Cleavage of target RNA by dCas13-RNase fusion protein

Abstract

The present invention provides proteins, nucleic acids, systems, and methods for regulating RNA. In some embodiments, the proteins, nucleic acids, systems, and methods are used for targeted RNA cleavage. In other embodiments, the proteins, nucleic acids, systems, and methods are used for trans-splicing of RNA molecules.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 052,121, filed July 15, 2020, and U.S. Provisional Patent Application No. 63 / 052,238, filed July 15, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Reference to a "Sequence Listing," table, or computer program listing appendix submitted as an ASCII text file This application incorporates by reference the entirety of the ASCII formatted text file named "204606-0132-00WO Sequence Listing.txt." The text file containing the sequence listing for this application was created on July 14, 2021, and is 961,054 bytes in size. [Background technology]

[0003] The RNA-targeted and RNA-activated CRISPR-Cas13 system generally consists of a targeting CRISPR guide RNA (crRNA) and a CRISPR-associated protein, Cas13, which functions as a programmable endoribonuclease (O'Connell, 2019, J Mol. Biol 431:66-87; Abudayyeh et al., 2018, Nature 550:280-84; Mohanraju et al., 2016, Science 343:aad5147). The Cas13 protein contains two higher eukaryotic and prokaryotic nucleotide-binding (HEPN) domains that enable it to cleave single-stranded RNA. In vitro and in bacteria, binding of a complementary target RNA to a guide RNA constitutively activates HEPN-RNase activity, resulting in target RNA degradation and nonspecific RNA cleavage (collateral cleavage) (Abudayyeh et al., 2018, Nature 550:280-84; Abudayyeh et al., 2016, Science 343:aaf5573; Konermann et al., 2018, Cell 173:665-76). In mammalian cells, guide RNA activation is more precise, resulting in cis cleavage of the target RNA at multiple sites, but no detectable trans / collateral cleavage (Figure 1A). Remarkably, catalytically inactivating mutations within the HEPN domain prevent the RNase activity of Cas13 while retaining its RNA-targeting function.

[0004] Rapid programmability, with little need for flanking crRNA sequence motifs (protospacer flanking motifs, PFSs), gives CRISPR-Cas13 superior capabilities for target RNA cleavage. However, CRISPR-Cas13 is not optimal in all situations because it can only cleave single-stranded RNA (ssRNA), resulting in complete degradation of the target RNA. Therefore, there is a need in the art for compositions and methods for modulating and / or cleaving RNA. [Brief explanation of the drawings]

[0005] [Figure 1] Figure 1 shows a schematic diagram of target RNA cleavage by dCas13-RNase fusion proteins. (A) A schematic diagram of the CRISPR-Cas13 cleavage mechanism. (B) A schematic diagram of the CRISPRase cleavage mechanism. (C) Experimental results showing the relative activity of CRISPR-Cas13 and different CRISPRase fusion proteins targeting luciferase mRNA in mammalian cells. [Figure 2A] 1 shows modifications of CRISPRase fusion proteins, 2 shows a schematic diagram showing the fusion of dCas13 with an RNase having different RNA substrate specificity, and 3 shows a schematic diagram showing the fusion of dCas13 with an RNase having substrate specificity for ssRNA. [Figure 2B] Figure 1 shows modifications of CRISPRase fusion proteins, Figure 2 shows a schematic diagram of fusion of dCas13 with RNases with different RNA substrate specificities, and Figure 3 shows a schematic diagram of fusion of dCas13 with RNase tandem dimers with substrate specificity for ssRNA or dsRNA. [Figure 2C] 1 shows the modification of CRISPRase fusion proteins. 2 shows a schematic diagram of fusion of dCas13 with RNases with different RNA substrate specificities. [Figure 2D]

[0023] Figure 1 shows modifications of CRISPRase fusion proteins.

[0024] Figure 2 shows a schematic diagram of the structural arrangement of the RNase domain with dCas13 to direct RNA cleavage or provide multiple cleavage sites.

[0025] Figure 3 shows a schematic diagram of an RNase-dCas13 fusion protein that allows cleavage 5' of the crRNA target site. [Figure 2E]

[0023] Figure 1 shows modifications of CRISPRase fusion proteins.

[0024] Figure 2 shows a schematic diagram of the structural arrangement of RNase domains with dCas13 to direct RNA cleavage or provide multiple cleavage sites.

[0025] Figure 3 shows a schematic diagram of an RNase-dCas13-RNase fusion protein that provides cleavage sites flanking the crRNA target site. [Figure 3A]

[0023] Figure 1 shows guide RNA modifications to prevent or enable specific CRISPRase activity. Extending the length of the guide RNA can be used to inhibit ssRNA-specific CRISPRase cleavage, or including a bulge at the unpaired base can provide nucleotide-specific cleavage. [Figure 3B] 1 shows modifications of guide RNAs to prevent or enable specific CRISPRase activity. A schematic diagram showing that extending the length of a guide RNA can either form a dsRNA substrate to allow cleavage by a dsRNA-specific RNase or focus cleavage by forming an adjacent bulge of unpaired residues. [Figure 3C] 1 shows the modification of guide RNAs to prevent or enable specific CRISPRase activity. Schematic diagram showing that the addition of single or multiple DNA oligos complementary to the target RNA allows cleavage by an RNAse specific for cleavage of RNA in an RNA:DNA hybrid substrate. [Figure 4A] Schematic showing inducible CRISPRase activity using complementation of split RNases. RNase1 / RNaseA ribonuclease is split into its distinct catalytically inactive S-peptide and S-protein components, which are reassembled in trans to regain catalytic activity. Fusion of dCas13 to the S protein is shown to confer "inducible" CRISPRase cleavage when complemented with the corresponding S peptide. Fusion of the S peptide to ERT2 and small molecule-responsive protein domains, such as tamoxifen (tmx), can be used to create a drug-activated CRISPRase cleavage system. [Figure 4B]Schematic showing inducible CRISPRase activity using split RNase complementation. RNase1 / RNaseA ribonuclease is split into its catalytically inactive S-peptide and S-protein components, which are reassembled in trans to regain catalytic activity. Fusion of dCas13 to the S-peptide is shown to confer "inducible" CRISPRase cleavage when complemented with the corresponding S-protein. Fusion of the S-protein to ERT2 and small molecule-responsive protein domains, such as tamoxifen (tmx), can be used to create a drug-activated CRISPRase cleavage system. [Figure 4C] Figure 1 shows a schematic diagram of inducible CRISPRase activity using split RNase complementation. RNase 1 / RNase A ribonuclease is split into its distinct catalytically inactive S-peptide and S-protein components, which are reassembled in trans to regain catalytic activity. Figure 2 shows a schematic diagram of fusion of dCas13 with multiple tandem S-peptide domains that can be used to promote RNA target cleavage by CRISPRase. [Figure 5A] Figure 1 shows experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression. Figure 2 shows a schematic diagram depicting a luciferase reporter gene encoding the 3'UTR sequence of human DMPK encoding 12 or 960 copies of CUG repeats (pGL3P-DT12a or pGL3P-DT960, respectively). [Figure 5B] Figure 1 shows experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression. Figure 2 shows the relative luciferase activity of the pGL3P-DT12a and pGL3P-DT960 luciferase reporter genes. [Figure 5C]Experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression. Luciferase activity of the pGL3P-DT960 reporter targeted with a CUG-targeting guide RNA (CAGcrRNA) by eraseR, dCas13, or CRISPRase is shown compared to a non-targeting negative control guide RNA. [Figure 5D] Experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression are shown. The number of RNA foci induced per cell by expression of an RNA containing the 3' UTR of human DMPK, which contains 960 CUG repeats, targeted by CRISPRase or CRISPRase with a CUG-targeting guide RNA (CAGcrRNA) or a non-targeting negative control guide RNA (NCcrRNA) is shown. [Figure 5E] We present experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression. [Figure 5F] We present experimental results demonstrating the therapeutic application of CRISPRase to degrade toxic RNA foci and maintain host gene expression. [Figure 6] Schematics showing the use of novel fusion editing proteins to perform trans-splicing of RNA by targeted RNAse cleavage. Targeted RNAse cleavage, as performed by CRISPRase, generates unique RNA ends that can undergo trans-RNA splicing in cells or, when catalyzed by RtcB ligase, in vitro. (A) shows the use of CRISPRase targeted by multiple guide RNAs to direct the assembly of independent RNAs. (B) shows the use of CRISPRase targeted by multiple guide RNAs to delete sequences within a single RNA. (C) shows the use of CRISPRase with RNAse fusions at both the N- and C-termini to be targeted by a single guide RNA to delete a specific internal RNA sequence. DETAILED DESCRIPTION OF THE INVENTION

[0006] In one aspect, the present disclosure is based on the development of novel fusion proteins that provide targeted RNA cleavage. In one embodiment, the fusion proteins comprise a catalytically deactivated CRISPR-associated (dCas) protein and an RNase protein. These fusion proteins combine the catalytic activity of the RNase protein with the programmable DNA targeting capabilities of catalytically deactivated Cas. In one embodiment, the RNase protein is txRNase 1, RNase T1, ribonuclease H1, PIN RNase, or RNase A. In one embodiment, the RNase is an RNase dimer. In one embodiment, the fusion protein further comprises a nuclear localization signal (NLS). In some embodiments, the fusion proteins do not contain an NLS, making them suitable for targeting RNA in the cytoplasm.

[0007] In one embodiment, the fusion protein comprises a catalytically inactive CRISPR-associated (dCas) protein and an s-protein. The dCas-s-protein fusion protein is delivered in trans with an s-peptide to confer RNase catalytic activity. Thus, in one embodiment, the present disclosure provides a composition comprising a dCas-s-protein fusion protein and an s-peptide.

[0008] In another aspect, the present invention includes novel fusions of editing proteins, compositions thereof, and methods of using them to trans-splice RNA molecules. In some embodiments, the fusion editing proteins generate 2',3' cyclic phosphate and 5' hydroxyl RNA termini. In one embodiment, 2',3' cyclic phosphate and 5' hydroxyl RNA termini can be ligated together. In one embodiment, ligation is mediated by RtcB ligase.

[0009] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0010] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

[0011] Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are generally carried out according to conventional methods in the art and various general references provided throughout the specification (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0012] The nomenclature used herein and the laboratory procedures utilized in analytical chemistry and organic synthesis described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0013] As used in the context of the present invention (particularly in the context of the claims), the terms "a," "an," and "the," and similar terms, are to be construed as encompassing both the singular and the plural, unless otherwise stated herein or clearly contradicted by context.

[0014] As used herein, "about," when referring to a measurable value such as an amount, temporal duration, etc., is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, where such variations are appropriate for practicing the disclosed methods.

[0015] "Antisense" refers specifically to a nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein, or a sequence that is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to a sequence of a double-stranded DNA molecule encoding a protein. It is not necessary for an antisense sequence to be complementary only to the coding portion of the coding strand of a DNA molecule. An antisense sequence may be complementary to a regulatory sequence identified on the coding strand of a DNA molecule encoding a protein, which regulatory sequence controls the expression of the coding sequence.

[0016] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the health of the animal continues to deteriorate if the disease is not ameliorated.

[0017] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is not better than it would be in the absence of the disorder. A disorder, if left untreated, does not necessarily cause a further deterioration in the animal's health.

[0018] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which a patient experiences such sign or symptom, or both, is reduced.

[0019] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, or a biological property resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0020] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell suitable for the methods described herein, whether in vitro or in vivo. In one embodiment, a subject includes vertebrates and invertebrates. Invertebrates include, but are not limited to, Drosophila melanogaster and Caenorhabditis elegans. Vertebrates include, but are not limited to, primates, rodents, domestic animals, or game animals. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include, but are not limited to, mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., zebrafish, trout, catfish, and salmon). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0021] The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a specific antigen in a sample but does not substantially recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific.

[0022] In some instances, the terms "specific binding" or "specific binding" may be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to particular protein structures rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0023] The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of an mRNA molecule produced by transcription of the gene.

[0024] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that coincide with the anti-codon region of a transfer RNA molecule or that encode a stop codon during translation of the mRNA molecule. Thus, a coding region can include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues of a protein export signal sequence).

[0025] As used herein, "complementary" refers to nucleic acids and the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in the second nucleic acid region that is antiparallel to the first region if that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand that is antiparallel to the first strand if that residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue in the first region can base pair with a residue in the second region when the two regions are arranged in an antiparallel fashion. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, such that when the first and second portions are arranged in an antiparallel manner, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion can base pair with nucleotide residues in the second portion. In one embodiment, all nucleotide residues of the first portion can base pair with nucleotide residues of the second portion.

[0026] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0027] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0028] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, etc. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions.

[0029] As used herein, the term "wild-type" is a term of the art understood by those skilled in the art and means the typical form of an organism, strain, gene, or characteristic occurring in nature, as distinguished from mutant or variant forms.

[0030] The term "homology" refers to the degree of complementarity. There may be partial or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software compares similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0031] "Nucleic acid" refers to any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sulfone bonds, and combinations of such bonds. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0032] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0033] The direction of 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand with the same sequence as the mRNA is referred to as the "coding strand," the sequence on the DNA strand that is 5' to that reference point on the DNA is referred to as the "upstream sequence," and the sequence on the DNA strand that is 3' to that reference point on the DNA is referred to as the "downstream sequence."

[0034] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0035] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0036] As used herein, the term "RNA" is defined as ribonucleic acid.

[0037] A "variant," as the term is used herein, refers to a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains the basic biological properties of the reference molecule. Sequence changes in a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, but may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are typically limited or conservative, such that the sequences of the reference peptide and variant are closely similar overall and, in many regions, identical. A variant and a reference peptide differ in amino acid sequence by any combination of one or more substitutions, additions, or deletions. Nucleic acid or peptide variants can be naturally occurring, such as allelic variants, or can be variants that are not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be generated by mutagenesis techniques or direct synthesis.

[0038] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to encompass non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0039] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0040] fusion proteins In one aspect, the present disclosure is based on the development of novel editing protein and RNase protein fusions that provide targeted RNA cleavage. In some embodiments, the fusion proteins are efficiently delivered to cells. These fusion proteins combine the catalytic activity of RNase proteins with the programmable DNA targeting capabilities of catalytically inactive Cas. In one embodiment, the present invention provides fusion proteins comprising a CRISPR-associated (Cas) protein and an RNase protein. In one embodiment, the fusion protein comprises a nuclear localization signal for targeting RNA within the nucleus. In one embodiment, the fusion protein does not comprise a nuclear export signal (NES) for targeting RNA in the cytoplasm. In other embodiments, the fusion protein does not comprise an NLS for targeting RNA in the cytoplasm. Other localization signals (known in the art) can also be used to target RNA within organelles such as mitochondria. In one embodiment, the fusion protein comprises a linker. In one embodiment, the linker connects the Cas protein and the RNase protein. In one embodiment, the fusion protein comprises a purification and / or detection tag.

[0041] In another aspect, the invention comprises novel fusions of editing proteins for trans-splicing RNA molecules in cells or in vitro, and compositions thereof. In one embodiment, the invention relates to a composition comprising one or more novel fusions of editing proteins described herein or nucleic acids encoding said novel fusions of editing proteins, one or more targeting nucleic acids described herein, and one or more RNA molecules. In one embodiment, the composition further comprises RtcB ligase or a nucleic acid encoding an RtcB ligase.

[0042] Edited proteins In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0043] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cm Examples of Cas proteins include r4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, and xCas9 3.7, their homologs, their orthologs, or modified forms thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein induces cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein induces cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9, Cas13, or Cpfl. In one embodiment, the Cas protein lacks catalytic activity (dCas).

[0044] In one embodiment, the Cas protein has RNA binding activity. In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is selected from the group consisting of PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, H heCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas1 3b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, Rcd Cas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dA spCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13 d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnC as13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b,dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, dUrCas13d, dCas13X.1, or mini-dCas13X.1. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, DBT, et al., Science, 2017, 358:1019-1027; Abudayyeh et al., Nature, 2017, 550:280-284, Gootenberg et al., Science, 2017, 356:438-442; and East-Seletsky et al., Mol Cell, 2017, 66:373-383 e3, which are incorporated herein by reference).

[0045] In one embodiment, the Cas protein comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48 and 826. In one embodiment, the Cas protein comprises the sequence of a variant of one of SEQ ID NOs: 1-48 and 826, wherein the variant catalytically inactivates the Cas protein. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 1-46 and 826 with one or more insertions, deletions, or substitutions, which catalytically inactivate the Cas protein. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 1-48 and 826. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 47-48.

[0046] RNase In one embodiment, the fusion protein comprises an RNase protein. In one embodiment, the fusion protein comprises two RNase proteins. In one embodiment, the fusion protein comprises three or more RNase proteins. In one embodiment, the fusion protein comprises two of the same RNase proteins. In one embodiment, the fusion protein comprises three or more of the same RNase proteins. In one embodiment, the fusion protein comprises two different RNase proteins. In one embodiment, the fusion protein comprises three or more different RNase proteins.

[0047] In one embodiment, the RNase protein is heterologous to the Cas protein. In one embodiment, the RNase can cleave phosphodiester bonds within a polynucleotide chain. In one embodiment, the RNase can cleave one or more of single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or RNA in a hybrid RNA:DNA complex. In one embodiment, the RNase comprises sequence-specific cleavage activity. In various embodiments, the RNase is an endonuclease.

[0048] In one embodiment, the RNase is RNase 1, RNase 2, RNase 3, RNase 4, RNase 5, RNase 6, RNase 7, RNase 8, RNase A, RNase 1, RNase 1B, txRNase 1 (RNase 1 R39D / N67D / N88A / G89D / R91D), txRNase A (RNase A D38R / R39D / N67R / G88R), RNase T1, RNase T2, Onconase, Erns (C171R), RNase U2, PIN RNase domain, bovine seminal ribonuclease (SRN), RNase V1, Mini RNase III (MiniR3), RNase III domain (DICER), ribonuclease HI (RNase HI*), or ribonuclease HI(D125N) (RNase HI*D125N).

[0049] In one embodiment, the RNase protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-89. In one embodiment, the RNase protein comprises the sequence of one of SEQ ID NOs: 49-89.

[0050] In one embodiment, the RNase is a dimer of RNase monomers. In one embodiment, the RNase dimer can cleave dsRNA or ssRNA. In one embodiment, the RNase dimers are linked to each other by a linker sequence. In one embodiment, the RNase dimer is a homodimer. In one embodiment, the RNase dimer is a heterodimer. In one embodiment, the RNase dimer is a naturally occurring dimer. In one embodiment, the RNase dimer is a synthetic dimer. In one embodiment, the RNase dimer is a synthetic tandem dimeric RNase 1 (tdRNase 1), a synthetic tandem PIN RNase domain (tdPIN), a synthetic tandem dimeric bovine seminal ribonuclease (tdSRN), a synthetic tandem dimeric Mini RNase III (tdMiniR3), a synthetic tandem dimeric RNase III domain (tdDICER), a synthetic tandem RNase III domain (tdRNC), a natural tandem RNase III domain (DROSHA), or a natural tandem RNase III domain dimer (giDICER). In one embodiment, the RNase dimer comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 57, 77, 79, 82, and 84-87. In one embodiment, the RNase dimer comprises the sequence of one of SEQ ID NOs: 57, 77, 79, 82, and 84-87.

[0051] In one embodiment, the RNase protein is a fragment of an RNase protein, hi one embodiment, the fragment of an RNase protein is capable of being complemented in trans with a second fragment of an RNase protein to confer inducible catalytic activity. In one embodiment, the fragment of an RNase protein is RNase 1, RNase 2, RNase 3, RNase 4, RNase 5, RNase 6, RNase 7, RNase 8, RNase A, RNase 1, RNase 1B, txRNase 1 (RNase 1 R39D / N67D / N88A / G89D / R91D), txRNase A (RNase A D38R / R39D / N67R / G88R), RNase T1, RNase T2, Onconase, Erns (C171R), RNase U2, PIN RNase domain, bovine seminal ribonuclease (SRN), RNase V1, Mini RNase III (MiniR3), RNase III domain (DICER), ribonuclease HI (RNase HI*), or ribonuclease HI (D125N) (RNase It is a fragment of HI*D125N.

[0052] In one embodiment, the fragment of the RNase protein is a fragment of RNase 1. In one embodiment, the fragment of the RNase protein is an s protein. In one embodiment, the s protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 90, 93, and 96. In one embodiment, the s protein comprises the sequence of one of SEQ ID NOs: 90, 93, and 96.

[0053] In one embodiment, the fragment of the RNase protein is an s-peptide. In one embodiment, the s-peptide comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 91, 92, 94, 95, and 97-99. In one embodiment, the s-peptide comprises the sequence of one of SEQ ID NOs: 91, 92, 94, 95, and 97-99.

[0054] Localization Signals In some embodiments, the fusion protein has a localization signal, such as a nuclear localization signal (NLS), a nuclear export signal (NES), or other localization signal, that allows it to localize to organelles such as mitochondria. In some embodiments, the localization signal causes the fusion protein to localize to the site where the target RNA is present.

[0055] Nuclear localization signal In one embodiment, the fusion protein comprises an NLS. In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from Ty1, yeast GAL4, SKI3, L29, or histone H2B protein, polyomavirus large T protein, VP1 or VP2 capsid protein, SV40VP1 or VP2 capsid protein, adenovirus Ela or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleophosmin (NPM1), or simian virus 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 110. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 111. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 112. In one embodiment, the NLS comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730. In one embodiment, the NLS protein comprises one of the sequences set forth in SEQ ID NOs: 110-730.

[0056] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX and a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises 20 to 40 amino acids. In one embodiment, the Ty1-like NLS comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 118-730. In one embodiment, the NLS comprises one of SEQ ID NOs: 118-730, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more insertions, deletions, or substitutions. In one embodiment, the Ty1-like NLS protein comprises one of SEQ ID NOs: 118-730.

[0057] In one embodiment, the NLS comprises two copies of the same NLS, for example, in one embodiment, the NLS comprises a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0058] Nuclear export signal In one embodiment, the fusion protein comprises a nuclear export signal (NES). In one embodiment, the NES is attached to the N-terminus of the Cas protein. In one embodiment, the NES localizes the fusion protein to the cytoplasm for targeting cytoplasmic RNA. In one embodiment, the NES comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 802 or 803. In one embodiment, the NES comprises the amino acid sequence of SEQ ID NO:802 or 803.

[0059] Organelle localization signals In one embodiment, the fusion protein comprises a localization signal that localizes the fusion protein to a cellular organelle. In one embodiment, the localization signal localizes the protein to the nucleolus, ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, or centriole. Many localization signals are known in the art.

[0060] In one embodiment, the fusion protein comprises a localization signal that localizes the fusion protein to an organelle or extracellularly, hi one embodiment, the localization signal localizes the protein to a nucleolus, ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondrion, vacuole, cytosol, lysosome, or centriole.

