gRNA sequence is a first gRNA sequence, and wherein the second spacer sequence is comprised within a second gRNA sequence.

12. The composition of claim 10, wherein the second target RNA sequence encodes a protein component of an adaptive immune response.

13. The composition of claim 10, wherein the second spacer sequence comprises a portion of a nucleic acid sequence encoding a protein component is selected from the group consisting of Beta-2-microglobulin (β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), and CC Chemokine Receptor 7 (CCR7).

14. The composition of claim 10, wherein the second spacer sequence comprises at least 1, 2, 3, 4, 5, 6, or 7 repeats of a nucleic acid sequence selected from the group consisting of: CUG (SEQ ID NO: 18), CCUG (SEQ ID NO: 19), CAG (SEQ ID NO: 80), GGGGCC (SEQ ID NO: 81), and a combination thereof.

15. A composition comprising a nucleic acid sequence comprising: (a) a first guide RNA (gRNA) sequence that specifically binds a first target RNA sequence, and (b) a second gRNA that specifically binds a second target RNA sequence, wherein the first target RNA sequence encodes a protein component of an adaptive immune response, and wherein the first gRNA sequence comprises a spacer sequence comprising a portion of a nucleic acid sequence encoding the protein component, and wherein the protein component is selected from the group consisting of Beta-2-microglobulin (β2M), Human Leukocyte Antigen A (HLA-A), Human Leukocyte Antigen B (HLA-B), Human Leukocyte Antigen C (HLA-C), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 86 (CD86), Inducible T-cell Costimulator (ICOS), ICOS Ligand (ICOSLG), OX40L, Interleukin 12 (IL12), and CC Chemokine Receptor 7 (CCR7).

16.-17. (canceled)

18. A composition comprising a nucleic acid sequence comprising: (a) a first guide RNA (gRNA) that specifically binds a first target RNA sequence within a first RNA molecule, wherein the first target RNA sequence encodes a protein component of an adaptive immune response (b) a second guide RNA (gRNA) that specifically binds a second target RNA sequence within a second RNA molecule and (c) a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a first RNA-binding polypeptide 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.

19. The composition of claim 18, wherein the first gRNA sequence comprises a spacer sequence comprising a portion of a nucleic acid sequence encoding a protein selected from the group consisting of Beta-2-microglobulin (β2M), HLA-A, HLA-B, HLA-C, CD28, CD80, CD86, ICOSLG, OX40L, IL12, and CCR7.

20.-26. (canceled)

27. A vector comprising the composition of claim 18.

28. The vector of claim 27, wherein the vector is selected from the group consisting of: adeno-associated virus, retrovirus, lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex, polymersome, polyplex, and dendrimer.

29. (canceled)

30. The composition of claim 18, wherein the second RNA-binding polypeptide is selected from the group consisting of: RNAse1, RNAse4, RNAse6, RNAse7, RNAse8, RNAse2, RNAse6PL, RNAseL, RNAseT2, RNAse11, RNAseT2-like, NOB1, ENDOV, ENDOG, ENDOD1, hFEN1, hSLFN14, hLACTB2, APEX2, ANG, HRSP12, ZC3H12A, RIDA, PDL6, NTHL, KIAA0391, APEX1, AGO2, EXOG, ZC3H12D, ERN2, PELO, YBEY, CPSF4L, hCG_2002731, ERCC1, RAC1, RAA1, RAB1, DNA2, F1135220, F1113173, ERCC4, RNAse1(K41R), RNAse1(K41R, D121E), RNAsel(K41R, D121E, H119N), RNAsel(H119N), RNAsel(R39D, N67D, N88A, G89D, R91D, H119N), RNAsel(R39D, N67D, N88A, G89D, R91D, H119N, K41R, D121E), RNAsel(R39D, N67D, N88A, G89D, R91D), TENM1, TENM2, RNAseK, TALEN, ZNF638, and hSMG6 PIN.