[0061] Numerous localization signals are known in the art. Exemplary localization signals include, but are not limited to, 1× mitochondrial targeting sequence, 4× mitochondrial targeting sequence, secretory signal sequence (IL-2), myristylation, calsequestrin leader, KDEL retention, and peroxisomal targeting sequence.

[0062] In one embodiment, the fusion protein comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 806-812. In one embodiment, the fusion protein comprises the sequence of SEQ ID NOs: 806-812.

[0063] Linker In one embodiment, the fusion protein comprises a linker. In one embodiment, the linker links the Cas protein and the RNase protein. In one embodiment, the linker is linked to the C-terminus of the Cas protein and the N-terminus of the RNase protein. In one embodiment, the linker is connected to the N-terminus of the Cas protein and the C-terminus of the RNase protein. The linker can be a flexible linker, such as a linker composed primarily of Gly and Ser amino acid residues, or a more rigid linker that may include amino acids such as Ala and Pro (among others). In one embodiment, the linker comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 100-108. In one embodiment, the linker comprises a sequence of one of SEQ ID NOs: 100-108.

[0064] Purification and / or detection tags In one embodiment, the fusion protein comprises a purification and / or detection tag. In one embodiment, the tag is at the N-terminus of the fusion protein. In one embodiment, the tag is a 3xFLAG tag. In one embodiment, the tag comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109. In one embodiment, the tag comprises the amino acid sequence of SEQ ID NO: 109.

[0065] Proteins, peptides and fusion proteins The proteins of the present disclosure can be produced using chemical methods. For example, proteins can be synthesized by solid-phase techniques (Roberge JY et al (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431A Peptide Synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0066] The protein of the present disclosure can be produced using recombinant protein expression. The recombinant expression vector of the present disclosure comprises the nucleic acid of the present invention in a form suitable for expression in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell used for expression. In the recombinant expression vector, "operably linked" is intended to mean that the target nucleotide sequence is linked to a regulatory sequence in a manner that allows the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell).

[0067] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector can depend on factors such as the choice of host cell to be transformed, the desired expression level of protein, and the like. The expression vectors of the present invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0068] The recombinant expression vectors of the invention can be designed for production of variant proteins in prokaryotic or eukaryotic cells. For example, proteins of the invention can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0069] Protein expression in prokaryotes is often carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add several amino acids to the encoded protein, either at the amino or C-terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) increase recombinant protein expression, (ii) increase the solubility of the recombinant protein, and (iii) aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, PreScission, TEV, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc., Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the target recombinant protein.

[0070] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89), although this is not exact; pET11a-d have an N-terminal T7 tag.

[0071] One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired ability to proteolytically cleave the recombinant protein. See, e.g., Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20:2111-2118). Such alterations of the nucleic acid sequences of the present invention can be performed by standard DNA synthesis techniques. Another strategy for resolving codon bias is to use the BL21 Codon Plus bacterial strain (Invitrogen) or the Rosetta bacterial strain (Novagen), which contain extra copies of rare E. coli tRNA genes.

[0072] In another embodiment, the expression vector encoding the protein of the present disclosure is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0073] Alternatively, the polypeptides of the present invention can be produced in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).

[0074] In yet another embodiment, the nucleic acids of the present disclosure are expressed in mammalian cells using mammalian expression vectors. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells, and many other cells. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen), pREP4 (Invitrogen), and pcDNA3 (Invitrogen). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous sarcoma virus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0075] In another embodiment, the recombinant mammalian expression vector can direct expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific, Pinkert, et al., 1987. Genes Dev. 1:268-277), lymphocyte-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of T-cell receptors (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic-specific promoters (Edlund, et al., 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), and the like. al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoter, U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters, such as the murinehox promoter (Kessel and Gruss, 1990, Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546), are also encompassed.

[0076] The present invention should also be construed to include any form of protein having substantial homology to the proteins disclosed herein. In one embodiment, a "substantially homologous" protein is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous, about 91% homologous, about 92% homologous, about 93% homologous, about 94% homologous, about 95% homologous, about 96% homologous, about 97% homologous, about 98% homologous, or about 99% homologous to the amino acid sequence of a fusion protein disclosed herein.

[0077] Alternatively, the protein may be made by recombinant means or by cleavage from a longer polypeptide. The composition of the protein may be confirmed by amino acid analysis or sequencing.

[0078] Variants of proteins according to the present invention can be (i) those in which one or more amino acid residues have been replaced with conserved or non-conserved amino acid residues, which may or may not be those encoded by the genetic code; (ii) those in which one or more modified amino acid residues have been present, e.g., residues modified by the attachment of a substituent group; (iii) those in which the peptide is an alternative splicing variant of a protein of the present invention; (iv) fragments of the peptide; and / or (v) those in which the protein is fused to another peptide, such as a leader sequence or secretory sequence, or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multiple-site proteolysis) of the original sequence. Variants can be post-translationally or chemically modified. Such variants are considered to be within the scope of those skilled in the art given the teachings herein.

[0079] As is known in the art, "similarity" between two fusion proteins is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. A variant is defined as comprising a peptide sequence that differs from the original sequence. In one embodiment, a variant differs from the original sequence by fewer than 40% of the residues per segment of interest, fewer than 25% of the residues per segment of interest, fewer than 10% of the residues per segment of interest, or only a small number of residues per segment of interest, while remaining sufficiently homologous to the original sequence to maintain the function of the original sequence and / or its ability to stimulate stem cell differentiation into osteoblastic lineages. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar or identical to the original amino acid sequence. The degree of identity between two peptides can be determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences can be determined using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)).

[0080] The proteins of the present disclosure may be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, proteolytic processing, and the like. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding canine microsomal membranes or Xenopus egg extract (U.S. Patent No. 6,103,489) to a standard translation reaction.

[0081] Proteins of the disclosure can include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of techniques are available for introducing unnatural amino acids during protein translation.

[0082] Proteins of the present disclosure can be phosphorylated using conventional methods, such as those described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0083] Cyclic derivatives of the fusion proteins of the present invention are also part of the present invention. Cyclization can allow proteins to assume a more favorable conformation for associating with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between the amino group of one component and the carboxyl group of another component. Cyclization can also be performed using azobenzene-containing amino acids as described in Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The bond-forming component can be an amino acid side chain, a non-amino acid component, or a combination of the two. In one embodiment of the present invention, the cyclic peptide can contain a beta turn at the correct position. A beta turn can be introduced into the peptides of the present invention by adding the amino acids Pro-Gly at the correct position.

[0084] It may be desirable to produce cyclic proteins that are more flexible than cyclic peptides containing peptide bond linkages such as those described above. A more flexible peptide can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are positioned so as not to distort the β-sheet and turn. This peptide is more flexible as a result of the length of the disulfide bond and the fewer hydrogen bonds in the β-sheet portion. The relative flexibility of cyclic peptides can be determined by molecular dynamics simulations.

[0085] The present invention also relates to peptides, including fusion proteins comprising Cas13 and an RNase protein, the fusion protein itself being fused to or integrated into a targeting protein and / or a targeting domain capable of directing the chimeric protein to a desired cellular component or cell type or tissue. Chimeric proteins may also contain additional amino acid sequences or domains. Chimeric proteins are recombinant in the sense that the various components are derived from different sources and, therefore, are not found together in nature (i.e., heterologous).

[0086] In one embodiment, the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence that directs the protein to associate with, for example, a vesicle or the nucleus. In one embodiment, the targeting domain can target the peptide to a specific cell type or tissue. For example, the targeting domain can be a cell surface ligand or an antibody against a cell surface antigen of the target tissue. The targeting domain can target the peptide of the present invention to a cellular component.

[0087] The peptides of the present invention can be synthesized by conventional techniques. For example, peptides or chimeric proteins can be synthesized by chemical synthesis using solid-phase peptide synthesis. These methods use either solid-phase synthesis or liquid-phase synthesis (see, for example, JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2002). nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254, and for classical solution synthesis, see M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1.) By way of example, the peptides of the invention may be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry to directly incorporate phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0088] N- or C-terminal fusion proteins comprising the peptides or chimeric proteins of the present invention conjugated with other molecules can be prepared by recombinantly fusing the N- or C-terminus of the peptide or chimeric protein with the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains a protein fused to the selected protein or marker protein described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0089] The peptides of the present invention can be developed using biological expression systems. The use of these systems allows for the generation of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to specific proteins. Libraries can be created by cloning synthetic DNA encoding random peptide sequences into an appropriate expression vector (see Christian et al. 1992, J. Mol. 227:711; Devlin et al. 1990 Science 249:404; Cwirla et al. 1990, Proc. Natl. Acad. Sci. USA, 87:6378). Libraries can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0090] The peptides and chimeric proteins of the present invention can be converted into pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0091] In one embodiment, the fusion protein comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 827, and is linked to an RNase at the C-terminus as described above. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 827, and is linked to an RNase at the C-terminus.

[0092] In one embodiment, the fusion protein comprises an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 828, and is linked to a first RNase at the N-terminus and a second RNase at the C-terminus. In one embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 828, and is linked to a first RNase at the N-terminus and a second RNase at the C-terminus.

[0093] Proteins for trans-splicing RNA molecules In some embodiments, the present invention relates to novel fusions of editing proteins for trans-splicing RNA molecules in cells or in vitro. In one embodiment, the present invention includes a composition comprising one or more novel fusions of editing proteins described herein, one or more targeting nucleic acids described herein, and one or more RNA molecules. In some embodiments, the one or more RNA molecules comprise a single RNA molecule. In some embodiments, the one or more RNA molecules comprise at least two RNA molecules. In one embodiment, the composition further comprises an RtcB ligase or a nucleic acid encoding an RtcB ligase. Exemplary RtcB ligase proteins and their corresponding amino acid sequences can be found in International Application No. PCT / US2021 / 016885, incorporated herein by reference in its entirety.

[0094] nucleic acid In one embodiment, the disclosure provides a nucleic acid encoding a fusion protein of the disclosure. In one embodiment, the nucleic acid encodes a fusion protein comprising an editing protein and an RNase protein. In one embodiment, the nucleic acid encodes a fusion protein comprising a Cas protein and an RNase protein. In one embodiment, the fusion protein combines the catalytic activity of an RNase protein with the programmable nucleic acid targeting capabilities of a catalytically inactive Cas.

[0095] The present disclosure also provides a targeting nucleic acid, including a CRISPR RNA (crRNA), for targeting the fusion protein of the present disclosure to a target RNA. In one embodiment, the crRNA is selected based on the RNase activity of the fusion protein. For example, the RNase of the fusion protein can cleave one or more of ssRNA, dsRNA, or RNA:DNA complexes. Thus, the present disclosure provides a crRNA that can be used with the fusion protein of the present disclosure to enable targeted cleavage of ssRNA, dsRNA, or RNA:DNA complexes.

[0096] Nucleic acid encoding the fusion protein In one aspect, the present disclosure is based on the development of novel nucleic acid molecules encoding fusions of an editing protein and an RNase protein that can be effectively delivered to cells to provide targeted RNA cleavage. These fusion proteins combine the catalytic activity of an RNase protein with the programmable nucleic acid targeting capabilities of a catalytically inactive Cas. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an editing protein and a nucleic acid sequence encoding an RNase protein. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a localization signal (e.g., an NLS). In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a linker. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a purification and / or detection tag.

[0097] Edited proteins In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an editing protein, including, but not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0098] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cm Examples of Cas proteins include r4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, and xCas9 3.7, their homologs, their orthologs, or modified forms thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein induces cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein induces cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9, Cas13, or Cpfl. In one embodiment, the Cas protein lacks catalytic activity (dCas).

[0099] In one embodiment, the Cas protein has RNA binding activity. In one embodiment, the Cas protein is Cas13. In one embodiment, the Cas protein is PspCas13b, PspCas13b truncated, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, Hh eCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13 b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdC as13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncated, dAdmCas13d, dA spCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13 d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnC as13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b,dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, dUrCas13d, dCas13X.1, or mini-dCas13X.1. Additional Cas proteins are known in the art (e.g., Konermann et al., Cell, 2018, 173:665-676 e14, Yan et al., Mol Cell, 2018, 7:327-339 e5; Cox, DBT, et al., Science, 2017, 358:1019-1027; Abudayyeh et al., Nature, 2017, 550:280-284, Gootenberg et al., Science, 2017, 356:438-442; and East-Seletsky et al., Mol Cell, 2017, 66:373-383 e3, which are incorporated herein by reference).

[0100] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-48 and 826. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of a variant of one of SEQ ID NOs: 1-48 and 826, wherein the variant catalytically inactivates the Cas protein. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 1-46 and 826 with one or more insertions, deletions, or substitutions, wherein the one or more insertions, deletions, or substitutions catalytically inactivate the Cas protein. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 1-48 and 826. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 47-48.

[0101] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 734-735 and 823. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a variant of one of SEQ ID NOs: 734-735 and 823, wherein the variant catalytically inactivates the encoded Cas protein. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises the nucleic acid sequence of one of SEQ ID NOs: 734-735 and 823. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises the nucleic acid sequence of one of SEQ ID NOs: 736-737.

[0102] RNase In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an RNase protein. In one embodiment, the nucleic acid molecule encodes two RNase proteins. In one embodiment, the nucleic acid molecule encodes three or more RNase proteins. In one embodiment, the nucleic acid molecule encodes two of the same RNase proteins. In one embodiment, the nucleic acid molecule encodes three or more of the same RNase proteins. In one embodiment, the nucleic acid molecule encodes two different RNase proteins. In one embodiment, the nucleic acid molecule encodes three or more different RNase proteins.

[0103] In one embodiment, the RNase protein is heterologous to the Cas protein. In one embodiment, the RNase can cleave phosphodiester bonds within a polynucleotide chain. In one embodiment, the RNase can cleave one or more of single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or RNA in a hybrid RNA:DNA complex. In one embodiment, the RNase comprises sequence-specific cleavage activity.

[0104] In one embodiment, the RNase is RNase 1, RNase 2, RNase 3, RNase 4, RNase 5, RNase 6, RNase 7, RNase 8, RNase A, RNase 1, RNase 1B, txRNase 1 (RNase 1 R39D / N67D / N88A / G89D / R91D), txRNase A (RNase A D38R / R39D / N67R / G88R), RNase T1, RNase T2, Onconase, Erns (C171R), RNase U2, PIN RNase domain, bovine seminal ribonuclease (SRN), RNase V1, Mini RNase III (MiniR3), RNase III domain (DICER), ribonuclease HI (RNase HI*), or ribonuclease HI(D125N) (RNase HI*D125N).

[0105] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an RNase having an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-89. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an RNase having the amino acid sequence of one of SEQ ID NOs: 49-89.

[0106] In one embodiment, the nucleic acid sequence encoding the RNase protein comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 738-779. In one embodiment, the nucleic acid sequence encoding the RNase protein comprises the nucleic acid sequence of one of SEQ ID NOs: 738-779.

[0107] In one embodiment, the nucleic acid molecule encodes a dimer of RNase monomers. In one embodiment, the RNase dimers are linked to each other by a linker sequence. In one embodiment, the RNase dimer is a homodimer. In one embodiment, the RNase dimer is a heterodimer. In one embodiment, the RNase dimer is a naturally occurring dimer. In one embodiment, the RNase dimer is a synthetic dimer. In one embodiment, the RNase dimer is a synthetic tandem dimeric RNase 1 (tdRNase 1), a synthetic tandem PIN RNase domain (tdPIN), a synthetic tandem dimeric bovine seminal ribonuclease (tdSRN), a synthetic tandem dimeric MiniRNase III (tdMiniR3), a synthetic tandem dimeric RNase III domain (tdDICER), a synthetic tandem RNase III domain (tdRNC), a natural tandem RNase III domain (DROSHA), or a natural tandem RNase III domain dimer (giDICER).

[0108] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an RNase dimer comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 57, 77, 79, 82, and 84-87. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an RNase dimer comprising the amino acid sequence of one of SEQ ID NOs: 57, 77, 79, 82, and 84-87.

[0109] In one embodiment, the nucleic acid sequence encoding the RNase dimer comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 746, 767, 769, 772, and 774-777. In one embodiment, the nucleic acid sequence encoding the RNase dimer comprises the nucleic acid sequence of one of SEQ ID NOs: 746, 767, 769, 772, and 774-777.

[0110] In one embodiment, the nucleic acid molecule encodes a fragment of an RNase protein, hi one embodiment, the fragment of the RNase protein is capable of being complemented in trans with a second fragment of the RNase protein to confer inducible catalytic activity. In one embodiment, the fragment of an RNase protein is RNase 1, RNase 2, RNase 3, RNase 4, RNase 5, RNase 6, RNase 7, RNase 8, RNase A, RNase 1, RNase 1B, txRNase 1 (RNase 1 R39D / N67D / N88A / G89D / R91D), txRNase A (RNase A D38R / R39D / N67R / G88R), RNase T1, RNase T2, Onconase, Erns (C171R), RNase U2, PIN RNase domain, bovine seminal ribonuclease (SRN), RNase V1, Mini RNase III (MiniR3), RNase III domain (DICER), ribonuclease HI (RNase HI*), or ribonuclease HI (D125N) (RNase It is a fragment of HI*D125N.

[0111] In one embodiment, the nucleic acid molecule encodes a fragment of RNase 1. In one embodiment, the nucleic acid molecule encodes an s protein. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an s protein comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 90, 93, and 96. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an s protein comprising the amino acid sequence of one of SEQ ID NOs: 90, 93, and 96.

[0112] In one embodiment, the nucleic acid sequence encoding the s protein comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 780, 783, and 786. In one embodiment, the nucleic acid sequence encoding the s protein comprises the nucleic acid sequence of one of SEQ ID NOs: 780, 783, and 786.

[0113] In one embodiment, the fragment of the RNase protein is an s-peptide. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an s-peptide comprising an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 91, 92, 94, 95, and 97-99. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an s-peptide comprising the amino acid sequence of one of SEQ ID NOs: 91, 92, 94, 95, and 97-99.

[0114] In one embodiment, the nucleic acid sequence encoding the s peptide comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 781, 782, 784, 785, and 787-789. In one embodiment, the nucleic acid sequence encoding the s peptide comprises the nucleic acid sequence of one of SEQ ID NOs: 781, 782, 784, 785, and 787-789.

[0115] Localization Signals In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a localization signal, such as a nuclear localization signal (NLS), a nuclear export signal (NES), or other localization signal for localization to organelles such as mitochondria. In some embodiments, the localization signal localizes the fusion protein to the site where the target RNA is present.

[0116] Nuclear localization signal In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a nuclear localization signal (NLS). In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from Ty1, yeast GAL4, SKI3, L29 or histone H2B protein, polyomavirus large T protein, VP1 or VP2 capsid protein, SV40VP1 or VP2 capsid protein, adenovirus Ela or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleophosmin (NPM1), or simian virus 40 ("SV40") T antigen.

[0117] In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 110. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 111. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 112. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 110-730.

[0118] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises KKRX and KKR motifs. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises 20 to 40 amino acids. In one embodiment, a nucleic acid sequence encoding a Ty1-like NLS comprises a nucleic acid sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:118-730. In one embodiment, the nucleic acid sequence encoding the Ty1-like NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 118-730, wherein the sequence comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more insertions, deletions, or substitutions. In one embodiment, the nucleic acid sequence encoding the Ty1-like NLS comprises a nucleic acid sequence encoding the amino acid sequence of one of SEQ ID NOs: 118-730.

[0119] In one embodiment, the nucleic acid sequence encoding the NLS encodes two copies of the same NLS, for example, in one embodiment, the nucleic acid sequence encodes a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0120] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 794. In one embodiment, the nucleic acid sequence encoding the NLS comprises the nucleic acid sequence of SEQ ID NO: 794.

[0121] Nuclear export signal In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a nuclear export signal (NES). In one embodiment, the NES localizes the fusion protein to the cytoplasm for targeting cytoplasmic RNA. In one embodiment, the nucleic acid sequence encoding the NES comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 802 or 803. In one embodiment, the nucleic acid sequence encoding the NES comprises a sequence encoding the amino acid sequence of SEQ ID NO:802 or 803.

[0122] In one embodiment, the nucleic acid sequence encoding the NES comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 804 or 805. In one embodiment, the nucleic acid sequence encoding the NES comprises the sequence of SEQ ID NO: 804 or 805.

[0123] Organelle and extracellular localization signals In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a localization signal that localizes the fusion protein to an organelle or extracellularly. In one embodiment, the localization signal localizes the protein to the nucleolus, ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, or centriole. Many localization signals are known in the art.

[0124] Exemplary localization signals include, but are not limited to, 1x mitochondrial targeting sequence, 4x mitochondrial targeting sequence, secretory signal sequence (IL-2), myristylation, calsequestrin leader, KDEL retention, and peroxisomal targeting sequence.

[0125] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a localization signal. In one embodiment, the localization signal localizes the fusion protein to an organelle or extracellularly. In one embodiment, the nucleic acid sequence encoding the localization signal comprises a sequence encoding an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 806-812. In one embodiment, the nucleic acid sequence encoding the localization signal comprises a sequence encoding the amino acid sequence of SEQ ID NOs: 806-812.

[0126] In one embodiment, the nucleic acid sequence encoding the localization signal comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 813-819. In one embodiment, the nucleic acid sequence encoding the localization signal comprises the sequence of SEQ ID NOs: 813-819.

[0127] Linker In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a linker peptide. In one embodiment, the linker connects the Cas protein and the RNase protein. In one embodiment, the linker is linked to the C-terminus of the Cas protein and the N-terminus of the RNase protein. In one embodiment, the linker is connected to the N-terminus of the Cas protein and the C-terminus of the RNase protein.

[0128] In one embodiment, the nucleic acid sequence encoding the linker peptide encodes an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 100-108. In one embodiment, the nucleic acid sequence encoding the linker peptide encodes the amino acid sequence of one of SEQ ID NOs: 100-108.

[0129] In one embodiment, the nucleic acid sequence encoding the linker peptide comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 790-792. In one embodiment, the nucleic acid sequence encoding the linker peptide comprises the sequence of one of SEQ ID NOs: 790-792.

[0130] Purification and / or detection tags In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a purification and / or detection tag. In one embodiment, the tag is at the N-terminus of the fusion protein. In one embodiment, the tag is a 3xFLAG tag. In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag encodes an amino acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 109. In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag encodes the amino acid sequence of SEQ ID NO: 109.

[0131] In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 793. In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag comprises the sequence of SEQ ID NO:793.

[0132] Targeting nucleic acids and CRISPR RNA (crRNA) In one aspect, the present invention provides a targeting nucleic acid comprising a CRISPR RNA (crRNA) for targeting Cas to a target RNA. In one embodiment, the targeting nucleic acid is a crRNA. In one embodiment, the crRNA comprises a guide sequence. In one embodiment, the crRNA comprises a direct repeat (DR) sequence. In one embodiment, the crRNA comprises a direct repeat sequence and a guide sequence fused or linked to a guide sequence or a spacer sequence. In one embodiment, the direct repeat sequence may be located upstream (i.e., 5') from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') from the guide sequence or spacer sequence.

[0133] In some embodiments, the crRNA comprises a stem-loop. In one embodiment, the crRNA comprises a single stem-loop. In one embodiment, the direct repeat sequence forms a stem-loop. In one embodiment, the direct repeat sequence forms a single stem-loop.

[0134] In one embodiment, the crRNA is complementary to the RNase of the fusion protein. For example, in one embodiment, the RNase can cleave ssRNA, and the crRNA guide sequence comprises a sequence that has sufficient complementarity to a sequence adjacent to the target sequence. In one embodiment, the RNase can cleave ssRNA, and the crRNA guide sequence comprises a sequence that has sufficient complementarity to the target sequence to form a bulge ssRNA at the target site.

[0135] In one embodiment, an RNase can cleave a dsRNA, and the crRNA guide sequence has sufficient complementarity to the target sequence to form a dsRNA that can be cleaved by the RNase. In one embodiment, an RNase can cleave a dsRNA, and the crRNA guide sequence has sufficient complementarity to the target sequence and comprises sequences that mismatch with the 5' and 3' sides of the target site to form bulges of ssRNA at the 5' and 3' sides of the target site, thereby forming a dsRNA at the target site that can be cleaved by the RNase.

[0136] In one embodiment, the target nucleic acid is a targeting DNA oligo. In one embodiment, the DNA oligo includes a guide sequence that includes a sequence having sufficient complementarity to the target sequence. In one embodiment, the targeting DNA oligo can be delivered in combination with a crRNA. In one embodiment, the crRNA guides a Cas13-RNase fusion protein to the target site. In one embodiment, the crRNA guides the Cas13-RNase fusion protein to the target site, where the RNase can cleave the RNA:DNA complex. In one embodiment, the DNA oligo binds to the target site, thereby enabling the RNase of the Cas13-RNase fusion protein to cleave the target site.

[0137] In one embodiment, the spacer length of the guide RNA is 15 to 35 nt. In one embodiment, the spacer length of the guide RNA is at least 15 nucleotides. In one embodiment, the spacer length is 15 to 17 nt, for example, 15, 16, or 17 nt, 17 to 20 nt, for example, 17, 18, 19, or 20 nt, 20 to 24 nt, for example, 20, 21, 22, 23, or 24 nt, 23 to 25 nt, for example, 23, 24, or 25 nt, 24 to 27 nt, for example, 24, 25, 26, or 27 nt, 27 to 30 nt, for example, 27, 28, 29, or 30 nt, 30 to 35 nt, for example, 30, 31, 32, 33, 34, or 35 nt, or 35 nt or more.

[0138] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50% or more, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about 5 nucleotides or more, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or more. In some embodiments, the guide sequence is less than about 75 nucleotides, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides or less. Preferably, the guide sequence is 10-30 nucleotides long. The ability of the guide sequence to direct the sequence-specific binding of CRISPR complexes to target sequences can be evaluated by any suitable assay.For example, components of a CRISPR system sufficient to form a CRISPR complex, including a test guide sequence, can be provided to a host cell having a corresponding target sequence, such as by transfection with a vector encoding the CRISPR sequence components, followed by assessment of preferential cleavage within the target sequence, such as by a surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence can be assessed in vitro by providing components of a CRISPR complex including the target sequence, the test guide sequence, and a control guide sequence different from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between reactions using the test guide sequence and the control guide sequence. Other assays are possible and will occur to those skilled in the art.

[0139] In some embodiments of the CRISPR-Cas system, the degree of complementarity between the guide sequence and its corresponding target sequence may be about 50% or more, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%, and the guide or RNA or sgRNA may be about 5 or more, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length, or the guide or RNA or sgRNA may be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length, and advantageously, the tracrRNA is 30 or 50 nucleotides in length. However, one aspect of the present invention is to reduce off-target interactions, e.g., to reduce guide interactions with target sequences with low complementarity. Indeed, in the examples, the present invention is shown to include mutations that result in a CRISPR-Cas system that can distinguish between target sequences and off-target sequences with greater than 80% to about 95% complementarity, e.g., 83% to 84%, or 88% to 89%, or 94% to 95% complementarity (e.g., distinguish between a target with 18 nucleotides and an 18-nucleotide off-target with 1, 2, or 3 mismatches). Thus, in the context of the present invention, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 99.9%, or 100%.An off-target is less than 100% or 99.9% or 99.5% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, and advantageously, an off-target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.

[0140] Ef1a2 promoter In one aspect, a nucleic acid molecule of the present disclosure comprises an Ef1a2 promoter that drives expression of a protein or gene described herein. In one embodiment, the promoter is an Ef1a2 promoter that can drive expression in heart, skeletal muscle, and neural tissues, such as the brain and motor neurons. In one embodiment, the Ef1a2 promoter comprises a sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 820-822. In one embodiment, the Ef1a2 promoter comprises one of the sequences set forth in SEQ ID NOs: 820-822.

[0141] nucleic acid The isolated nucleic acid sequences of the present disclosure can be obtained using any of a number of recombinant methods known in the art, such as, for example, by screening libraries from cells which express the gene, or by deriving the gene from a vector known to contain it, or by direct isolation using standard techniques from cells and tissues which contain it. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0142] The isolated nucleic acid can include any type of nucleic acid, including, but not limited to, DNA and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule, including, for example, an isolated cDNA molecule, encoding a protein of the present disclosure. In one embodiment, the composition includes an isolated RNA molecule encoding a fusion of the present disclosure, or a functional fragment thereof.

[0143] The nucleic acid molecules of the present invention can be modified to improve stability in serum or cell culture growth medium. Modifications can be added to improve stability, functionality, and / or specificity while minimizing the immunostimulatory properties of the nucleic acid molecules of the present invention. For example, to enhance stability, 3' residues can be stabilized against degradation. For example, they can be selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, such as uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0144] In one embodiment of the present invention, the nucleic acid molecule may contain at least one modified nucleotide analogue, for example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0145] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate group attached to adjacent ribonucleotides is replaced with a modified group, such as a phosphorothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, or ON, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0146] Another example of modification is ribonucleotide with modified nucleobase, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.The base can be modified to inhibit the activity of adenosine deaminase.Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine.It should be noted that the above modifications can be combined.

[0147] In some cases, nucleic acid molecules contain at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modifications of one or more nucleotides. In certain embodiments, nucleic acid molecules of the present invention may have enhanced resistance to nucleases. To increase nuclease resistance, nucleic acid molecules may contain, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) may be modified or replaced with a number of different "oxy" or "deoxy" substituents. To increase nuclease resistance, nucleic acid molecules of the present invention may contain 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, e.g., 2-amino-A modifications, 2-thio (e.g., 2-thio-U) modifications, G-clamp modifications, can also increase binding affinity to the target.

[0148] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0149] In certain embodiments, nucleic acid molecules of the invention have one or more of the following properties:

[0150] The nucleic acid agents described herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the nucleic acid components, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those occurring in nature or naturally occurring in the human body. The art has referred to rare or unusual, but naturally occurring, RNA as modified RNA. See, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs are often referred to as modified RNAs, typically the result of post-transcriptional modifications, and are included within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to molecules in which one or more of the nucleic acid components, i.e., sugar, base, and phosphate moieties, are different from those occurring in nature or naturally occurring in the human body. They are referred to as "modified RNAs," although they do, of course, include molecules that, due to the modifications, are not, strictly speaking, RNA. Nucleoside surrogates are molecules in which the ribose phosphate backbone has been replaced with a non-ribose phosphate structure, e.g., an uncharged mimic of the ribose phosphate backbone, that allows the bases to be presented in the correct spatial relationship so that hybridization is substantially similar to that seen with the ribose phosphate backbone.

[0151] Modifications of the nucleic acids of the invention can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0152] The invention also includes vectors into which the isolated nucleic acids of the invention have been inserted. The art is replete with suitable vectors useful in the present invention.

[0153] Briefly, expression of natural or synthetic nucleic acids encoding proteins of the present disclosure is typically achieved by operably linking the nucleic acid encoding the protein of the present disclosure, or a portion thereof, to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0154] The vectors of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466 (incorporated herein by reference in their entirety). In another embodiment, the present invention provides a gene therapy vector.

[0155] The isolated nucleic acids of the present invention can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0156] Furthermore, the vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors have a replication origin that functions in at least one organism, a promoter sequence, a useful restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0157] In one embodiment, a nucleic acid encoding one or more fusion proteins of the invention comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 824 linked at its 3' end to a nucleic acid sequence encoding an RNase, as described above. In one embodiment, a nucleic acid encoding one or more fusion proteins comprises the nucleic acid sequence of SEQ ID NO: 824 linked at its 3' end to a nucleic acid sequence encoding an RNase.

[0158] In one embodiment, a nucleic acid encoding one or more fusion proteins of the invention comprises a nucleic acid sequence at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 825 linked at its 5' end to a nucleic acid sequence encoding a first RNase and linked at its 3' end to a nucleic acid sequence encoding a second RNase. In one embodiment, the nucleic acid encoding the one or more fusion proteins comprises the nucleic acid sequence of SEQ ID NO: 825 linked at its 5' end to a nucleic acid sequence encoding a first RNase and linked at its 3' end to a nucleic acid sequence encoding a second RNase.

[0159] Nucleic acids that encode proteins for trans-splicing RNA molecules In some embodiments, the present invention relates to nucleic acids encoding novel fusions of editing proteins for trans-splicing RNA molecules in cells or in vitro. In one embodiment, the present invention includes a composition comprising one or more nucleic acids encoding one or more novel fusions of editing proteins described herein, one or more targeting nucleic acids described herein, and one or more RNA molecules. In some embodiments, the one or more RNA molecules comprise a single RNA molecule. In some embodiments, the one or more RNA molecules comprise at least two RNA molecules. In one embodiment, the composition further comprises an RtcB ligase or a nucleic acid encoding an RtcB ligase. Exemplary RtcB ligases and corresponding nucleic acid sequences encoding the RtcB ligases described above can be found in International Application No. PCT / US2021 / 016885, the entire contents of which are incorporated herein by reference.

[0160] Delivery Systems and Methods In one aspect, the present invention relates to the development of a novel lentiviral packaging and delivery system. Lentiviral particles deliver viral enzymes as proteins. This makes the lentiviral enzymes short-lived, limiting the possibility of off-target editing due to long-term expression throughout the life of the cell. Incorporation of editing components or conventional CRISPR-Cas editing components as proteins in lentiviral particles is advantageous, given that their required activity is only required for a short period of time. Thus, in one embodiment, the present invention provides a lentiviral delivery system, as well as methods for delivering compositions of the present invention, methods for editing genetic material, and methods for delivering nucleic acids using a lentiviral delivery system.

[0161] In one embodiment, the delivery system comprises (1) a packaging plasmid, (2) a transfer plasmid, and (3) an envelope plasmid. In one embodiment, the delivery system comprises (1) a packaging plasmid, (2) an envelope plasmid, and (3) a VPR plasmid. In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a mutation selected from D116N and D64V. In one embodiment, the transfer plasmid comprises a nucleic acid sequence encoding a crRNA sequence and a Cas protein of the present disclosure.

[0162] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g protein (VSV-g) envelope protein. In one embodiment, the envelope protein can be selected based on the desired cell type.

[0163] In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein, and an RNase protein. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein, an RNase protein, and an NLS. In one embodiment, the VPR plasmid comprises a nucleic acid sequence encoding a fusion protein comprising VPR, a Cas protein, an RNase protein, and an NES. In one embodiment, the fusion protein comprises a protease cleavage site between the VPR and the Cas protein, and an RNase protein. In one embodiment, the VPR plasmid packaging plasmid further comprises a sequence encoding a targeting nucleic acid sequence.

[0164] In one embodiment, a packaging plasmid, a transfer plasmid, an envelope plasmid, and a VPR plasmid are introduced into a cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes and translates a fusion protein to produce a VPR-fusion protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding a guide RNA. In one embodiment, the transcribed transfer plasmid and gag-pol protein are packaged into a lentiviral vector. In one embodiment, the lentiviral vector is recovered from the cell culture medium. In one embodiment, the viral particle transduces a target cell, and the transcribed crRNA and Cas protein are cleaved and translated to produce the Cas protein and crRNA, and the crRNA binds to the Cas protein and guides the Cas protein to an RNA having a sequence substantially complementary to the crRNA sequence.

[0165] In one embodiment, the gag-pol protein, envelope polyprotein, and VPR-fusion protein bound to the guide RNA are packaged into a viral particle. In one embodiment, the viral particle is recovered from the cell culture medium. In one embodiment, the VPR is cleaved from the fusion protein by a protease site within the viral particle to yield the Cas-fusion protein. In one embodiment, the viral particle transduces a target cell, where the guide RNA targets the Cas-fusion protein by binding to the target region of the RNA.

[0166] Many additional virus-based systems have been developed for gene transfer into mammalian cells.For example, retroviruses provide a convenient platform for gene delivery systems.Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles.Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo.Many retroviral systems are well known in the art.In some embodiments, adenoviral vectors are used.Many adenoviral vectors are well known in the art.In some embodiments, lentiviral vectors are used.

[0167] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells. Compared with vectors derived from oncoretroviruses such as murine leukemia viruses, lentiviral vectors have the additional advantage of being able to transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of being less immunogenic.

[0168] In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). The term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, and AAV-9. AAV vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess several characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a specific gene contained within an AAV vector can be specifically targeted to one or more cell types by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0169] AAV vectors can have deletions of one or more of the AAV wild-type genes, preferably the rep and / or cap genes, in whole or in part, but retain functional flanking ITR sequences. Despite their high degree of homology, different serotypes have distinct tissue tropisms. While the receptor for AAV1 is unknown, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Since many studies have been conducted with pseudotyped vectors in which vector DNA flanked by AAV2 ITRs is packaged within capsids of alternative serotypes, it is clear that biological differences are related to the capsid rather than the genome. Recent evidence indicates that DNA expression cassettes packaged in AAV1 capsids are at least 1 log higher in transducing cardiomyocytes than those packaged in AAV2 capsids. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).

[0170] Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3, and hypervariable regions; rep proteins, including rep78, rep68, rep52, and rep40; and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences that may be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Thus, exemplary AAVs, or artificial AAVs, suitable for expression of one or more proteins include AAV2 / 8 (see U.S. Patent No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication No. WO2005 / 033321), AAV2 / 6 (U.S. Patent No. 6,156,303), and AAVrh8 (International Patent Publication No. WO2003 / 042397), among others.

[0171] In certain embodiments, the vector also contains conventional control elements operably linked to the transgene in a manner that allows its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with a virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or remotely to regulate the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Many expression control sequences, including naturally occurring, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized.

[0172] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. Often, the spacing between promoter elements is flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. In some promoters, individual elements appear to be able to activate transcription either cooperatively or independently.

[0173] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any operably linked polynucleotide sequence. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention need not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when its expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0174] Enhancer sequences found on vectors also regulate the expression of genes contained in the vector. Typically, enhancers bind to protein factors to enhance gene transcription. Enhancers can be located upstream or downstream of the gene they regulate. Enhancers can also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vector of the present invention contains one or more enhancers to promote the transcription of genes present in the vector.

[0175] To assess the expression of the fusion protein of the present invention, the expression vector introduced into cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a cell population to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on a separate DNA and used in a co-transfection procedure. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0176] Reporter genes are used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are neither present nor expressed in the recipient organism or tissue, and whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially obtained. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0177] Methods for introducing and expressing genes into cells are known in the art. With regard to expression vectors, the vector can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, the expression vector can be introduced into host cells by physical, chemical, or biological means.

[0178] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An exemplary method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0179] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0180] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0181] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into a host (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in a lipid as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or as a "collapsed" structure. They may also simply be dispersed in the solution, possibly forming aggregates that are not uniform in size or shape.Lipid refers to fatty substances that can be naturally occurring lipids or synthetic lipids.For example, lipids include the naturally occurring lipid droplets in cytoplasm, and the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0182] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vehicles formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess amount of aqueous solution. The lipid components self-reorganize before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0183] Regardless of the method used to introduce exogenous nucleic acid into host cells, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR, as well as "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0184] nanoparticles In some embodiments, the present disclosure provides a fusion protein, nucleic acid, or combination thereof of any of the above paragraphs formulated in a nanoparticle (e.g., a lipid nanoparticle).

[0185] In some embodiments, the fusion protein, nucleic acid, or combination thereof is formulated in a lipid nanoparticle. In some embodiments, the fusion protein, nucleic acid, or combination thereof is formulated in a lipid-polycation complex called a cationic lipid nanoparticle. By way of non-limiting example, the polycation may include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the fusion protein, nucleic acid, or combination thereof is formulated in a lipid nanoparticle comprising a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0186] Lipid nanoparticle formulations can be influenced by biophysical parameters, including, but not limited to, the cationic lipid component selected, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and size. In one example by Semple et al. (Nature Biotech. 2010 28:172-176), a lipid nanoparticle formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. In another example, varying the composition of the cationic lipid can more effectively deliver siRNA to various antigen-presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200).

[0187] In some embodiments, the lipid nanoparticle formulation may comprise 35-45% cationic lipid, 40-50% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the lipid to RNA (e.g., mRNA) ratio in the lipid nanoparticle may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0188] In some embodiments, the proportion of PEG in the lipid nanoparticle formulation can be increased or decreased, and / or the carbon chain length of the PEG lipid can be changed from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, the lipid nanoparticle formulation can contain PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) at a lipid molar ratio of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0%, and / or 3.0% to 6.0% relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, PEG-c-DOMG can be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid can be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

[0189] In some embodiments, the fusion protein, nucleic acid, or combination thereof is formulated as nanoparticles containing at least one lipid selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipid can be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid can be a lipid described in U.S. Patent Publication No. US20130150625, the entire contents of which are incorporated herein by reference, and / or prepared by the methods described therein. Non-limiting examples of cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 described in US20130150625), 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 described in US20130150625), 2-amino-3-[(

[0039] 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 described in US20130150625), and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1ol (compound 4 described in US20130150625), or any pharmaceutically acceptable salt or stereoisomer thereof.

[0190] Lipid nanoparticle formulations typically contain lipids, particularly ionizable cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), as well as neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids.

[0191] In some embodiments, the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM, (iii) a sterol, e.g., cholesterol, and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

[0192] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% on a molar basis, e.g., 35% to 65%, 45% to 65%, 60%, 57.5%, 50%, or 40% on a molar basis, of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0193] In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% by molar basis, e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% by molar basis, of a neutral lipid. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the lipid nanoparticle formulation comprises 5% to 50% by molar basis (e.g., 15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% by molar basis) of a neutral lipid. A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 20% by molar basis (e.g., 0.5% to 10%, 0.5% to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% by molar basis) of PEG or PEG-modified lipid. In some embodiments, PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2000 Da. In some embodiments, PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2000, for example, about 1,500 Da, about 1000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipid include PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (further described in Reyes et al. J. Controlled Release, 107, 276-287 (2005) which is incorporated herein by reference in its entirety).

[0194] In some embodiments, the molar ratio of lipids is 50 / 10 / 38.5 / 1.5 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG, PEG-DSG, or PEG-DPG)), 57.2 / 7.1 / 34.3 / 1.4 (mol% of cationic lipid / neutral lipid (e.g., DPPC) / cholesterol / PEG-modified lipid (e.g., PEG-cDMA)), 40 / 15 / 40 / 5 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DSG)), 50 / 10 / 35 / 4.5 / 0.5 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DSG)), ), 50 / 10 / 35 / 5 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG)), 40 / 10 / 40 / 10 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), 35 / 15 / 40 / 10 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)), or 52 / 13 / 30 / 5 (mol% of cationic lipid / neutral lipid (e.g., DSPC) / cholesterol / PEG-modified lipid (e.g., PEG-DMG or PEG-cDMA)).

[0195] Non-limiting examples of lipid nanoparticle compositions and methods for preparing them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., S1:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578, which are incorporated herein by reference in their entireties.

[0196] In some embodiments, lipid nanoparticle formulations may comprise cationic lipids, PEG lipids, and structured lipids, and may optionally comprise non-cationic lipids. As a non-limiting example, lipid nanoparticles may comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEG lipids, and 30-50% structured lipids. As another non-limiting example, lipid nanoparticles may comprise 50% cationic lipids, 10% non-cationic lipids, 1.5% PEG lipids, and 38.5% structured lipids. As yet another non-limiting example, lipid nanoparticles may comprise 55% cationic lipids, 10% non-cationic lipids, 2.5% PEG lipids, and 32.5% structured lipids. In some embodiments, the cationic lipid may be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0197] In some embodiments, the lipid nanoparticle formulations described herein can be four-component lipid nanoparticles. The lipid nanoparticles can comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles can comprise 40-60% cationic lipid, 5-15% non-cationic lipid, 1-2% PEG lipid, and 30-50% structured lipid. As another non-limiting example, the lipid nanoparticles can comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid, and 38.5% structured lipid. As yet another non-limiting example, the lipid nanoparticles can comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid, and 32.5% structured lipid. In some embodiments, the cationic lipid can be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0198] In some embodiments, the lipid nanoparticle formulations described herein can comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-KC2-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structured lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise 55% cationic lipid L319, 10% non-cationic lipid DSPC, 2.5% PEG lipid PEG-DMG, and 32.5% structural lipid cholesterol.

[0199] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, or 40 to 200 nm. In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0200] system In one aspect, the present invention provides a system for reducing the number of RNA transcripts in a subject. In one embodiment, the system includes, in one or more vectors, a nucleic acid sequence encoding a fusion protein comprising a CRISPR-associated (Cas) protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and a nucleic acid sequence encoding a CRISPR-Cas system crRNA. In one embodiment, the system further includes, on the same or a different vector, a nucleic acid sequence encoding a second targeting nucleic acid. In one embodiment, the CRISPR-Cas system crRNA substantially hybridizes to a target RNA sequence in the RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the CRISPR-Cas system crRNA are on the same vector. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the CRISPR-Cas system crRNA are on different vectors.

[0201] In one embodiment, the nucleic acid sequence encoding the fusion protein is (1) at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% or more of a nucleic acid sequence selected from the group consisting of: (1) a nucleic acid sequence selected from the group consisting of: %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-89; and (2) a nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, (3) a nucleic acid sequence encoding an amino acid sequence that is at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730; and and nucleic acid sequences encoding amino acid sequences that are at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.In one embodiment, the nucleic acid sequence encoding the fusion protein includes (1) a nucleic acid sequence encoding one amino acid selected from SEQ ID NOs: 47-48, (2) a nucleic acid sequence encoding one amino acid selected from SEQ ID NOs: 49-89, and (3) a nucleic acid sequence encoding one amino acid selected from SEQ ID NOs: 110-730.

[0202] In one embodiment, the system includes, in one or more vectors, a nucleic acid sequence encoding a fusion protein comprising a CRISPR-associated (Cas) protein, an s protein, and optionally a localization signal such as an NLS or NES, a nucleic acid sequence encoding a CRISPR-Cas system crRNA, and a nucleic acid sequence encoding an s peptide. In one embodiment, the s peptide further comprises ERT2. In one embodiment, the nucleic acid sequences encoding the s peptide are on different vectors. In one embodiment, the s peptide forms a catalytically active RNase by binding to the s protein of the fusion protein. In one embodiment, the crRNA of the CRISPR-Cas system substantially hybridizes to a target RNA sequence within the RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the crRNA of the CRISPR-Cas system are on the same vector. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the crRNA of the CRISPR-Cas system are on different vectors.

[0203] In one embodiment, the system includes, in one or more vectors, a nucleic acid sequence encoding a fusion protein comprising a CRISPR-associated (Cas) protein, an s-peptide, and optionally a localization sequence such as an NLS or NES, a nucleic acid sequence encoding a crRNA, and a nucleic acid sequence encoding the s-protein. In one embodiment, the s-peptide further comprises ERT2. In one embodiment, the nucleic acid sequences encoding the s-protein are on different vectors. In one embodiment, the s-protein forms a catalytically active RNase by binding to the s-peptide of the fusion protein. In one embodiment, the crRNA of the CRISPR-Cas system substantially hybridizes to a target RNA sequence within the RNA transcript. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the crRNA of the CRISPR-Cas system are on the same vector. In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the crRNA of the CRISPR-Cas system are on different vectors.

[0204] In one embodiment, the nucleic acid sequence encoding the fusion protein is (1) at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least (2) a nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, or at least 94% identical to one of SEQ ID NOs: 90, 93, and 96; (3) a nucleic acid sequence encoding an amino acid sequence that is at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 83, 84, 86, 87, 89, and 90; a nucleic acid sequence encoding an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of the present invention;(4) A nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730. In one embodiment, the nucleic acid sequence encoding the fusion protein includes (1) a nucleic acid sequence encoding one amino acid of SEQ ID NOs: 47 to 48, (2) a nucleic acid sequence encoding one amino acid of SEQ ID NOs: 90, 93, and 96, (3) a nucleic acid sequence encoding one amino acid of SEQ ID NOs: 83, 84, 86, 87, 89, and 90, and (4) a nucleic acid sequence encoding one amino acid of SEQ ID NOs: 110 to 730.

[0205] Compositions and Formulations In one aspect, the present invention provides a composition for reducing the number of RNA transcripts in a subject. In one embodiment, the composition comprises a fusion protein comprising a CRISPR-associated (Cas) protein, an RNase protein, and optionally a localization sequence such as an NLS or NES. In one embodiment, the composition comprises a crRNA of a CRISPR-Cas system. In one embodiment, the composition is a second targeting nucleic acid. In one embodiment, the crRNA of the CRISPR-Cas system substantially hybridizes to a target RNA sequence within the RNA transcript.

[0206] In one embodiment, the composition comprises: (1) one of SEQ ID NOs: 47-48 at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least (2) an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% identical to one of SEQ ID NOs: 49 to 89; %, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730; and (3) an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, and fusion proteins comprising an amino acid sequence at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the target gene.In one embodiment, the composition comprises a fusion protein comprising (1) one amino acid of SEQ ID NOs: 47-48, (2) one amino acid of SEQ ID NOs: 49-89, and (3) one amino acid of SEQ ID NOs: 110-730.

[0207] In one embodiment, the composition comprises a fusion protein comprising a CRISPR-associated (Cas) protein, an s protein, and optionally a localization sequence such as an NLS or NES, a crRNA of the CRISPR-Cas system, and an s peptide. In one embodiment, the s peptide further comprises ERT2. In one embodiment, the s peptide forms a catalytically active RNase upon binding to the s protein of the fusion protein. In one embodiment, the crRNA of the CRISPR-Cas system substantially hybridizes to a target RNA sequence within the RNA transcript. In one embodiment, the fusion protein and the crRNA of the CRISPR-Cas system are separated from the s peptide. In one embodiment, the s peptide is subsequently added to a composition comprising the fusion protein and the crRNA of the CRISPR-Cas system to confer inducible catalytic activity.

[0208] In one embodiment, the composition comprises a fusion protein comprising a CRISPR-associated (Cas) protein, an s peptide, and optionally a localization sequence such as an NLS or NES, a crRNA of the CRISPR-Cas system, and the s protein. In one embodiment, the s peptide further comprises ERT2. In one embodiment, the s protein forms a catalytically active RNase by binding to the s peptide of the fusion protein. In one embodiment, the crRNA of the CRISPR-Cas system substantially hybridizes to a target RNA sequence within the RNA transcript. In one embodiment, the fusion protein and the crRNA of the CRISPR-Cas system are separated from the s protein. In one embodiment, the s protein is subsequently added to a composition comprising the fusion protein and the crRNA of the CRISPR-Cas system to confer inducible catalytic activity.

[0209] In one embodiment, the fusion protein comprises: (1) a fusion protein comprising at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 201%, at least 202%, at least 203%, at least 204%, at least 205%, at least 206%, at least 207%, at least 208%, at least 209%, at least 300%, at least 310%, at least 311%, at least 312%, at least 313%, at least 314%, at least 315%, at least 316%, at least 317%, at least 318%, at least 319%, at least 320%, at least 321%, at least 322%, at least 323%, at least 324%, at least 325%, at least 326%, at least 327%, at least 328%, at least 329%, at least 330%, at least 331%, at least 332, at least 33 3%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 90, 93, and 96; and (2) an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, or at least identical to one of SEQ ID NOs: 90, 93, and 96. (3) an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79% identical to one of SEQ ID NOs: 83, 84, 86, 87, 89, and 90; (3) an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 110-730; and (4) an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%,and an amino acid sequence at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 47-48. In one embodiment, the composition comprises a fusion protein comprising: (1) an amino acid sequence of one of SEQ ID NOs: 47-48; (2) an amino acid sequence of one of SEQ ID NOs: 90, 93, and 96; (3) an amino acid sequence of one of SEQ ID NOs: 83, 84, 86, 87, 89, and 90; and (4) an amino acid sequence of one of SEQ ID NOs: 110-730.

[0210] The present disclosure also encompasses the use of the pharmaceutical composition of the present disclosure for carrying out the method of the present disclosure. Such pharmaceutical compositions may consist of at least one modulator (e.g., inhibitor or activator) composition of the present invention or its salt in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one modulator (e.g., inhibitor or activator) composition of the present invention or its salt, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The compound of the present invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as by combining with a physiologically acceptable cation or anion, as is well known in the art.

[0211] In one embodiment, pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day, hi another embodiment, pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0212] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, and also depending on the route by which the composition is administered. As an example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0213] Pharmaceutical compositions useful in the methods of the present invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or other routes of administration. Compositions useful in the methods of the present invention may be administered directly to the skin or any other tissue of a mammal. Other contemplated formulations include liposomal formulations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to those skilled in the art and will depend on several factors, including the type and severity of the disease being treated, the species and age of the veterinary or human subject being treated, etc.

[0214] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the field of pharmacology. Generally, such preparation methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage unit.

[0215] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient administered to a subject, or a convenient fraction of such a dosage, e.g., one-half or one-third of such a dosage. The unit dosage form can be a single daily dose or multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0216] In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound or conjugate of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0217] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are often included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.

[0218] The formulations may be used in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration known in the art. Pharmaceutical preparations are sterilized and, if desired, may be mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, colorants, flavorings, and / or fragrances. They may also be combined with other active agents, such as other analgesics, if desired.

[0219] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients, surface active agents, dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0220] The compositions of the present invention may contain about 0.005% to 2.0% by weight of a preservative, based on the total weight of the composition. Preservatives are used to prevent spoilage when exposed to environmental contaminants. Examples of preservatives useful in accordance with the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. An exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0221] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits degradation of the compound. Exemplary antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid in the range of about 0.01% to 0.3% by weight of the total composition, and BHT in the range of 0.03% to 0.1% by weight. In one embodiment, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total composition. Exemplary chelating agents include edetate (e.g., edetate disodium) and citric acid in the range of about 0.01% to 0.20% by weight. In some embodiments, the chelating agent is present in an amount of 0.02% to 0.10% by weight of the total composition. Chelating agents are useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. BHT and edetate disodium are exemplary antioxidants and chelating agents, respectively, for some compounds, however, other suitable and equivalent antioxidants and chelating agents may be substituted as would be known to one skilled in the art.

[0222] Liquid suspensions can be prepared using conventional methods to suspend active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions can further contain one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweeteners. Oily suspensions can further contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phospholipids such as lecithin, condensation products of alkylene oxides with fatty acids, long-chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, or n-propyl parahydroxybenzoate, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0223] Liquid solutions of active ingredients in aqueous or oily solvents can be prepared in essentially the same manner as liquid suspensions, with the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, "oily" liquids comprise liquid molecules containing carbon and exhibit lower polarity than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, but it should be understood that suspending agents do not necessarily aid in dissolving the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0224] Powder and granular formulations of the pharmaceutical preparations of the present invention can be prepared using known methods. Such formulations can be administered directly to a subject or can be used, for example, to form tablets, fill capsules, or to prepare aqueous or oily suspensions or solutions by adding an aqueous or oily vehicle thereto. Each of these formulations can further include one or more of dispersing or wetting agents, suspending agents, and preservatives. Additional excipients, such as fillers and sweeteners, flavorings, or coloring agents, can also be included in these formulations.

[0225] The pharmaceutical composition of the present invention can also be prepared, packaged, or sold in the form of an oil-in-water emulsion or a water-in-oil emulsion.The oil phase can be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof.Such compositions can further contain one or more emulsifiers, such as naturally occurring gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soybean or lecithin phosphatides, esters or partial esters derived from the combination of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.These emulsions can also contain additional ingredients, including, for example, sweeteners or flavoring agents.

[0226] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of depositing or bonding the chemical composition onto a surface, methods of incorporating the chemical composition into the structure of the material during its synthesis (i.e., in physiologically degradable materials), and methods of absorbing aqueous or oily solutions or suspensions into absorbent materials with or without subsequent drying.

[0227] The dosage regimen can affect what constitutes an effective amount. The therapeutic formulation can be administered to a subject either before or after diagnosis of the disease. Furthermore, several divided doses and staggered doses can be administered daily or continuously, or the dose can be continuously infused or bolus injected. Furthermore, the dosage of the therapeutic formulation can be increased or decreased accordingly when the exigencies of the therapeutic or prophylactic situation are indicated.

[0228] Administration of the compositions of the present invention to subjects, including mammals, e.g., humans, can be carried out using known procedures at dosages and for periods effective to prevent or treat disease. The effective amount of therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the activity of the particular compound employed, the time of administration, the rate of compound excretion, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the state of the disease or disorder being treated, the age, sex, weight, condition, general health, and previous medical history of the subject, as well as similar factors well known in the medical field. Dosage regimens can be adjusted to obtain the optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be reduced accordingly if the exigencies of the therapeutic situation indicate. A non-limiting example of an effective dose range for the therapeutic compounds of the present invention is approximately 1 to 5,000 mg / kg body weight / day. Those of ordinary skill in the art will be able to study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0229] The compound may be administered to a subject as frequently as several times a day, or less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months, or once a year or less. It is understood that the daily compound dosage can be administered, in non-limiting examples, every day, every other day, every two days, every three days, every four days, or every five days. For example, in every other day administration, a 5 mg / day dose can be administered starting on Monday, the first subsequent 5 mg / day dose is administered on Wednesday, the second subsequent 5 mg / day dose is administered on Friday, and so on. The frequency of administration is readily apparent to those skilled in the art and will depend on several factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0230] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve a desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0231] A physician, e.g., a doctor or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of the compounds of the present invention employed in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0232] In certain embodiments, it is particularly advantageous to formulate compounds into unit dosage forms for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of therapeutic compound calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical vehicle. The unit dosage forms of the present invention are governed by and directly depend on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding / formulating such therapeutic compounds for the treatment of disease in subjects.

[0233] In one embodiment, the compositions of the present invention are administered to a subject at a dose ranging from once to five or more times per day. In another embodiment, the compositions of the present invention are administered to a subject at a dosage ranging from once daily, once every two days, once every three days, to once weekly, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will vary from subject to subject depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, overall health, and other factors. Therefore, the present invention should not be construed as limited to any particular dosing regimen, and the exact dosage and composition to be administered to any subject will be determined by the attending physician, taking into account all other factors relevant to the subject.

[0234] The compounds of the present invention for administration may be administered in the following dosages: about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3,050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 2,000 mg to about 5 ... The range may be from about 100 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any whole or partial increment therebetween.

[0235] In some embodiments, the dosage of the compound of the invention is about 1 mg to about 2,500 mg. In some embodiments, the dosage of the compound of the invention used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of a second compound described herein (i.e., a drug used to treat the same disease as that treated by the composition of the present invention or a different disease) is less than about 1,000 mg, less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any whole or partial increments thereof.

[0236] In one embodiment, the invention relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second agent, and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0237] The term "container" includes any receptacle for containing a pharmaceutical composition. For example, in one embodiment, the container is packaging containing the pharmaceutical composition. In another embodiment, the container is not packaging containing the pharmaceutical composition. That is, the container is a container such as a box or vial containing a packaged or unpackaged pharmaceutical composition and instructions for using the pharmaceutical composition. Furthermore, packaging technology is well known in the art. It should be understood that instructions for use of the pharmaceutical composition may be included in the packaging containing the pharmaceutical composition, and thus the instructions form a strong functional relationship with the packaged product. However, it should be understood that the instructions may also include information regarding the ability of the compound to perform its intended function, such as, for example, treating or preventing a disease in a subject or delivering an imaging or diagnostic agent to a subject.

[0238] Routes of administration of any of the compositions of the present invention include oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, and nasal), intravesical, intraduodenal, intragastric, rectal, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, or intravenous administration.

[0239] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel capsules, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, electuaries, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions contemplated as useful in the present invention are not limited to the specific formulations and compositions described herein.

[0240] RNA reduction methods and therapeutic methods In one aspect, the present invention provides a method for reducing the number of nuclear RNAs in a subject. In one embodiment, the nuclear RNA is abnormal nuclear RNA. In one embodiment, the method includes administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the RNA.

[0241] In some embodiments, the RNA comprises cytoplasmic RNA. In such embodiments, the method comprises administering to a subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein and an RNase protein, or a fusion protein of the present disclosure comprising a Cas protein and an RNase protein, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence.

[0242] In some embodiments, the method includes administering to a subject: (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and an NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and an NES; and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence.

[0243] In some embodiments, the RNA comprises nuclear RNA. In such embodiments, the method comprises administering to a subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and an NLS, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and an NLS, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence.

[0244] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic or eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal, or fungal cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is an animal cell. In one embodiment, the cell is a yeast cell.

[0245] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0246] In one embodiment, the amount of nuclear RNA is reduced in vitro. In one embodiment, the amount of nuclear RNA is reduced in vivo.

[0247] In one embodiment, the nuclear RNA is a nuclear RNA focus. In one embodiment, the nuclear RNA focus comprises a CUG repeat. In one embodiment, the guide nucleic acid comprises a sequence complementary to a CUG repeat expansion. In one embodiment, the guide nucleic acid comprises a sequence complementary to a CTG repeat expansion. In one embodiment, the guide nucleic acid comprises a sequence complementary to a CTG repeat expansion in the 3'UTR of the human myotonic dystrophy protein kinase (DMPK) gene. In one embodiment, the guide nucleic acid comprises the sequence of one of SEQ ID NOs: 798-800.

[0248] In one aspect, the present invention provides a method of treating a subject having a disease or disorder associated with abnormal nuclear RNA. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the nuclear RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the nuclear RNA.

[0249] In one embodiment, the disease or disorder associated with abnormal nuclear RNA is selected from the group consisting of myotonic dystrophy type 2 (DM2), amyotrophic lateral sclerosis (ALS), Huntington's disease-like 2 (HDL2), spinocerebellar ataxias 8, 31, and 10 (SCA8, SCA31, SCA10), and fragile X-associated tremor ataxia syndrome (FXTAS).

[0250] In one embodiment, the abnormal nuclear RNA is a toxic nuclear RNA foci. In one embodiment, the disease or disorder associated with toxic nuclear RNA foci is myotonic dystrophy type 1. In one embodiment, the targeting nucleotide sequence comprises a sequence complementary to a CTG repeat expansion in the 3'UTR of the human myotonic dystrophy protein kinase (DMPK) gene. In one embodiment, the targeting nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 798-800.

[0251] In one aspect, the present invention provides a method for cleaving nuclear RNA in a subject. In one embodiment, the method comprises administering to the subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the nuclear RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the nuclear RNA.

[0252] In one aspect, the present invention provides a method for treating a disease or disorder associated with increased gene expression. In one embodiment, the method comprises administering to a subject: (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence within an RNA transcript of a gene, or a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence within an RNA transcript of a gene. In one embodiment, the Cas protein cleaves the RNA transcript, thereby preventing translation and protein expression.

[0253] In one aspect, the present invention provides a method for treating an RNA-related disease or disorder. For example, in one embodiment, the present invention provides a method for treating an RNA virus infection. In one embodiment, the method includes administering to a subject (1) a nucleic acid molecule encoding a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, or a fusion protein of the present disclosure comprising a Cas protein, an RNase protein, and optionally a localization sequence such as an NLS or NES, and (2) a nucleic acid molecule encoding a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the viral RNA, or a guide nucleic acid molecule comprising a targeting nucleotide sequence complementary to a target RNA sequence in the viral RNA. In one embodiment, the Cas protein binds to the crRNA, the crRNA binds to the target RNA sequence, and the RNase cleaves the RNA sequence, thereby preventing translation and expression of the viral protein.

[0254] Treatment and Use The present invention provides methods for treating, reducing symptoms of, and / or reducing the risk of developing a disease or disorder in a subject. For example, in one embodiment, the method of the present invention can be used to treat, reduce symptoms of, and / or reduce the risk of developing a mammalian disease or disorder. In one embodiment, the method of the present invention can be used to treat, reduce symptoms of, and / or reduce the risk of developing a plant disease or disorder. In one embodiment, the method of the present invention treats, reduces symptoms of, and / or reduces the risk of developing a yeast disease or disorder.

[0255] In one embodiment, the subject is a cell. In one embodiment, the cell is a prokaryotic or eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In one embodiment, the cell is a plant, animal, or fungal cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is an animal cell. In one embodiment, the cell is a yeast cell.

[0256] In one embodiment, the subject is a mammal. For example, in one embodiment, the subject is a human, a non-human primate, a dog, a cat, a horse, a cow, a goat, a sheep, a rabbit, a pig, a rat, or a mouse. In one embodiment, the subject is a non-mammalian subject. For example, in one embodiment, the subject is a zebrafish, a fruit fly, or a roundworm.

[0257] In one embodiment, disease or disorder is caused by one or more mutations in gene locus.Therefore, in one embodiment, disease or disorder can be treated, reduced, or risk reduced by preventing or reducing mRNA transcription or by preventing or reducing protein translation.Therefore, in one embodiment, the method comprises manipulating RNA transcription.

[0258] In one embodiment, disease or disorder is caused by abnormal RNA.Therefore, in one embodiment, disease or disorder can be treated, reduced, or risk reduced through the agent that prevents or reduces RNA transcription.Therefore, in one embodiment, the method comprises the manipulation of RNA transcription.

[0259] In one embodiment, the method includes administering to a subject (1) a fusion protein of the present disclosure or a nucleic acid molecule encoding the fusion protein of the present disclosure, and (2) one or more targeting nucleic acid molecules comprising a targeting nucleotide sequence complementary to a target region within a gene, wherein the gene encodes an RNA transcript. In one embodiment, the RNase cleaves the RNA transcript.

[0260] In one embodiment, the method includes administering to a subject (1) a fusion protein of the present disclosure or a nucleic acid molecule encoding the fusion protein of the present disclosure, and (2) one or more targeting nucleic acid molecules comprising a targeting nucleotide sequence complementary to a target region within the RNA transcript. In one embodiment, the RNase cleaves the RNA transcript.

[0261] In one embodiment, the disease or disorder is associated with abnormal RNA or increased RNA transcription. For example, in one embodiment, the disease or disorder is an endocrine disease. For example, in one embodiment, the endocrine disease includes, but is not limited to, beta-thalassemia, neonatal diabetes, IPEX syndrome, Mayer-Rokitanski-Kuster-Hauser syndrome, hypothalamic-pituitary-adrenal axis dysregulation, adrenal dysfunction, gonadal dysfunction, ectopic Cushing's syndrome, preeclampsia, diabetic nephropathy, type I diabetes, type II diabetes, and IGF-1 deficiency.

[0262] In one embodiment, the disease or disorder is an oncogenic disease, for example, in one embodiment, oncogenic diseases include, but are not limited to, mantle cell lymphoma, hereditary and sporadic parathyroid tumors, medullary thyroid carcinoma, proliferative conditions, colorectal cancer, glioblastoma, chronic lymphocytic leukemia, and breast cancer.

[0263] In one embodiment, the disease or disorder is a neurological disease or disorder. For example, in one embodiment, the neurological disease includes, but is not limited to, Parkinson's disease, oculopharyngeal muscular dystrophy, Huntington's disease, Fabry's disease, fragile X syndrome, spinal muscular atrophy, amyotrophic lateral sclerosis, spinocerebellar ataxia, spinocerebellar ataxia 1, spinocerebellar ataxia 2, spinocerebellar ataxia 3, spinocerebellar ataxia 6, spinocerebellar ataxia 7, spinocerebellar ataxia 8, spinocerebellar ataxia 10, spinocerebellar ataxia 17, spinocerebellar ataxia 31, and Alzheimer's disease.

[0264] In one embodiment, the disease or disorder is a hematological disease or disorder. For example, in one embodiment, hematological diseases include, but are not limited to, beta thalassemia and alpha thalassemia.

[0265] In one embodiment, the disease or disorder is an infectious disease or an immunological disease or disorder, for example, in one embodiment, the infectious disease or immunological disease includes, but is not limited to, B cell differentiation, T cell activation, systemic lupus erythematosus, Wiskott-Aldrich syndrome, osteoarthritis, scleroderma, and IPEX syndrome.

[0266] In one embodiment, the disease or disorder is a musculoskeletal disease or disorder, for example, in one embodiment, the infectious or immunological disease includes myotonic dystrophy type 1, spinal-bulbar muscular atrophy, and dentatorubral-pallidoluysian atrophy.

[0267] Exemplary diseases or disorders and corresponding targets include, but are not limited to, those listed in Table 1. Additional diseases and disorders and corresponding genes are known in the art, for example, Rehfeld et al., Alternations in Polyadenylation and Its Implications for Endocrine Disease, Front. Endocrinol. 4:53 (2013), Chang et al., Alternative Polyadenylation in Human Diseases, Endocrinol Metab. 32:413-421 (2017), and Curinha et al., Implications of polyadenylation in health and disease, Nucleus 5:508-519 (2014), which are incorporated herein by reference in their entireties. [Table 1-1] [Table 1-2]

[0268] In one embodiment, the disease or disorder is a viral infection.Therefore, in one embodiment, the disease or disorder can be treated, reduced, or risk reduced through the agent that prevents or reduces viral mRNA transcription or prevents or reduces viral protein translation.Therefore, in one embodiment, the method comprises manipulating viral RNA transcription.

[0269] In one embodiment, the method includes administering to a subject (1) a fusion protein of the present disclosure or a nucleic acid molecule encoding the fusion protein of the present disclosure, and (2) one or more targeting nucleic acid molecules comprising a targeting nucleotide sequence complementary to a viral RNA transcript. In one embodiment, the RNase cleaves the viral RNA transcript.

[0270] In one embodiment, the virus is an RNA virus. In one embodiment, the virus produces RNA during its life cycle. In one embodiment, the virus is a human virus, a plant virus, or an animal virus. Exemplary viruses include Adenoviridae, Adenoviridae, Alphaflexiviridae, Anelloviridae, Arenaviridae, Arteriviridae, Asfarviridae, Astroviridae, Beniviridae, Betaflexiviridae, Birnaviridae, Bornaviridae, Bromoviridae, Caliciviridae, Caulimoviridae, Circoviridae, Closteroviridae, Coronaviridae, Filoviridae, Flaviviridae, Geminiviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, These include, but are not limited to, viruses from the families Herpesviridae, Kitaviridae, Luteoviridae, Nairoviridae, Nanoviridae, Nimaviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Polyomaviridae, Pospiviridae, Potyviridae, Poxviridae, Reoviridae, Retroviridae, Retroviruses, Rhabdoviridae, Secoviridae, Togaviridae, Tombusviridae, Tospoviridae, Tymoviridae, and Virugaviridae.For example, exemplary viruses include African swine fever, avian hepatitis E, infectious avian laryngotracheitis, avian nephritis virus, bamboo mosaic virus, banana bunchy top virus, wheat stripe mosaic virus, barley yellow dwarf virus, potato leaf curl virus, Borna disease, brome mosaic virus, wheat, cauliflower mosaic virus, chikungunya, eastern equine encephalitis virus, citrus leprosy, leprosis), Citrus sudden death associated virus, Citrus tristeza virus, Coconut cadang cadang viroid, Curly top virus, African cassava mosaic virus, Cytomegalovirus, Epstein-Barr virus, Dengue fever, Yellow fever, West Nile virus, Zika virus, Ebola virus, Marburg virus, Equine arteritis virus, Porcine reproductive and respiratory syndrome virus, Equine infectious anemia, Foot and mouth disease, Enterovirus, Rhinovirus, Hepatitis B virus, Hepatitis E virus, HIV, HIV-1, HIV-2, Infectious bursal disease virus (poultry), Infectious pancreatic necrosis (salmon), Infectious canine hepatitis, Avian adenovirus of poultry, Influenza virus, Lassa virus, Lymphocytic choriomeningitis virus, Monkeypox, Nairobi sheep disease, Neisseria gonorrhoeae Numerous examples of viruses that harm crops include, but are not limited to, Castle disease virus (poultry), Norwalk virus, Potato virus Y, Porcine circovirus 2, Beak and feather disease virus (poultry), Potato virus M, Rabies virus, Respiratory and enteric adenoviruses, Respiratory syncytial virus, Rice stripe necrosis virus, Rift Valley fever, Rotavirus, SARS-CoV-2, MERS, Sheep pox virus, Lumpy skin disease virus, Sin Nombre virus, Andes virus, SV40, Tobacco ring spot virus, Tomato bushy stunt virus, Tomato spotted wilt virus, Torque teno virus, Venezuelan equine encephalitis virus, Vesicular stomatitis Indiana virus, Viral hemorrhagic septicemia (trout), and White spot syndrome virus (shrimp).

[0271] In one embodiment, exemplary viruses include primate T-lymphotropic virus 1, primate T-lymphotropic virus 2, primate T-lymphotropic virus 3, human immunodeficiency virus 1, human immunodeficiency virus 2, simian foamy virus, human picobirnavirus, Colorado tick fever virus, Changuinola virus, Great Island virus, Lebombo virus, Orungo virus, rotavirus A, rotavirus B, rotavirus C, Banna virus, Borna disease virus, Lake Victoria Marburg virus, Reston Ebola virus, Sudan Ebola virus, Virus, Tai Forest Ebola virus, Zaire virus, Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus, Newcastle disease virus, Human parainfluenza virus 1, Human parainfluenza virus 3, Hendra virus, Nipah virus, Measles virus, Human respiratory syncytial virus, Human metapneumovirus, Chandipura virus, Isfahan virus, Pili virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis Indiana virus, Vesicular stomatitis New Jersey virus, O Australian bat lyssavirus, Dubenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Mokola virus, rabies virus, Guanarito virus, Junin virus, Lassa virus, lymphocytic choriomeningitis virus, Machupo virus, Pichinde virus, Sabia virus, Whitewater Arroyo virus, Bunyamwera virus, Bwamba virus, California encephalitis virus, Carapal virus, Katu virus, Guama virus, Guaroa virus, Kaili virus, Maritubawi virus Rus, Olivocavirus, Oropauchevirus, Shunivirus, Takaiumavirus, Weomiavirus, Andesvirus, Bayouvirus, Black Creek Canalvirus, Dobrava-Belgradevirus, Hantanvirus, Laguna Negravirus, New Yorkvirus, Puumalavirus, Seoulvirus, Sin Nombrevirus, Crimean-Congo hemorrhagic fevervirus, Dugbevirus, Kandilvirus, Punta Torovirus, Rift Valley fevervirus, Sandfly fever Naplesvirus, Influenza Avirus,Influenza B virus, Influenza C virus, Dohorivirus, Thogotovirus, Hepatitis Delta virus, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1, Human coronavirus OC43, SARS coronavirus, Human Torovirus, Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Encephalomyocarditis virus, Theilovirus, Equine rhinovirus A, Foot-and-mouth disease virus, Hepatitis A virus, Human parechovirus, Ljung virus, Aichi virus, Human astrovirus, Human astrovirus 2, Human astrovirus 3, Human astrovirus 4, Human astrovirus 5, Human astrovirus 6, Human astrovirus 7, Human astrovirus 8, Norwalk virus, Sapporo virus These include, but are not limited to, Aroa virus, Banzi virus, Dengue virus, Ilheus virus, Japanese encephalitis virus, Kocobera virus, Kyasanuru Forest disease virus, Loopingir virus, Murray Valley encephalitis virus, Untaya virus, Omsk hemorrhagic fever virus, Powassan virus, Rio Bravo virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Ustu virus, Wesselsbron virus, West Nile virus, yellow fever virus, Zika virus, hepatitis C virus, hepatitis E virus, Barmah Forest virus, chikungunya virus, eastern equine encephalitis virus, Everglades virus, Getah virus, Mayaro virus, Mucambo virus, O'nyong-nyong virus, Pixuna virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, western equine encephalitis virus, Wataroa virus, and rubella virus.

[0272] In one embodiment, exemplary viruses include, but are not limited to, frog herpesvirus 1, frog herpesvirus 2, frog herpesvirus 3, eel herpesvirus 1, carp herpesvirus 1, carp herpesvirus 2, carp herpesvirus 3, sturgeon herpesvirus 2, channel catfish herpesvirus 1, channel catfish herpesvirus 2, salmon herpesvirus 1, salmon herpesvirus 2, salmon herpesvirus 3, avian alphaherpesvirus (Gallid alphaherpesvirus)1, Parakeet Alphaherpesvirus 1, Duck Alphaherpesvirus 1, Pigeon Alphaherpesvirus 1, Avian Alphaherpesvirus 2, Avian Alphaherpesvirus 3, Turkey Alphaherpesvirus 1, Penguin Alphaherpesvirus 1, Sea Turtle Alphaherpesvirus 5, Tortoise Alphaherpesvirus 3, Spider Monkey Alphaherpesvirus 1, Bovine Alphaherpesvirus 2, Cercopithecine Alphaherpesvirus 2, Human Alphaherpesvirus 1, Human Alphaherpesvirus 2, Rabbit Alphaherpesvirus 4, Macaque Alphaherpesvirus 1, Kangaroo Alphaherpesvirus 1, Kangaroo Alphaherpesvirus 2, Chimpanzee Alphaherpesvirus 3, Baboon Alphaherpesvirus 2, Fruit Bat Alphaherpesvirus rupesvirus 1, squirrel monkey alphaherpesvirus 1, bovine alphaherpesvirus 1, bovine alphaherpesvirus 5, buffalo alphaherpesvirus 1, canine alphaherpesvirus 1, caprine alphaherpesvirus 1, long-tailed monkey alphaherpesvirus 9, deer alphaherpesvirus 1, deer alphaherpesvirus 2, equine alphaherpesvirus 1, equine alphaherpesvirus 3, equine alphaherpesvirus 4, equine alphaherpesvirus 8, equine alphaherpesvirus 9, feline alphaherpesvirus 1, human alphaherpesvirus 3, whale alphaherpesvirus 1, seal alphaherpesvirus 1, wild boar alphaherpesvirus 1, sea turtle alphaherpesvirus 6, owl monkey betaherpesvirus 1, capuchin monkey betaherpesvirus 1,Cercopithecus betaherpesvirus 5, Human betaherpesvirus 5, Macaque betaherpesvirus 3, Macaque betaherpesvirus 8, Mandrill betaherpesvirus 1, Chimpanzee betaherpesvirus 2, Baboon betaherpesvirus 3, Baboon betaherpesvirus 4, Squirrel monkey betaherpesvirus 4, Mouse betaherpesvirus 1, Mouse betaherpesvirus 2, Mouse betaherpesvirus 8, Elephant betaherpesvirus 1, Elephant betaherpesvirus 4, Elephant betaherpesvirus 5, Human betaherpesvirus 7, Human betaherpesvirus 6A, Human betaherpesvirus 6B, Macaque betaherpesvirus 9, Mouse betaherpesvirus 3, Wild boar betaherpesvirus 2, Guinea pig betaherpesvirus 2, Tree shrew betaherpesvirus 1, Common marmoset gammaherpesvirus 3, Cercopithecus gammaherpesvirus 14, Gorilla gammaherpesvirus gammaherpesvirus) 1, human gammaherpesvirus 4, macaque gammaherpesvirus 4, macaque gammaherpesvirus 10, chimpanzee gammaherpesvirus 1, baboon gammaherpesvirus 1, orangutan gammaherpesvirus 2, arcellafin gammaherpesvirus 1, arcellafin gammaherpesvirus 2, bovine gammaherpesvirus 6, caprine gammaherpesvirus 2, antelope gammaherpesvirus 1, ovine gammaherpesvirus 2, wild boar gammaherpesvirus 3, wild boar gammaherpesvirus 4, wild boar gammaherpesvirus 5, and swine gammaherpesvirus 6. Horse gammaherpesvirus 2, equine gammaherpesvirus 5, feline gammaherpesvirus 1, badger gammaherpesvirus 1, seal gammaherpesvirus 3, eastern bat gammaherpesvirus 1, spider monkey gammaherpesvirus 2, spider monkey gammaherpesvirus 3, bovine gammaherpesvirus 4, crescendo rat gammaherpesvirus 2, human gammaherpesvirus 8, macaque gammaherpesvirus 5, macaque gammaherpesvirus 8, macaque gammaherpesvirus 11, macaque gammaherpesvirus 12, mouse gammaherpesvirus 4, mouse gammaherpesvirus 7,Squirrel monkey gammaherpesvirus 2, Equine gammaherpesvirus 7, Seal gammaherpesvirus 2, Saguinine gammaherpesvirus 1, Iguana herpesvirus 2, Haliotid herpesvirus 1, Ihodagaki herpesvirus 1, Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus Rus38, Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus KWBSE43-6, Klebsiella virus 0507KN21, Klebsiella virus KpS110, Klebsiella virus May, Klebsiella virus Menlow, Serratia virus IME250, Erw inia virus Ea2809, Serratia virus MAM1, Acinetobacter virus Acibel007, Acinetobacter virus AB3, Acinetobacter virus AbKT21III, Acinetobacter virus Abp1, Acinetobacter virus Aci07, Acinetobacter virus Aci08, Acinetobacter virus AS11, Acinetobacter virus AS12, Acinetobacter virus Fri1, Acinetob Acinetobacter virus IME200, Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobacter virus phiAB1, Acinetobacter virus SH-Ab15519, Acinetobacter virus SWHAb1, Acinetobacter virus SWHAb3, Acinetobacter virus WCHABP5, Acintetobacter virus B1, Acintetobacter virus B2, Acintetobacter virus B5,Acintetobacter virus D2, Acintetobacter virus P1, Acintetobacter virus P2, Acintetobacter virus phiAB6, Acintetobacter virus Petty, Vibrio virus Vc1, Vibrio virus A318, Vibrio virus AS51, Vibrio virus Vp670, Marinomonas virus CB5A, Marinomonas virus CPP1m, Vibrio virus VEN, Pseudomonas virus Achelous, Pseudomonas virus Alpheus, Pseudomonas virus Nerthus, Pseudomonas virus Njord, Pseudomonas virus uligo, Pseudomonas virus C171, Pectobacterium virus PP16, Pectobacterium virus PPWS1, Pectobacterium virus PPWS2, Pectobacterium virus CB5, Pectobacterium virus Clickz, Pectobacterium virus RusfM1, Pectobacterium virus Gaspode, Pectobacterium virus Khlen, Pectobacterium virus Koot, Pectobacterium virus Lelidair, Pectobacterium virus Nobby, Pectobacterium virus Peat1, Pectobacterium virus Phoria, Pectobacterium virus PP90, Pectobacterium virus Zenivior, Dickeya virus BF25-1 2, Pseudomonas virus NV3, Pseudomonas virus 130-113, Pseudomonas virus 15pyo, Pseudomonas virus Ab05, Pseudomonas virus ABTNL, Pseudomonas virus DL62, Pseudomonas virus kF77, Pseudomonas virus LKD16, Pseudomonas virus LUZ19, Pseudomonas virus MPK6, Pseudomonas virus MPK7, Pseudomonas virus NFS,Pseudomonas virus PAXYB1, Pseudomonas virus phiKMV, Pseudomonas virus PT2, Pseudomonas virus PT5, Pseudomonas virus RLP, Pseudomonas virus LKA1, Pseudomonas virus f2, Aeromonas virus 25AhydR2PP, Aeromonas virus AS7, Aeromonas virus ZPAH7, Yersinia virus ISAO8, Aeromonas virus Ahp1, Aeromonas virus CF7, Cronobacter virus DevCD23823, Cronobacter virus GAP227, Salmonella virus Spp16, Yersinia virus R8-01, Yersinia virus fHeYen301, Yersinia virus Phi80-18, Pectobacterium virus Arno160, Pectobacterium virus PP2, Proteus virus PM85, Proteus virus PM93, Proteus virus PM116, Proteus virus Pm5460, Pectobacterium virus PP1, Erwinia virus Era103, Erwinia virus S2, Lelliottia virus phD2B, Citrobacter CrRp3, Escherichia virus LL11, Escherichia virus AAPEc6, Escherichia virus ACGC91, Escherichia virus B, Escherichia virus C, Escherichia virus K, Escherichia virus K1-5, Escherichia virus K1E, Escherichia virus mutPK1A2, Escherichia virus VEC3, Escherichia virus UAB78, Salmonella virus BP12B, Salmonella virus SP6, Burkholderia virus BpAMP1, Ralstonia virus RSPI1, Ralstonia virus RSB1, Ralstonia virus RsoP1IDN, Burkholderia virus JG068, Ralstonia virus RSJ2, Ralstonia virus RSJ5,Ralstonia virus RSPII1, Shigella virus Buco, Escherichia virus Minorna, Klebsiella virus AltoGao, Klebsiella virus BO1E, Klebsiella virus F19, Klebsiella virus K244, Klebsiella virus Kp2, Klebs, iella virus KP34, Klebsiella virus KPRio2015, Klebsiella virus KpS2, Klebsiella virus KpV41, Klebsiella virus KpV48, Klebsiella virus KpV71, Klebsiella virus KpV74, Klebsiella virus KpV475, Klebsiella virus KPV811, Klebsiella virus myPSH1235, Klebsiella virus SU503, Klebsiella virus SU552A, S higella virus SFN6B, Enterobacter virus KDA1, Proteus virus PM16, Proteus virus PM75, Dickeya virus Dagda, Dickeya virus Katbat, Dickeya virus Luksen, Dickeya virus Mysterion, Yersinia virus AP10, Erwinia virus FE44, Escherichia virus 285P, Escherichia virus BA14, Escherichia virus P483, Escherichia ia virus P694, Escherichia virus S523, Kluyvera virus Kvp1, Pectobacterium virus PP74, Salmonella virus BP12A, Salmonella virus BSP161, Shigella virus A7, Yersinia virus Berlin, Yersinia virus PYPS50, Yersinia virus Yepe2, Yersinia virus Yepf, Citrobacter virus CR8, Vibrio virus ICP3, Vibrio virus N4 , Vibrio virus VP4, Enterobacter virus Eap1, Erwinia virus L1, Escherichia virus SRT7, Pseudomonas virus 17A, Pseudomonas virus gh1, Pseudomonas virus Henninger, Pseudomonas virus KNP, Pseudomonas virus Pf1ERZ2017, Pseudomonas virus PhiPSA2, Pseudomonas virus PhiPsa17, Pseudomonas virus PPPL1,Pseudomonas virus shl2, Pseudomonas virus WRT, Yersinia virus fPS9, Yersinia virus fPS53, Yersinia virus fPS59, Yersinia virus fPS54ocr, Pectobacterium virus Jarilo, Citrobacter virus CR44b, Citrobacter virus SH3, Citrobacter virus SH4, Cronobacter virus Dev2, Cronobacter virus GW1, Enterobacter r virus EcpYZU01, Escherichia virus EcoDS1, Escherichia virus F, Escherichia virus GA2A, Escherichia virus IMM002, Escherichia virus K1F, Escherichia virus LM33P1, Escherichia virus PE3-1, Escherichia virus Ro45lw, Escherichia virus ST31, Escherichia virus Vec13, Escherichia virus YZ1, Escherichia hia virus ZG49, Shigella virus SFPH2, Morganella virus MmP1, Morganella virus MP2, Dickeya virus JA10, Dickeya virus Ninurta, Pectobacterium virus PP47, Pectobacterium virus PP81, Pectobacterium virus PPWS4, Pseudomonas virus PPpW4, Pseudomonas virus 22PfluR64PP, Pseudomonas virus IBBPF7A, Pseu Pseudomonas virus Pf10, Pseudomonas virus PFP1, Pseudomonas virus PhiS1, Pseudomonas virus UNOSLW1, Pseudomonas virus PspYZU08, Escherichia virus K30, Klebsiella virus 2044-307w, Klebsiella virus BIS33, Klebsiella virus Henu1, Klebsiella virus IL33, Klebsiella virus IME205, Klebsiella virus IME321,Klebsiella virus K5, Klebsiella virus K11, Klebsiella virus K5-2, Klebsiella virus K5-4, Klebsiella virus KN1-1, Klebsiella virus KN3-1, Klebsiella virus KN4-1, Klebsiella virus Kp1, Klebsiella virus KP32, Klebsiella virus KP32i192, Klebsiella virus KP32i194, Klebsiella virus KP32i195, Klebsiella lla virus KP32i196, Klebsiella virus kpssk3, Klebsiella virus KpV289, Klebsiella virus KpV763, Klebsiella virus KpV766, Klebsiella virus KpV767, Klebsiella virus Pharr, Klebsiella virus PRA33, Klebsiella virus SHKp152234, Klebsiella virus SHKp152410, Citrobacter virus CFP1, Citrobacter virus SH1, Citrobacter virus SH2, Enterobacter virus E2, Enterobacter virus E3, Enterobacter virus KPN3, Enterobacteria virus T7M, Escherichia virus ECA2, Escherichia virus LL2, Escherichia virus T3, Escherichia virus T3Luria, Leclercia virus 10164-302, Salmonella virus SG-JL2, Serratia virus 2050H2 , Serratia virus SM9-3Y, Yersinia virus AP5, Yersinia virus YeF10, Yersinia virus YeO3-12, Enterobacteria virus IME390, Escherichia virus 13a, Escherichia virus 64795ec1, Escherichia virus C5, Escherichia virus CICC80001, Escherichia virus Ebrios, Escherichia virus EG1, Escherichia virus HZ2R8,Escherichia virus HZP2, Escherichia virus N30, Escherichia virus NCA, Escherichia virus T7, Salmonella virus 3A8767, Salmonella virus Vi06, Stenotrophomonas virus IME15, Yersinia virus YpPY, Yersinia virus YpsPG, Pseudomonas virus Phi15, Pectobacterium virus DUPPII, Synechococcus virus SCBP42, Aquamicrobium virus P14, Ashi virus S45C4, Agrobacterium virus Atuph02, Agrobacterium virus Atuph03, Ralstonia virus Ap1, Ayaq virus S45C18, Prochlorococcus virus SS120-1, Pseudomonas virus Phi15 omonas virus Andromeda, Pseudomonas virus Bf7, Escherichia virus J8-65, Escherichia virus Lidtsur, Prochlorococcus virus NATL1A7, Chos virus KM23C739, Rhizobium virus RHEph02, Rhizobium virus RHEph08, Rhizobium virus RHEph09, Vibrio virus Cyclit, Escherichia virus PGT2, Escherichia virus PhiKT, Alteromonas virus H4-4, Fouss virus S46C10, Fuss virus S30C28, Escherichia virus ECBP5, Pectobacterium virus PP99, Ralstonia virus DURPI, Ralstonia virus RsoP1EGY, Synechococcus STIP37, Jalka virus S08C159, Ralstonia virus RSB3, Kawa virus SWcelC56, Synechococcus virus SRIP1, Providencia virus PS3, Curvibacter virus P26059B, Ralstonia virus RSB2, Synechococcus virus SCBP2, Krak virus S39C11, Podo virus Lau218,Pantoea virus LIMElight, Prochlorococcus virus PGSP1, Synechococcus virus SCBP3, Caulobacter virus Lullwater, Vibrio virus KF1, Vibrio virus KF2, Vibrio virus OWB, Vibrio virus VP93, Pseudomonas virus VSW3, Nohi virus S31C1, Oinez virus S37C6, Rhizobium virus RHEph01, Paga virus S05C849, Mesorhizobiu m virus Lo5R7ANS, Pedos virus S28C3, Pekhit virus S04C24, Pelagibacter virus HTVC019P, Pelagi virus S35C6, Caulobacter virus Percy, Delftia virus IMEDE1, Podi virus S05C243, Pseudomonas virus PollyC, Synechococcus virus SCBP4, Pow virus S08C41, Xanthomonas virus f20, Xanthomonas virus f30, Xanthomonas s virus XAJ24, Xanthomonas virus Xc10, Xylella virus Prado, Synechococcus virus SB28, Sphingomonas virus Scott, Synechococcus virus SRIP2, Ralstonia virus ITL1, Sieq virus S42C7, Ralstonia virus RPSC1, Stopala virus S38C3, Pelagibacter virus HTVC011P, Stupnya virus KM16C193, Prochlorococcus virus 95 1510a, Prochlorococcus virus NATL2A133, Prochlorococcus virus PSSP10, Vibrio virus JSF7, Prochlorococcus virus PSSP7, Synechococcus virus P60, Prochlorococcus virus PSSP3, Synechococcus virus PSSP2, Synechococcus virus Syn5, Votkov virus S28C10, Pantoea virus LIMEzero, Pasteurella virus PHB01,Pasteurella virus PHB02, Escherichia virus GJ1, Escherichia virus ST32, Erwinia virus Faunus, Erwinia virus Y2, Aeromonas virus pAh6C, Pectobacterium virus PM1, Pectobacterium virus PP101, Shewanella virus Spp001, Shewanella virus SppYZU05, Vibrio virus Ceto, Vibrio virus Thalassa, Vibrio virus JSF10, Vibrio virus JSF12, Vibrio virus phi3, Vibrio virus pVp1, Escherichia virus EPS7, Escherichia virus mar003J3, Escherichia virus saus132, Salmonella virus 123, Salmonella virus 329, Salmonella virus 118970sal2, S Salmonella virus LVR16A, Salmonella virus S113, Salmonella virus S114, Salmonella virus S116, Salmonella virus S124, Salmonella virus S126, Salmonella virus S132, Salmonella virus S133, Salmonella virus S147, Salmonella virus Seafire, Salmonella virus SH9, Salmonella virus STG2, Salmonella virus Stit ch, Salmonella virus Sw2, Yersinia virus phiR201, Escherichia virus AKFV33, Escherichia virus BF23, Escherichia virus chee24, Escherichia virus DT5712, Escherichia virus DT57C, Escherichia virus FFH1, Escherichia virus Gostya9, Escherichia virus H8, Escherichia virus mar004NP2, Escherichia virus SOSSYSP, Escherichia virus phiAPCEc03, Escherichia virus phiLLS, Escherichia virus slur09, Escherichia virus T5, Salmonella virus NR01, Salmonella virus S131, Salmonella virus Shivani, Salmonella virus SP01, Salmonella virus SP3, Salmonella virus SPC35, Shigella virus SHSML45, Shigella virus SSP1,Pectobacterium virus DUPPV, Pectobacterium virus My1, Proteus virus PM135, Proteus virus Stubb, Vibrio virus PG07, Vibrio virus VspSw1, Aeromonas virus AhSzq1, Aeromonas virus AhSzw1, Klebsiella virus IME260, Klebsiella virus Sugarland, Escherichia virus IME542, Escherichia virus ACGM12, Escherichia virus Shigella virus EC3a, Escherichia virus DTL, Escherichia virus IME253, Escherichia virus Rtp, Shigella virus Sf12, Escherichia virus phiEB49, Escherichia virus AHP42, Escherichia virus AHS24, Escherichia virus AKS96, Escherichia virus C119, Escherichia virus E41c, Escherichia virus Eb49, Escherichia virus RusJk06, Escherichia virus KP26, Escherichia virus phiJLA23, Escherichia virus Rogue1, Shigella virus Sd1, Shigella virus pSf1, Citrobacter virus DK2017, Citrobacter virus Sazh, Citrobacter virus Stevie, Escherichia virus LL5, Escherichia virus TLS, Salmonella virus 36, Salmonella virus PHB07, Salm onella virus phSE2, Salmonella virus SP126, Salmonella virus YSP2, Escherichia virus 95, Escherichia virus mar001J1, Escherichia virus mar002J2, Escherichia virus SECphi27, Escherichia virus swan01, Escherichia virus IME347, Escherichia virus SRT8, Escherichia virus ADB2, Escherichia virus BIFF,Escherichia virus IME18, Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus SH2, Escherichia virus T1, Shigella virus 008, Shigella virus ISF001, Shigella virus PSf2, Shigella virus Sfin1, Shigella virus SH6, Shigella virus Shfl1, Shigella virus ISF002, Cronobacter virus Esp2949-1, Enterobacter virus E cL1, Cronobacter virus PhiCS01, Escherichia virus ESCO41, Pantoea virus AAS23, Escherichia virus NBD2, Enterobacter virus F20, Klebsiella virus 1513, Klebsiella virus GHK3, Klebsiella virus KLPN1, Klebsiella virus KOX1, Klebsiella virus KP36, Klebsiella virus KpCol1, Klebsiella virus KpKT21phi1, Klebsiella virus KPN N141, Klebsiella virus KpV522, Klebsiella virus MezzoGao, Klebsiella virus NJR15, Klebsiella virus NJS1, Klebsiella virus NJS2, Klebsiella virus PKP126, Klebsiella virus Sushi, Klebsiella virus TAH8, Klebsiella virus TSK1, Bacillus virus Agate, Bacillus virus Bobb, Bacillus virus Bp8pC, Bacillus virus Bastille, Bacillus virus CAM003, Bacillus virus Evoli, Bacillus virus HoodyT, Bacillus virus AvesoBmore, Bacillus virus B4, Bacillus virus Bigbertha, Bacillus virus Riley, Bacillus virus Spock, Bacillus virus Troll, Bacillus virus Bc431, Bacillus virus Bcp1,Bacillus virus BCP82, Bacillus virus BM15, Bacillus virus Deepblue, Bacillus virus JBP901, Bacillus virus Grass, Bacillus virus NIT1, Bacillus virus SPG24, Bacillus virus BCP78, Bacillus virus TsarBomba, Bacillus virus BPS13, Bacillus virus BPS10C, Bacillus virus Hakuna, Bacillus virus Megatron, Bacillus virus WPh, Bacillus virus Mater, Bacillus virus Moonbeam, Bacillus virus SIOphi, Enterococcus virus ECP3, Enterococcus virus EF24C, Enterococcus virus EFLK1, Enterococcus virus EFDG1, Enterococcus virus EFP01, Enterococcus virus EfV12, Listeria virus A511, Listeria virus AG20, Listeria virus L ist36, Listeria virus LMSP25, Listeria virus LMTA34, Listeria virus LMTA148, Listeria virus LP048, Listeria virus LP064, Listeria virus LP083-2, Listeria virus P100, Listeria virus WIL1, Bacillus virus Camphawk, Bacillus virus SPO1, Bacillus virus CP51, Bacillus virus JL, Bacillus virus Shanette, Staphylococcus virus BS1, Staphylococcus virus BS2, Lactobacillus virus Bacchae, Lactobacillus virus Bromius, Lactobacillus virus Iacchus, Lactobacillus virus Lpa804, Lactobacillus virus Semele, Staphylococcus virus G1, Staphylococcus virus G15, Staphylococcus virus JD7, Staphylococcus virus K,Staphylococcus virus MCE2014, Staphylococcus virus P108, Staphylococcus virus Rodi, Staphylococcus virus S253, Staphylococcus virus S25-4, Staphylococcus virus SA12, Staphylococcus virus Sb1, Staphylococcus virus SscM1, Staphylococcus virus IPLAC1C, Staphylococcus virus SEP1, Staphyloc occus virus Remus, Staphylococcus virus SA11, Staphylococcus virus Stau2, Staphylococcus virus Twort, Brochothrix virus A9, Lactobacillus virus Lb338-1, Lactobacillus virus LP65, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus IBB35, Camp Campylobacter virus CP81, Campylobacter virus CP30A, Campylobacter virus CPX, Campylobacter virus Los1, Campylobacter virus NCTC12673, Escherichia virus Alf5, Escherichia virus AYO145A, Escherichia virus EC6, Escherichia virus HY02, Escherichia virus JH2, Escherichia virus TP1, Escherichia virus VpaE1, Escherichia virus wV8, Salmonella virus BPS15Q2, Salmonella virus BPS17L1, Salmonella virus BPS17W1, Salmonella virus FelixO1, Salmonella virus Mushroom, Salmonella virus Si3, Salmonella virus SP116, Salmonella virus UAB87, Erwinia virus Ea214, Erwinia virus M7, Citrobacter virus Moogle,Citrobacter virus Mordin, Shigella virus Sf13, Shigella virus Sf14, Shigella virus Sf17, Escherichia virus SUSP1, Escherichia virus SUSP2, Ralstonia virus RSA1, Ralstonia virus RSY1, Mannhe, imia virus 1127AP1, Mannheimia virus PHL101, Aeromonas virus phiO18P, Vibrio virus Canoe, Pseudoalteromonas virus C5a, Pseudomonas virus Dobby, Pseudomonas virus phiCTX, Erwinia virus EtG, Escherichia virus 186, Salmonella virus PsP3, Salmonella virus SEN1, Erwinia virus ENT90, Klebsiella virus 4LV2017, Salmonella virus Fels2, Salmonella virus RE2010, Salmonella virus SEN8, Salmonella virus SopEphi, Haemophilus virus HP1, Haemophilus virus HP2, Vibrio virus Kappa, Pasteurella virus F108, Burkholderia virus KS14, Burkholderia virus AP3, Burkholderia virus KS5, Vibrio virus K139, Burkholderia ia virus ST79, Escherichia virus fiAA91ss, Escherichia virus P2, Escherichia virus pro147, Escherichia virus pro483, Escherichia virus Wphi, Yersinia virus L413C, Pseudomonas virus phi3, Salinivibrio virus SMHB1, Klebsiella virus 3LV2017, Salmonella virus SEN4, Cronobacter virus ESSI2, Stenotrop homonas virus Smp131, Salmonella virus FSLSP004, Burkholderia virus KL3, Burkholderia virus phi52237, Burkholderia virus phiE122, Burkholderia virus phiE202, Vibrio virus PV94, Escherichia virus P88, Escherichia virus Bp7, Escherichia virus IME08, Escherichia virus JS10, Escherichia virus JS98,Escherichia virus MX01, Escherichia virus QL01, Escherichia virus VR5, Escherichia virus WG01, Escherichia virus VR7, Escherichia virus VR20, Escherichia virus VR25, Escherichia virus VR26, Shigella virus SP18, Salmonella virus Melville, Salmonella virus S16, Salmonella virus STML198, Salmonella virus STP4a, Klebsiella virus JD18, Klebsiella virus PKO111, Enterobacter virus PG7, Escherichia virus CC31, Escherichia virus ECD7, Escherichia virus GEC3S, Escherichia virus JSE, Escherichia virus phi1, Escherichia virus RB49, Citrobacter virus CF1, Citrobacter virus Merlin, Citrobacter virus Moo n, Escherichia virus APCEc01, Escherichia virus HP3, Escherichia virus HX01, Escherichia virus JS09, Escherichia virus O157tp3, Escherichia virus O157tp6, Escherichia virus PhAPEC2, Escherichia virus RB69, Escherichia virus ST0, Shigella virus SHSML521, Shigella virus UTAM, Vibrio virus KVP40, Vi brio virus nt1, Vibrio virus ValKK3, Enterobacter virus Eap3, Klebsiella virus KP15, Klebsiella virus KP27, Klebsiella virus Matisse, Klebsiella virus Miro, Klebsiella virus PMBT1, Escherichia virus AR1, Escherichia virus C40, Escherichia virus CF2, Escherichia virus E112, Escherichia virus ECML134,Escherichia virus HY01, Escherichia virus HY03, Escherichia virus Ime09, Escherichia virus RB3, Escherichia virus RB14, Escherichia virus slur03, Escherichia virus slur04, Escherichia virus T4, Shigella virus Pss1, Shigella virus Sf21, Shigella virus Sf22, Shigella virus Sf24, Shigella virus SHBML 501, Shigella virus Shfl2, Yersinia virus D1, Yersinia virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas virus Aeh1, Escherichia virus RB16, Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus 42, Escherichia virus Av05, Cronobacter virus CR3, Cronobacter virus CR8, Cro nobacter virus CR9, Cronobacter virus PBES02, Pectobacterium virus phiTE, Cronobacter virus GAP31, Escherichia virus 4MG, Salmonella virus PVPSE1, Salmonella virus SSE121, Escherichia virus APECc02, Escherichia virus FFH2, Escherichia virus FV3, Escherichia virus JES2013, Escherichia virus Mur ica, Escherichia virus slur16, Escherichia virus V5, Escherichia virus V18, Brevibacillus virus Abouo, Brevibacillus virus Davies, Synechococcus virus SMbCM100, Erwinia virus Deimos, Erwinia virus Desertfox, Erwinia virus Ea35-70, Erwinia virus RAY, Erwinia virus Simmy50, Erwinia virus SpecialG,Synechococcus virus SShM2, Klebsiella virus K64-1, Klebsiella virus RaK2, Dickeya virus AD1, Erwinia virus Alexandra, Lactobacillus virus LBR48, Synechococcus virus SCAM1, Synechococcus virus SCBWM1, Vibrio virus Aphrodite1, Escherichia virus 121Q, Eschierichia virus PBECO4, Synechococcus virus Virus AC2014fSyn7803C8, Synechococcus virus ACG2014f, Synechococcus virus ACG2014fSyn7803US26, Synechococcus virus STIM5, Pseudomonas virus PaBG, Rheinheimera virus Barba18A, Rheinheimera virus Barba19A, Rheinheimera virus Barba21A, Rheinheimera virus Barba5S, Rheinheimera virus Barba8 S, Burkholderia virus BcepMu, Burkholderia virus phiE255, Synechococcus virus Bellamy, Gordonia virus GMA6, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacterium virus Bxz1, Mycobacterium virus Dandelion, Mycobacterium virus HyRo, Mycobacterium virus I3, Mycobacterium virus Lukilu, Mycobacterium virus Nappy, Mycobacterium virus Sebata, Faecalibacterium virus Brigit, Prochlorococcus virus Syn33, Synechococcus virus SRIM12-01, Synechococcus virus SRIM12-06, Synechococcus virus SRIM12-08, Salmonella virus SEN34, Acidovorax virus ACP17, Xanthomonas virus Carpasina,Xanthomonas virus XcP1, Pseudomonas virus pf16, Synechococcus virus SCAM3, Ralstonia virus RSF1, Ralstonia virus RSL2, Synechococcus virus SWAM2, Erwinia virus Derbicus, Pseudomonas virus EL, Sinorhizobium virus M7, Sinorhizobium virus M12, Sinorhizobium virus N3, Serratia virus BF, Yersinia virus Ye n9-04, Faecalibacterium virus Epona, Erwinia virus Asesino, Erwinia virus EaH2, Prochlorococcus virus MED4-213, Prochlorococcus virus PHM1, Prochlorococcus virus PHM2, Flavobacterium virus FCL2, Flavobacterium virus FCV1, Pseudomonas virus KIL2, Pseudomonas virus KIL4, Edwardsiella virus GF2, E Escherichia virus Goslar, Halomonas virus HAP1, Vibrio virus VP882, Lactobacillus virus Lb, Erwinia virus EaH1, Iodobacter virus PLPE, Delftia virus PhiW14, Klebsiella virus JD001, Klebsiella virus KpV52, Klebsiella virus KpV80, Escherichia virus CVM10, Escherichia virus ECOO78, Escherichia virus ep3, Brevibacillus virus Jimmer, Brevibacillus virus Osiris, Synechococcus virus SCAM9, Rhizobium virus RHEph4, Faecalibacterium virus Lagaffe, Synechococcus virus SP4, Synechococcus virus Syn30, Prochlorococcus virus PTIM40, Synechococcus virus SSKS1, Salmonella virus ZCSE2, Clostridium virus phiC2,Clostridium virus phiCD27, Clostridium virus phiCD119, Erwinia virus Machina, Arthrobacter virus BarretLemon, Arthrobacter virus Beans, Arthrobacter virus Brent, Arthrobacter virus Jawnsk, i, Arthrobacter virus Martha, Arthrobacter virus Piccoletto, Arthrobacter virus Shade, Arthrobacter virus Sonny, Synechococcus virus SCAM7, Acinetobacter virus ME3, Ralstonia virus RSL1, Cronobacter virus GAP32, Pectinobacterium virus CBB, Faecalibacterium virus Mushu, Escherichia virus Mu, Shigella virus SfMu, Halobacterium virus phiH, Burkholderia virus Bcep1, Burkholderia virus Bcep43, Burkholderia virus Bcep781, Burkholderia virus BcepNY3, Xanthomonas virus OP2, Synechococcus virus SMbCM6, Pseudomonas virus Ab03, Pseudomonas virus G1, Pseudomonas virus KPP10, Pseudomonas virus PAKP3, Pseudomonas virus PS24, Synechococcus virus SRIM8, Synechococcus virus SRIM50, Synechococcus virus ACG2014bSyn7803C61, Synechococcus virus ACG2014bSyn9311C4, Synechococcus virus SRIM2, Synechococcus virus SPM2, Pseudomonas virus Noxifer, Acinetobacter virus AB1, Acinetobacter virus Rus AB2, Acinetobacter virus AbC62, Acinetobacter virus AbP2, Acinetobacter virus AP22, Acinetobacter virus LZ35, Acinetobacter virus WCHABP1, Acinetobacter virus WCHABP12, Pseudomonas virus Psa374, Pseudomonas virus VCM, Pseudomonas virus CAb1, Pseudomonas virus CAb02, Pseudomonas virus JG004,Pseudomonas virus MAG1, Pseudomonas virus PA10, Pseudomonas virus PAKP1, Pseudomonas virus PAKP2, Pseudomonas virus PAKP4, Pseudomonas virus PaP1, Pseudomonas virus phiMK, Pseudomonas virus Zigelbrucke, Prochlorococcus virus PSSM7, Burkholderia virus BcepF1, Pseudomonas virus 141, Pseudomonas virus Virus Ab28, Pseudomonas virus CEBDP1, Pseudomonas virus DL60, Pseudomonas virus DL68, Pseudomonas virus E215, Pseudomonas virus E217, Pseudomonas virus F8, Pseudomonas virus JG024, Pseudomonas virus KPP12, Pseudomonas virus KTN6, Pseudomonas virus LBL3, Pseudomonas virus LMA2, Pseudomonas virus NH4, P Pseudomonas virus PA5, Pseudomonas virus PB1, Pseudomonas virus PS44, Pseudomonas virus SN, Pectinobacterium virus PEAT2, Edwardsiella virus pEtSU, Bordetella virus PHB04, Escherichia phage ESCO13, Escherichia virus ESCO5, Escherichia virus phAPEC8, Escherichia virus Schickermooser, Klebsiella virus ZCKP1, Pseudomonas virus PA7, Pseudomonas virus phiKZ, Pseudomonas virus SL2, Pseudomonas virus PMW, Agrobacterium virus Atuph07, Synechococcus virus Syn19, Aeromonas virus 56, Aeromonas virus 43, Escherichia virus P1, Escherichia virus RCS47, Salmonella virus SJ46, Pseudoalteromonas virus J2-1,Arthrobacter virus ArV1, Arthrobacter virus Colucci, Arthrobacter virus Trina, Ralstonia virus RP12, Erwinia virus Risingsun, Salmonella virus BP63, Acinetobacter virus Aci05, Acinetobacter virus Aci01-1, Acinetobacter virus Aci02-2, Prochlorococcus virus PSSM2, Dickeya virus JA11, Dickeya virus JA29, Erwinia virus Y3, Agrobacterium virus 7-7-1, Salmonella virus SPN3US, Bacillus virus Shbh1, Bacillus virus 1, Geobacillus virus GBSV1, Pseudomonas virus tabernarius, Synechococcus virus ST4, Faecalibacterium virus Taranis, Synechococcus virus SIOM18, Yersinia virus R1RT, Yersinia virus TG1 , Synechococcus virus STIM4, Synechococcus virus SSM1, Bacillus virus SP15, Vibrio virus pTD1, Vibrio virus VP4B, Tetrasphaera virus TJE1, Faecalibacterium virus Toutatis, Aeromonas virus 25, Aeromonas virus Aes12, Aeromonas virus Aes508, Aeromonas virus AS4, Aeromonas virus Asgz, Stenotrophomonas virus IME13, Prochlorococcus virus Syn1, Synechococcus virus SRIM44, Vibrio virus MAR, Vibrio virus VHML, Vibrio virus VP585, Escherichia virus ECML4, Salmonella virus Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus ViI, Erwinia virus Wellington,Escherichia virus ECML-117, Escherichia virus FEC19, Escherichia virus WFC, Escherichia virus WFH, Serratia virus CHI14, Edwardsiella virus MSW3, Edwardsiella virus PEi21, Erwinia virus Yoloswag, Bacillus virus G, Bacillus virus PBS1, Microcystis virus Ma-LMM01, Streptococcus virus Cp1, Streptococcus virus Cp7, Lactococcus virus WP2, Bacillus virus B103, Bacillus virus GA1, Bacillus virus phi29, Kurthia virus 6, Actinomyces virus Av1, Mycoplasma virus P1, Staphylococcus virus Andhra, Staphylococcus virus St134, Staphylococcus virus 66, Staphylococcus s virus 44AHJD, Staphylococcus virus BP39, Staphylococcus virus CSA13, Staphylococcus virus GRCS, Staphylococcus virus Pabna, Staphylococcus virus phiAGO13, Staphylococcus virus PSa3, Staphylococcus virus S24-1, Staphylococcus virus SAP2, Staphylococcus virus SCH1, Staphylococcus virus SLPW, Shigella virus 7502Stx, Shigella virus POCJ13, Escherichia virus 191, Escherichia virus PA2, Escherichia virus TL2011, Shigella virus VASD, Escherichia virus 24B, Escherichia virus 933W, Escherichia virus Min27, Escherichia virus PA28, Escherichia virus Stx2 II, Dinoroseobacter virus DFL12, Pseudomonas virus Bjorn, Pseudomonas virus Ab22,Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonas virus PAA2, Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonas virus TL, Vibrio virus VC8, Vibrio virus VP2, Vibrio virus VP5, Escherichia virus N4, Flavobacterium virus Fpv1, Flavobacterium virus Fpv4, Streptococcus virus C1, Escherichia hia virus APEC5, Escherichia virus APEC7, Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virus ECBP1, Escherichia virus G7C, Escherichia virus IME11, Shigella virus Sb1, Escherichia virus C1302, Pseudomonas virus F116, Pseudomonas virus H66, Escherichia virus Pollock, Salmonella virus FSL SP-058, Salmonella virus FSL SP-076, Arthrobacter virus Adat, Arthrobacter virus Jasmine, Erwinia virus Ea9-2, Erwinia virus Frozen, Achromobacter virus Axp3, Achromobacter virus JWAlpha, Edwardsiella virus KF1, Burkholderia virus KL4, Pseudomonas virus KPP25, Pseudomonas virus R18, Pseudomonas virus tf, Escherichia virus 172-1, Escherichia virus ECB2, Escherichia virus NJ01, Escherichia virus phiEco32, Escherichia virus Septima11, Escherichia virus SU10, Escherichia virus HK620, Salmonella virus BTP1, Salmonella virus P22, Salmonella virus SE1Spa, Salmonella virus ST64T,Shigella virus Sf6, Burkholderia virus Bcep22, Burkholderia virus Bcepil02, Burkholderia virus Bcepmigl, Burkholderia virus DC1, Cellulophaga virus Cba41, Cellulophaga, virus Cba172, Pseudomonas virus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26, Pseudomonas virus KPP21, Pseudomonas virus LUZ7, Vibrio virus 48B1, Vibrio virus 51A6, Vibrio virus 51A7, Vibrio virus 52B1, Myxococcus virus Mx8, Bacillus virus Page, Bacillus virus Palmer, Bacillus virus Pascal, Bacillus virus Pony, Bacillus virus Pookie, Brucella virus Pr, Brucella virus Tb, Bordetella virus BPP1, Burkholderia virus BcepC6B, Helicobacter virus 1961P, Helicobacter virus KHP30, Helicobacter virus KHP40, Pseudomonas virus phCDa, Escherichia virus Skarpretter, Escherichia virus Sortsne, Klebsiella virus IME279, Escherichia virus phiV10, Salmonella virus Epsilon15, Salmonella virus SPN1S, Pseudomonas virus NV1, Pseudomonas virus UFVP2, Escherichia virus PTXU04, Hamiltonella virus APSE1, Lactococcus virus KSY1, Phormidium virus WMP3, Phormidium virus WMP4, Pseudomonas virus 119X, Roseobacter virus SIO1, Vibrio virus VpV262, Streptomyces virus ELB20, Streptomyces virus R4, Streptomyces virus Amela, Streptomyces virus phiCAM, Streptomyces virus Aaronocolus, Streptomyces virus Caliburn, Streptomyces virus Danzina, Streptomyces virus Hydra, Streptomyces virus Izzy,Streptomyces virus Lannister, Streptomyces virus Lika, Streptomyces virus Sujidade, Streptomyces virus Zemlya, Streptomyces virus phiHau3, Mycobacterium virus Acadian, Mycobacterium virus Baee, Mycobacterium virus Reprobate, Mycobacterium virus Adawi, Mycobacterium virus Bane1, Mycobacterium virus BrownCNA, Mycobacterium virus Chrisnmich, Mycobacterium virus Cooper, Mycobacterium virus JAMaL, Mycobacterium virus Nigel, Mycobacterium virus Stinger, Mycobacterium virus Vincenzo, Mycobacterium virus Zemanar, Mycobacterium virus Apizium, Mycobacterium virus Manad, Mycobacterium virus Mycobacterium virus Oline, Mycobacterium virus Osmaximus, Mycobacterium virus Pg1, Mycobacterium virus Soto, Mycobacterium virus Suffolk, Mycobacterium virus Athena, Mycobacterium virus Bernardo, Mycobacterium virus Gadjet, Mycobacterium virus Pipefish, Mycobacterium virus Godines, Mycobacterium virus Rosebush, Mycobacterium virus TA17a, Mycobacterium virus Babsiella, Mycobacterium virus Brujita, Mycobacterium virus Hawkeye, Mycobacterium virus Plot, Caulobacterium virus CcrBL9, Caulobacterium virus CcrSC, Caulobacterium virus CcrColossus, Caulobacterium virus CcrPW, Caulobacterium virus CcrBL10,Caulobacter virus CcrRogue, Caulobacter virus phiCbK, Caulobacter virus Swift, Salmonella virus SP31, Salmonella virus AG11, Salmonella virus Ent1, Salmonella virus f18SE, Salmonella virus Jersey, Salmonella virus L13, Salmonella virus LSPA1, Salmonella virus SE2, Salmonella virus SETP3, Salmonella virus SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonella virus SS3e, Salmonella virus wksl3, Escherichia virus K1G, Escherichia virus K1H, Escherichia virus K1ind1, Escherichia virus K1ind2, Escherichia virus Golestan, Raoultella virus RP180, Gordonia virus Asapag, Gordonia virus BE Ntherdunthat, Gordonia virus Getalong, Gordonia virus Kenna, Gordonia virus Horus, Gordonia virus Phistory, Leuconostoc virus Lmd1, Leuconostoc virus LN03, Leuconostoc virus LN04, Leuconostoc virus LN12, Leuconostoc virus LN6B, Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8, Leuconostoc virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34, Leuconostoc virus LNTR3, Mycobacterium virus Bongo, Mycobacterium virus Rey, Mycobacterium virus Butters, Mycobacterium virus Michelle, Mycobacterium virus Charlie, Mycobacterium virus Pipsqueaks, Mycobacterium virus Xeno,Mycobacterium virus Panchino, Mycobacterium virus Phrann, Mycobacterium virus Redi, Mycobacterium virus Skinnyp, Gordonia virus BaxterFox, Gordonia virus Yeezy, Gordonia virus Kita, Gordonia virus Nymphadora, Gordonia virus Zirinka, Mycobacterium virus Bignuz, Mycobacterium virus Brusacoram, Mycobacterium virus Donovan, Mycobacterium virus Fishburne, Mycobacterium virus Jebeks, Mycobacterium virus Malithi, Mycobacterium virus Phayonce, Lactobacillus virus B2, Lactobacillus virus Lenus, Lactobacillus virus Nyseid, Lactobacillus virus SAC12, Lactobacillus virus Ldl1, Lactobacillus lus virus ViSo2018a, Lactobacillus virus Maenad, Lactobacillus virus P1, Lactobacillus virus Satyr, Streptomyces virus AbbeyMikolon, Pseudomonas virus Ab18, Pseudomonas virus Ab19, Pseudomonas virus PaMx11, Burkholderia virus AH2, Arthrobacterium virus Amigo, Arthrobacteria virus Molivia, Propionibacterium virus Anatole, Propionibacterium virus B3, Arthrobacterium virus Andrew, Bacillus virus Andromeda, Bacillus virus Blastoid, Bacillus virus Curly, Bacillus virus Eoghan, Bacillus virus Finn, Bacillus virus Glittering, Bacillus virus Riggi, Bacillus virus Taylor, Microbacterium virus Appa,Gordonia virus Apricot, Microbacterium virus Armstrong, Gordonia virus Attis, Streptomyces virus Attoomi, Streptomyces virus Austintatious, Streptomyces virus Ididsumtinwong, Streptomyces virus PapayaSalad, Gordonia virus Bantam, Mycobacterium virus Barnyard, Mycobacterium virus Konstantine, Mycobacterium virus Predator, Pseudomonas virus B3, Pseudomonas virus JBD67, Pseudomonas virus JD18, Pseudomonas virus PM105, Mycobacterium virus Bernal13, Gordonia virus BetterKatz, Streptomyces virus Bing, Staphylococcus virus 13, Staphylococcus virus 77, Staphylococcus virus 108PVL, G Gordonia virus Bowser, Arthrobacter virus Bridgette, Arthrobacter virus Constance, Arthrobacter virus Eileen, Arthrobacter virus Judy, Arthrobacter virus Peas, Gordonia virus Britbrat, Mycobacterium virus Bron, Mycobacterium virus Faith1, Mycobacterium virus JoeDirt, Mycobacterium virus Rumpelstiltskin, Streptococcus virus 858, Streptococcus virus 2972, Streptococcus virus ALQ132, Streptococcus virus O1205, Streptococcus virus Sfi11, Pseudomonas virus D3112, Pseudomonas virus DMS3, Pseudomonas virus FHA0480, Pseudomonas virus LPB1, Pseudomonas virus MP22, Pseudomonas virus MP29Pseudomonas virus MP38, Pseudomonas virus PA1KOR, Cellulophaga virus ST, Bacillus virus 250, Bacillus virus IEBH, Lactococcus virus bIL67, Lactococcus virus c2, Corynebacterium virus C3PO, Corynebacterium virus Darwin, Corynebacterium virus Zion, Lactobacillus virus c5, Lactobacillus virus Ld3, Lactobacillus virus Ld17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu, Lactobacillus virus phiLdb, Mycobacterium virus Che9c, Mycobacterium virus Sbash, Mycobacterium virus Ardmore, Mycobacterium virus Avani, Mycobacterium virus Boomer, Mycobacterium virus Che8, Mycobacterium virus Che9d, Mycobacterium virus DeadP, Mycobacterium virus Drane, Mycobacterium virus Dorothy, Mycobacterium virus DotProduct, Mycobacterium virus Drago, Mycobacterium virus Fruitloo p, Mycobacterium virus GUmbie, Mycobacterium virus Ibhubesi, Mycobacterium virus Llij, Mycobacterium virus Mozy, Mycobacterium virus Mutaforma13, Mycobacterium virus Pacc40, Mycobacterium virus PMC, Mycobacterium virus Ramsey, Mycobacterium virus RockyHorror, Mycobacterium virus SG4, Mycobacterium virus Shauna1, Mycobacterium virus Shilan, Mycobacterium virus Spartacus, Mycobacterium virus Taj, Mycobacterium virus Tweety, Mycobacterium virus Wee, Mycobacterium virus Yoshi, Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonella virus iEPS5,Salmonella virus SPN19, Corynebacterium virus P1201, Clavibacter virus CMP1, Clavibacter virus CN1A, Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcus virus cIP1, Arthrobacter virus Coral, Arthrobacter virus Kepler, Mycobacterium virus Corndog, Mycobacterium virus Firecracker, Rhodobacter virus RcCronus, Gordonia virus DareDevil, Arthrobacter virus Decurro, Stenotrophomonas virus DLP5, Gordonia virus Demosthenes, Gordonia virus Katyusha, Gordonia virus Kvothe, Pseudomonas virus D3, Pseudomonas virus PMG1, Escherichia virus EK99P1, Escherichia virus HK578 , Escherichia virus JL1, Escherichia virus SSL2009a, Escherichia virus YD2008s, Shigella virus EP23, Sodalis virus SO1, Microbacterium virus Dismas, Propionibacterium virus B22, Propionibacterium virus Doucette, Propionibacterium virus E6, Propionibacterium virus G4, Microbacterium virus Eden, Enterococcus virus AL2, Enterococcus virus AL3, Enterococcus virus AUEF3, Enterococcus virus EcZZ2, Enterococcus virus EF3, Enterococcus virus EF4, Enterococcus virus EfaCPT1, Enterococcus virus IME196, Enterococcus virus LY0322, Enterococcus virus phiSHEF2, Enterococcus virus phiSHEF4,Enterococcus virus phiSHEF5, Enterococcus virus PMBT2, Enterococcus virus SANTOR1, Edwardsiella virus eiAU, Xanthomonas virus PhiL7, Microbacterium virus Eleri, Gordonia virus Cozz, Gordonia virus Emalyn, Gordonia virus GTE2, Gordonia virus Troje, Gordonia virus Eyre, Gordonia virus Fairfaxidum virus, Microbacterium virus ISF9, Erwinia virus Eho49, Erwinia virus Eho59, Staphylococcus virus 2638A, Staphylococcus virus QT1, Colwellia virus 9A, Mycobacterium virus Alma, Mycobacterium virus Arturo, Mycobacterium virus Astro, Mycobacterium virus Backyardigan, Mycobacterium virus Be nedict, Mycobacterium virus Bethlehem, Mycobacterium virus Billknuckles, Mycobacterium virus BPBiebs31, Mycobacterium virus Bruns, Mycobacterium virus Bxb1, Mycobacterium virus Bxz2, Mycobacterium virus Che12, Mycobacterium virus Cuco, Mycobacterium virus D29, Mycobacterium virus Doom, Mycobacterium virus Ericb, Mycobacterium virus Euphoria, Mycobacterium virus George, Mycobacterium virus Gladiator, Mycobacterium virus Goose, Mycobacterium virus Hammer, Mycobacterium virus Heldan, Mycobacterium virus Jasper, Mycobacterium virus JC27, Mycobacterium virus Jeffabunny,Mycobacterium virus JHC117, Mycobacterium virus KBG, Mycobacterium virus Kssjeb, Mycobacterium virus Kugel, Mycobacterium virus L5, Mycobacterium virus Lesedi, Mycobacterium virus LHTSCC, Mycobacterium virus lockley, Mycobacterium virus Marcell, Mycobacterium virus Microwolf, Mycobacterium virus Mrgordo, Mycobacterium virus Museum, Mycobacterium virus Nepal, Mycobacterium virus Packman, Mycobacterium virus Peaches, Mycobacterium virus Perseus, Mycobacterium virus Pukovnik, Mycobacterium virus Rebeuca, Mycobacterium virus Redrock, Mycobacterium virus Ridgecb, Mycobacterium Virus Rockstar, Mycobacterium virus Saintus, Mycobacterium virus Skipole, Mycobacterium virus Solon, Mycobacterium virus Switzer, Mycobacterium virus SWU1, Mycobacterium virus Tiger, Mycobacterium virus Timshel, Mycobacterium virus Trixie, Mycobacterium virus Turbido, Mycobacterium virus Twister, Mycobacterium virus U2, Mycobacterium virus Violet, Mycobacterium virus Wonder, Mycobacterium virus Gaia, Arthrobacterium virus Abidatro, Arthrobacterium virus Galaxy, Gordonia virus GAL1, Gordonia virus GMA3, Gordonia virus Gsput1, Gordonia virus GMA7, Gordonia virus GTE7, Gordonia virus Ghobes,Mycobacterium virus Giles, Microbacterium virus OneinaGillian, Gordonia virus GodonK, Microbacterium virus Goodman, Arthrobacter virus Captnmurica, Arthrobacter virus Gordon, Gordonia virus GordTnk2, Proteus virus Isfahan, Gordonia virus Jumbo, Gordonia virus Gustav, Gordonia virus Mahdia, Paenibacillus virus Harrison, Gordonia virus Hedwig, Cellulophaga virus Cba121, Cellulophaga virus Cba171, Cellulophaga virus Cba181, Escherichia virus HK022, Escherichia virus HK75, Escherichia virus HK97, Escherichia virus HK106, Escherichia virus HK446, Escherichia virus HK542, Escherichia virus HK646 ... herichia virus HK544, Escherichia virus HK633, Escherichia virus mEp234, Escherichia virus mEpX1, Escherichia virus mEpX2, Streptomyces virus Hiyaa, Salinibacter virus M1EM1, Salinibacter virus M8CR30-2, Listeria virus LP26, Listeria virus LP37, Listeria virus LP110, Listeria virus LP114, Li steria virus P70, Corynebacterium virus phi673, Corynebacterium virus phi674, Microbacterium virus Hamlet, Microbacterium virus Ilzat, Polaribacter virus P12002L, Polaribacter virus P12002S, Nonlabens virus P12024L, Nonlabens virus P12024S, Gordonia virus Jace, Brevibacillus virus Jenst,Corynebacterium virus Juicebox, Salinibacter virus M31CR41-2, Salinibacter virus SRUTV1, Arthrobacter virus Kellezzio, Arthrobacter virus Kitkat, Burkholderia virus KL1, Xanthomonas virus CP1, Microbacterium virus Golden, Micr, Mycobacterium virus Koji, Arthrobacterium virus Bennie, Arthrobacterium virus DrRobert, Arthrobacterium virus Glenn, Arthrobacterium virus HunterDalle, Arthrobacterium virus Joann, Arthrobacterium virus Korra, Arthrobacterium virus Preamble, Arthrobacterium virus Pumancara, Arthrobacterium virus Wayne, Mycobacterium virus 244, Mycobacterium virus Bask21, Mycobacterium virus CJW1, Mycobacterium virus Eureka, Mycobacterium virus Kostya, Mycobacterium virus Porky, Mycobacterium virus Pumpkin, Mycobacterium virus Sirduracell, Mycobacterium virus Toto, Microbacterium virus Krampus, Salinibacterium virus M8CC19, Sal Inibacter virus M8CRM1, Sphingobium virus Lacusarx, Escherichia virus DE3, Escherichia virus HK629, Escherichia virus HK630, Escherichia virus Lambda, Pseudomonas virus Lana, Arthrobacter virus Laroye, Eggerthella virus PMBT5, Arthobacter virus Liebe, Mycobacterium virus Halo, Mycobacterium virus Liefie, Acinetobacter virus IMEAB3, Acinetobacter virus Loki, Streptomyces virus phiBT1, Streptomyces virus phiC31, Brevibacterium virus LuckyBarnes, Gordonia virus Lucky10, Faecalibacterium virus Lugh, Bacillus virus BMBtp2, Bacillus virus TP21, Bacillus virus Mgbh1, Arthrobacter virus Maja,Arthrobacterium virus DrManhattan, Mycobacterium virus Ff47, Mycobacterium virus Muddy, Vibrio virus MAR10, Vibrio virus SSP002, Mycobacterium virus Marvin, Mycobacterium virus Mosmoris, Pseudomonas virus PMBT3, Microbacterium virus MementoMori, Microbacterium virus Fireman, Microbacterium virus Metamorphoo, Microbacterium virus RobsFeet, Microbacterium virus Min1, Streptococcus virus 7201, Streptococcus virus DT1, Streptococcus virus phiAbc2, Streptococcus virus Sfi19, Streptococcus virus Sfi21, Gordinia virus Birksandsocks, Gordonia virus Flaky, Gordonia virus Monty, Gordonia virus Stevefrench, Arthrobacter virus Circum, Arthrobacter virus Mudcat, Escherichia virus EC2, Salmonella virus Lumpael, Dinoroseobacter virus D5C, Burkholderia virus BcepNazgul, Microbacterium virus Neferthena, Pseudomonas virus nickie, Pseudomonas virus NP1, Pseudomonas virus PaMx25, Escherichia virus 9g, Escherichia virus JenK1, Escherichia virus JenP1, Escherichia virus JenP2, Salmonella virus SE1Kor, Salmonella virus 9NA, Salmonella virus SP069, Gordonia virus Nyceirae, Faecalibacterium virus Oengus, Mycobacterium virus Baka, Mycobacterium virus Courthouse,Mycobacterium virus Littlee, Mycobacterium virus Omega, Mycobacterium virus Optimus, Mycobacterium virus Thibault, Gordonia virus BrutonGaster, Gordonia virus OneUp, Gordonia virus Orchid, Thermus virus P23-45, Thermus virus P74-26, Propionibacterium virus ATCC29399BC, Propionibacterium virus ATCC29399BT, Propionibacterium virus Attacne, Propionibacterium virus Keiki, Propionibacterium virus Kubed, Propionibacterium virus Lauchelly, Propionibacterium virus MrAK, Propionibacterium virus Ouroboros, Propionibacterium virus P91, Propionibacterium virus P105, Propionibacterium virus P144, Propionibacterium virus P1001, Propionibacterium virus P1.1, Propionibacterium virus P100A, Propionibacterium virus P100D, Propionibacterium virus P101A, Propionibacterium virus P104A, Propionibacterium virus PA6, Propionibacterium virus Pacnes2012 15, Propionibacterium virus PAD20, Propionibacterium virus PAS50, Propionibacterium virus PHL009M11, Propionibacterium virus PHL025M00, Propionibacterium virus PHL037M02, Propionibacterium virus PHL041M10, Propionibacterium virus PHL060L00, Propionibacterium virus PHL067M01,Propionibacterium virus PHL070N00, Propionibacterium virus PHL071N05, Propionibacterium virus PHL082M03, Propionibacterium virus PHL092M00, Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01, Propionibacterium virus PHL112N0, Propionibacterium virus PHL113M 01, Propionibacterium virus PHL114L00, Propionibacterium virus PHL116M00, Propionibacterium virus PHL117M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL132N00, Propionibacterium virus PHL141N00, Propionibacterium virus PHL151M00, Propionibacterium virus PHL15 1N00, Propionibacterium virus PHL152M00, Propionibacterium virus PHL163M00, Propionibacterium virus PHL171M01, Propionibacterium virus PHL179M00, Propionibacterium virus PHL194M00, Propionibacterium virus PHL199M00, Propionibacterium virus PHL301M00, Propionibacterium virus PHL 308M00, Propionibacterium virus Pirate, Propionibacterium virus Procrass1, Propionibacterium virus SKKY, Propionibacterium virus Solid, Propionibacterium virus Stormborn, Propionibacterium virus Wizzo, Pseudomonas virus PaMx28, Pseudomonas virus PaMx74, Mycobacterium virus Papyrus,Mycobacterium virus Send513, Mycobacterium virus Patience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcus virus Poco6, Staphylococcus virus 11, Staphylococcus virus 29, Staphylococcus virus 37, Staphylococcus virus 53, Staphylococcus virus 55, Staphylococcus virus 69, Staphylococcus virus 71, Staphylococcus virus 80, Staphylococcus virus 85, Staphylococcus virus 88, Staphylococcus virus 92, Staphylococcus virus 96, Staphylococcus virus 187, Staphylococcus virus 52a, Staphylococcus virus 80alpha, Staphylococcus virus CNPH82, Staphylococcus virus EW, Staphylococcus virus Staphylococcus virus IPLA5, Staphylococcus virus IPLA7, Staphylococcus virus IPLA88, Staphylococcus virus PH15, Staphylococcus virus phiETA, Staphylococcus virus phiETA2, Staphylococcus virus phiETA3, Staphylococcus virus phiMR11, Staphylococcus virus phiMR25, Staphylococcus virus phiNM1, Staphylococcus virus phiNM2, Staphylococcus virus phiNM4, Staphylococcus virus SAP26, Staphylococcus virus X2, Enterococcus virus FL1, Enterococcus virus FL2, Enterococcus virus FL3, Streptomyces virus Picard, Microbacterium virus Pikmin, Corynebacterium virus Poushou, Providencia virus PR1, Listeria virus LP302,Listeria virus PSA, Psimuna virus psiM2, Propionibacterium virus PFR1, Microbacterium phage KaiHaiDragon, Microbacterium phage Paschalis, Microbacterium phage Quhwah, Streptomyces virus Darolandstone, Streptomyces u, Virus Raleigh, Escherichia virus N15, Rhodococcus virus RER2, Rhizobium virus P106B, Strepomyces virus Drgrey, Strepomyces virus Rima, Microbacterium virus Hendrix, Gordonia virus Fryberger, Gordonia virus Ronaldo, Aeromonas virus pIS4A, Streptomyces virus Rowa, Gordonia virus Ruthy, Streptomyces virus Jay2Jay, Streptomyces virus Mildred21, Streptomyces virus NootNoot, Streptomyces virus Paradiddles, Streptomyces virus Peebs, Streptomyces virus Samisti12, Pseudomonas virus SM1, Corynebacterium virus SamW, Xylella virus Salvo, Xylella virus Sano, Caulobacter virus Sansa, Ente rococcus virus BC611, Enterococcus virus IMEEF1, Enterococcus virus SAP6, Enterococcus virus VD13, Streptococcus virus SPQS1, Salmonella virus Sasha, Corynebacterium virus BFK20, Geobacillus virus Tp84, Streptomyces virus Scap1, Gordonia virus Schnabeltier, Microbacterium virus Schubert, Pseudomonas virus 73, Pseudomonas virus Ab26, Pseudomonas virus Kakheti25, Escherichia virus Cajan, Escherichia virus Seurat, Caulobacter virus Seuss, Staphylococcus virus SEP9, Staphylococcus virus Sextaec, Paenibacillus virus Diva, Paenibacillus virus Hb10c2, Paenibacillus virus Rani,Paenibacillus virus Shelly, Paenibacillus virus Sitara, Paenibacillus virus Willow, Lactococcus virus 712, Lactococcus virus ASCC191, Lactococcus virus ASCC273, Lactococcus virus ASCC281, Lactococcus virus ASCC465, Lactococcus virus ASCC532, Lactococcus virus Bibb29, Lactococcus virus bIL17 0, Lactococcus virus CB13, Lactococcus virus CB14, Lactococcus virus CB19, Lactococcus virus CB20, Lactococcus virus jj50, Lactococcus virus P2, Lactococcus virus P008, Lactococcus virus sk1, Lactococcus virus Sl4, Bacillus virus Slash, Bacillus virus Stahl, Bacillus virus Staley, Bacillus virus Sti lls, Gordonia virus Bachita, Gordonia virus ClubL, Gordonia virus Smoothie, Arthobacter virus Sonali, Gordonia virus Soups, Gordonia virus Strosahl, Gordonia virus Wait, Gordonia virus Sour, Bacillus virus SPbeta, Microbacterium virus Hyperion, Microbacterium virus Squash, Burkholderia virus phi6442, Burkholderia virus phi1026b, Burkholderia virus phiE125, Achromobacter virus 83-24, Achromobacter virus JWX, Arthrobacter virus Tank, Gordonia virus Suzy, Gordonia virus Terapin, Streptomyces virus TG1, Mycobacterium virus Anaya, Mycobacterium virus Angelica, Mycobacterium virus CrimD,Mycobacterium virus Fionnbharth, Mycobacterium virus JAWS, Mycobacterium virus Larva, Mycobacterium virus MacnCheese, Mycobacterium virus Pixie, Mycobacterium virus TM4, Tsukamurella virus TIN2, Tsukamurella virus TIN3, Tsukamurella virus TIN4, Rhodobacterium virus RcSpartan, Rhodobacterium virus RcTitan, Mycobacterium virus Tortellini, Staphylococcus virus 47, Staphylococcus virus 3a, Staphylococcus virus 42e, Staphylococcus virus IPLA35, Staphylococcus virus phi12, Staphylococcus virus phiSLT, Mycobacterium virus 32HC, Rhodococcus virus Trina, Gordonia virus Trine, Paeniba cillus virus Tripp, Flavobacterium virus 1H, Flavobacterium virus 23T, Flavobacterium virus 2A, Flavobacterium virus 6H, Streptomyces virus Lilbooboo, Streptomyces virus Vash, Paenibacillus virus Vegas, Gordonia virus Vendetta, Paracoccus virus Shpa, Pantoea virus Vid5, Acinetobacter virus B1251, Acinetobacter virus R3177, Gordonia virus Brandonk123, Gordonia virus Lennon, Gordonia virus Vivi2, Bordetella virus CN1, Bordetella virus CN2, Bordetella virus FP1, Bordetella virus MW2, Bacillus virus Wbeta, Rhodococcus virus Weasel, Mycobacterium virus Wildcat, Gordonia virus BillnyeGordonia virus Twister6, Gordonia virus Wizard, Gordonia virus Hotorobo, Gordonia virus Woes, Streptomyces virus TP1604, Streptomyces virus YDN12, Roseobacter virus RDJL1, Roseobacter virus RDJL2, Xanthomonas virus OP1, Xanthomonas virus Xop411, Xanthomonas virus Xp10, Arthobacter virus Yang, Alphaproteobacteria virus phiJl001, Pseudomonas virus LKO4, Pseudomonas virus M6, Pseudomonas virus MP1412, Pseudomonas virus PAE1, Pseudomonas virus Yua, Gordonia virus Yvonnetastic, Microbacterium virus Zeta1847, Rhodococcus virus RGL3, Paenibacillus virus Lily, Vibrio virus CTXphi, Propio Nibacterium virus B5, Vibrio virus KSF1, Xanthomonas virus Cf1c, Vibrio virus fs1, Vibrio virus VGJ, Ralstonia virus RS551, Ralstonia virus RS603, Ralstonia virus RSM1, Ralstonia virus RSM3, Escherichia virus If1, Escherichia virus M13, Escherichia virus I22, Salmonella virus IKe, Ralstonia virus PE22 6, Pseudomonas virus Pf1, Stenotrophomonas virus PSH1, Ralstonia virus RSS1, Vibrio virus fs2, Vibrio virus VFJ, Stenotrophomonas virus SMA6, Stenotrophomonas virus SMA9, Stenotrophomonas virus SMA7, Pseudomonas virus Pf3, Thermus virus OH3, Vibrio virus VfO3K6, Vibrio virus VCY, Vibrio virus Vf33,Xanthomonas virus Xf109, Acholeplasma virus L51, Spiroplasma virus SVTS2, Spiroplasma virus C74, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x, Escherichia virus alpha3, Escherichia virus ID21, Escherichia virus ID32, Escherichia virus ID62, Escherichia virus NC28, Escherichia virus NC29, Escherichia virus NC35, Escherichia chia virus phiK, Escherichia virus St1, Escherichia virus WA45, Escherichia virus G4, Escherichia virus ID52, Escherichia virus Talmos, Escherichia virus phiX174, Bdellovibrio virus MAC1, Bdellovibrio virus MH2K, Chlamydia virus Chp1, Chlamydia virus Chp2, Chlamydia virus CPAR39, Chlamydia virus CPG1, Spiroplasma virus SpV4, Bombyx mori bidensovirus, Acerodon celebensis polyomavirus 1, Artibeus planirostris polyomavirus 2, Artibeus planirostris polyomavirus 3, Ateles paniscus polyomavirus 1, Cardioderma cor polyomavirus 1, Carollia perspicillata polyomavirus 1, Chlorocebus pygerythrus polyomavirus 1, Chlorocebus pygerythrus polyomavirus 3, Dobsonia moluccensis polyomavirus 1, Eidolon helvum polyomavirus 1, Gorilla gorilla polyomavirus 1, Human polyomavirus 5, Human polyomavirus 8, Human polyomavirus 9, Human polyomavirus 13, Human polyomavirus 14, Macaca fascicularis polyomavirus 1,Mesocricetus auratus polyomavirus 1, Miniopterus schreibersii polyomavirus 1, Miniopterus schreibersii polyomavirus 2, Molossus molossus polyomavirus 1, Mus musculus polyomavirus 1, Otomops martiensseni polyomavirus 1, Otomo, ps martiensseni polyomavirus 2, Pan troglodytes polyomavirus 1, Pan troglodytes polyomavirus 2, Pan troglodytes polyomavirus 3, Pan troglodytes polyomavirus 4, Pan troglodytes polyomavirus 5, Pan troglodytes polyomavirus 6, Pan troglodytes polyomavirus 7, Papio cynocephalus polyomavirus 1, Piliocolobus badius polyomavirus 1, Piliocolobus rufomitratus polyomavirus 1, Pongo abelii polyomavirus 1, Pongo pygmaeus polyomavirus 1, Procyon lotor polyomavirus 1, Fruit bat vampyrus polyomavirus 1, Rattus norvegicus polyomavirus 1, Sorex araneus polyomavirus 1, Sorex coronatus polyomavirus 1, Sorex minutus polyomavirus 1, Sturnira lilium polyomavirus 1, Tupaia belangeri polyomavirus 1, Acerodon celebensis polyomavirus 2, Artibeus planirostris polyomavirus 1, Canis familiaris polyomavirus 1, Cebus albifrons polyomavirus 1, Cercopithecus erythrotis polyomavirus 1, Chlorocebus pygerythrus polyomavirus 2, Desmodus rotundus polyomavirus 1, Dobsonia moluccensis polyomavirus 2, Dobsonia moluccensis polyomavirus 3, Enhydra lutris polyomavirus 1, Equus caballus polyomavirus 1, Human polyomavirus 1, Human polyomavirus 2, Human polyomavirus 3, Human polyomavirus 4, Leptonychotes weddellii polyomavirus 1, Loxodonta africana polyomavirus 1,Macaca mulatta polyomavirus 1, Mastomys natalensis polyomavirus 1, Meles meles polyomavirus 1, Microtus arvalis polyomavirus 1, Miniopterus africanus polyomavirus 1, Mus musculus polyomavirus 2, Mus musculus polyomavirus 3, Myodes glareolus polyomavirus 1, Myotis lucifugus polyomavirus 1, Pan troglodytes polyomavirus 8, Papio cynocephalus polyomavirus 2, Pteronotus davyi polyomavirus 1, Pteronotus parnellii polyomavirus 1, Rattus norvegicus polyomavirus 2, Rousettus aegyptiacus polyomavirus 1, Saimiri boliviensis polyomavirus 1, Saimiri sciureus polyomavirus 1, Vicugna pacos polyomavirus 1, Zalophus californianus polyomavirus 1, Human polyomavirus 6, Human polyomavirus 7, Human polyomavirus 10, Human polyomavirus 11, Anser anser polyomavirus 1, Avian polyomavirus 1, Corvus monedula polyomavirus 1, Cracticus torquatus polyomavirus 1, Erythrura gouldiae polyomavirus 1, Lonchura maja polyomavirus 1, Pygoscelis adeliae polyomavirus 1, Pyrrhula pyrrhula polyomavirus 1, Serinus canaria polyomavirus 1, Ailuropoda melanoleuca polyomavirus 1, Bos taurus polyomavirus 1, Centropristis striata polyomavirus 1, Delphinus delphis polyomavirus 1, Procyon lotor polyomavirus 2, Rhynchobatus djiddensis polyomavirus 1, Sparus aurata polyomavirus 1,Trematomus bernacchii polyomavirus 1, Trematomus pennellii polyomavirus 1, Alphapapillomavirus 1, Alphapapillomavirus 2, Alphapapillomavirus 3, Alphapapillomavirus 4, Alphapapillomavirus 5, Alphapapillomavirus 6, Alphapapillomavirus 7, Alphapapillomavirus 8, Alphapapillomavirus 9, Alphapapillomavirus 10, Alphapapillomavirus 11, Alphapapillomavirus 12, Alphapapillomavirus 13, Alphapapillomavirus 14, Betapapillomavirus 1, Betapapillomavirus 2, Betapapillomavirus 3, Betapapillomavirus 4, Betapapillomavirus 5, Betapapillomavirus 6, Kaipapillomavirus 1, Kaipapillomavirus 2, Kaipapillomavirus 3, Deltapapillomavirus 1, Deltapapillomavirus 2, Deltapapillomavirus 3, Deltapapillomavirus 4, Deltapapillomavirus 5, Deltapapillomavirus 6, Deltapapillomavirus 7, Diokaipapillomavirus 1, Diode Rutapapillomavirus 1, Dioepsilonpapillomavirus 1, Dioetapapillomavirus 1, Dioiotapapillomavirus 1, Dioiotapapillomavirus 2, Diokappapapillomavirus 1, Diokappapapillomavirus 2, Diokappapapillomavirus 3, Diokappapapillomavirus 4, Diokappapapillomavirus 5, Diolamdapapillomavirus 1, Diompapillomavirus 1, Dionupapillomavirus 1, Diomegapapillomavirus 1, Diomicropapillomavirus 1 Diopapillomavirus 1, Diopipapillomavirus 1, Diophipapillomavirus 1, Diolopapillomavirus 1, Diosigmapapillomavirus 1, Diotaupapillomavirus 1, Diothetapapillomavirus 1, Dioupsilonpapillomavirus 1, Dioxsaipapillomavirus 1, Dioxsaipapillomavirus 2, Diozetapapillomavirus 1, Epsilonpapillomavirus 1, Epsilonpapillomavirus 2, Etapapillomavirus 1,Gamma papillomavirus 1, Gamma papillomavirus 2, Gamma papillomavirus 3, Gamma papillomavirus 4, Gamma papillomavirus 5, Gamma papillomavirus 6, Gamma papillomavirus 7, Gamma papillomavirus 8, Gamma papillomavirus 9, Gamma papillomavirus 10, Gamma papillomavirus 11, Gamma papillomavirus 12, Gamma papillomavirus 13, Gamma papillomavirus 14, Gamma papillomavirus 15, Gamma papillomavirus 16, Gamma papillomavirus 17, Gamma Papillomavirus 18, Gammapapillomavirus 19, Gammapapillomavirus 20, Gammapapillomavirus 21, Gammapapillomavirus 22, Gammapapillomavirus 23, Gammapapillomavirus 24, Gammapapillomavirus 25, Gammapapillomavirus 26, Gammapapillomavirus 27, Iotapapillomavirus 1, Iotapapillomavirus 2, Kappapapillomavirus 1, Kappapapillomavirus 2, Lambdapapillomavirus 1, Lambdapapillomavirus 2, Lambdapapillomavirus 3, Lambdapapillomavirus papillomavirus 4, lambdopapillomavirus 5, mupapillomavirus 1, mupapillomavirus 2, mupapillomavirus 3, neupapillomavirus 1, omegapapillomavirus 1, omicronpapillomavirus 1, phipapillomavirus 1, phipapillomavirus 2, psipapillomavirus 1, psipapillomavirus 2, psipapillomavirus 3, rhopapillomavirus 1, rhopapillomavirus 2, sigmapapillomavirus 1, taupapillomavirus 1, taupapillomavirus 2 Mavirus 2, Tau papillomavirus 3, Tau papillomavirus 4, Theta papillomavirus 1, Treis delta papillomavirus 1, Treis epsilon papillomavirus 1, Treis eta papillomavirus 1, Treis ciota papillomavirus 1, Treis cappa papillomavirus 1, Treis theta papillomavirus 1, Treis zeta papillomavirus 1, Upsilon papillomavirus 1, Upsilon papillomavirus 2, Upsilon papillomavirus 3, Quasi papillomavirus 1, Quasi papillomavirus 2Rhinoceros papillomavirus 3, Rhinoceros papillomavirus 4, Rhinoceros papillomavirus 5, Zeta papillomavirus 1, Aleph papillomavirus 1, Asteroid aquavidensovirus 1, Decapod aquavidensovirus 1, Cockroach grasshopper aquavidensovirus 1, Hemiptera hemiambidensovirus 1, Hemiptera hemiambidensovirus 2, Lepidoptera iteradensovirus 1, Lepidoptera iteradensovirus 2, Lepidoptera iteradensovirus 3, Lepidoptera iteradensovirus 4, Lepidoptera iteradensovirus 5, Orthoptera miniambidensovirus 1, Cockroach pef Ambidensovirus 1, Dipteran Protoambidensovirus 1, Lepidoptera Protoambidensovirus 1, Hemiptera Sindhaavidensovirus 1, Hymenoptera Sindhaavidensovirus 1, Orthoptera Sindhaavidensovirus 1, Dipteran Brevihama parvovirus 1, Dipteran Brevihama parvovirus 2, Carnivore Chafama parvovirus 1, Chiroptera Chafama parvovirus 1, Galliformes Chafama parvovirus 1, Galliformes Chafama parvovirus 2, Galliformes Chafama parvovirus 3, Rodent Chafama parvovirus 1, Rodents Chafamaparvovirus 2, Ungulate Chafamaparvovirus 1, Decapod Hepanhamaparvovirus 1, Syngnathidae Ictamaparvovirus 1, Decapod Penstylhamaparvovirus 1, Carnivore Amdoparvovirus 1, Carnivore Amdoparvovirus 2, Carnivore Amdoparvovirus 3, Carnivore Amdoparvovirus 4, Carnivore Amdoparvovirus 5, Chiropteran Altiparvovirus 1, Galliformes Abeparvovirus 1, Gruiformes Abeparvovirus 1, Carnivore Bocaparvovirus 1, Carnivore Bocaparvovirus 2, Carnivore Bocaparvovirus Bocavirus 3, Carnivore bocaparvovirus 4, Carnivore bocaparvovirus 5, Carnivore bocaparvovirus 6, Chiroptera bocaparvovirus 1, Chiroptera bocaparvovirus 2, Chiroptera bocaparvovirus 3, Chiroptera bocaparvovirus 4, Lagomorph bocaparvovirus 1, Pinniped bocaparvovirus 1, Pinniped bocaparvovirus 2, Primate bocaparvovirus 1, Primate bocaparvovirus 2, Rodent bocaparvovirus 1, Rodent bocaparvovirus 2, Ungulate Parvovirus 1, Ungulate Parvovirus 2Ungulate Parvovirus 3, Ungulate Parvovirus 4, Ungulate Parvovirus 5, Ungulate Parvovirus 6, Ungulate Parvovirus 7, Ungulate Parvovirus 8, Pinniped Copiparvovirus 1, Ungulate Copiparvovirus 1, Ungulate Copiparvovirus 2, Ungulate Copiparboui, Rus 3, Ungulate Copiparvovirus 4, Ungulate Copiparvovirus 5, Ungulate Copiparvovirus 6, Adeno-associated Dependvirus A, Adeno-associated Dependvirus B, Anseriform Dependparvovirus 1, Avian Dependparvovirus 1, Chiroptera Dependparvovirus 1, Pinniped Dependparvovirus 1, Rodent Dependparvovirus 1, Rodent Dependparvovirus 2, Squamate Dependparvovirus 1, Squamate Dependparvovirus 2, Pinniped Erythroparvovirus 1, Primate Erythroparvovirus erythroparvovirus 1, primate erythroparvovirus 2, primate erythroparvovirus 3, primate erythroparvovirus 4, rodent erythroparvovirus 1, ungulate erythroparvovirus 1, primate roriparvovirus 1, carnivore protoparvovirus, carnivore protoparvovirus 1, chiropteran protoparvovirus 1, eucaenid protoparvovirus 1, primate protoparvovirus 1, primate protoparvovirus 2, primate protoparvovirus 3, primate protoparvovirus 4, rodent protoparvovirus 1, rodent Rodent protoparvovirus 2, Rodent protoparvovirus 3, Ungulate protoparvovirus 1, Ungulate protoparvovirus 2, Chiropteran tetraparvovirus 1, Primate tetraparvovirus 1, Ungulate tetraparvovirus 1, Ungulate tetraparvovirus 2, Ungulate tetraparvovirus 3, Ungulate tetraparvovirus 4, Chaetocero diathodonavirus 1, Avon-Heathcote estuary-associated key serradnavirus, Chaetocero protobacilladnavirus 1, Chaetocero pro Tobasiladonavirus 2, Chaetocero protobasiladonavirus 3, Chaetocero protobasiladonavirus 4, Marine protobasiladonavirus 1, Snail-associated protobasiladonavirus 1, Snail-associated protobasiladonavirus 2, Koi circovirus, Bat-associated circovirus 1, Bat-associated circovirus 2, Bat-associated circovirus 3, Bat-associated circovirus 4, Bat-associated circovirus 5, Bat-associated circovirus 6, Bat-associated circovirus 7, Bat-associated circovirus 8,Bat-associated circovirus 9, Bat-associated circovirus 10, Bat-associated circovirus 11, Bat-associated circovirus 12, Beak and feather disease virus, Canary circovirus, Canine circovirus, Chimpanzee-associated circovirus 1, Civet circovirus, Duck circovirus, European catfish circovirus, Finch circovirus, Goose circovirus, Gull circovirus, Human-associated circovirus 1, Mink circovirus, Mosquito-associated circovirus 1, Pigeon circovirus, Porcine circovirus 1, Porcine Circovirus 2, Porcine Circovirus 3, Crow Circovirus, Rodent-associated Circovirus 1, Rodent-associated Circovirus 2, Rodent-associated Circovirus 3, Rodent-associated Circovirus 4, Rodent-associated Circovirus 5, Rodent-associated Circovirus 6, Rodent-associated Circovirus 7, Starling Circovirus, Swan Circovirus, Tick-associated Circovirus 1, Tick-associated Circovirus 2, Zebra Finch Circovirus, Ant-associated Cyclovirus 1, Bat-associated Cyclovirus 1, Bat-associated Cyclovirus 2, Bat-associated cyclovirus 3, Bat-associated cyclovirus 4, Bat-associated cyclovirus 5, Bat-associated cyclovirus 6, Bat-associated cyclovirus 7, Bat-associated cyclovirus 8, Bat-associated cyclovirus 9, Bat-associated cyclovirus 10, Bat-associated cyclovirus 11, Bat-associated cyclovirus 12, Bat-associated cyclovirus 13, Bat-associated cyclovirus 14, Bat-associated cyclovirus 15, Bat-associated cyclovirus 16, Bovine-associated cyclovirus 1, Chicken Li-associated cyclovirus 1, Chicken-associated cyclovirus 2, Chimpanzee-associated cyclovirus 1, Cockroach-associated cyclovirus 1, Dragonfly-associated cyclovirus 1, Dragonfly-associated cyclovirus 2, Dragonfly-associated cyclovirus 3, Dragonfly-associated cyclovirus 4, Dragonfly-associated cyclovirus 5, Dragonfly-associated cyclovirus 6, Dragonfly-associated cyclovirus 7, Dragonfly-associated cyclovirus 8, Duck-associated cyclovirus 1, Cat-associated cyclovirus 1, Goat-associated cyclovirus 1, Horse-associated cyclovirus 1, Human-associated cyclovirus 1,Human-associated cyclovirus 2, Human-associated cyclovirus 3, Human-associated cyclovirus 4, Human-associated cyclovirus 5, Human-associated cyclovirus 6, Human-associated cyclovirus 7, Human-associated cyclovirus 8, Human-associated cyclovirus 9, Human-associated cyclovirus 10, Human-associated cyclovirus 11, Human-associated cyclovirus 12, Mouse-associated cyclovirus 1, Rodent-associated cyclovirus 1, Rodent-associated cyclovirus 2, Spider-associated cyclovirus 1, Squirrel-associated cyclovirus 1, Bovine-associated bovis makovirus 1, Bovine-associated bovis makovirus 2, Tongue-associated cyclovirus 1, Tongue-associated cyclovirus 2, Tongue-associated cyclovirus 3, Tongue-associated cyclovirus 4, Tongue-associated cyclovirus 5, Tongue-associated cyclovirus 6, Tongue-associated cyclovirus 7, Tongue-associated cyclovirus 8, Tongue-associated cyclovirus 9, Tongue-associated cyclovirus 10, Tongue-associated cyclovirus 11, Tongue-associated cyclovirus 12, Tongue-associated cyclovirus 1 ... Bovine-associated bovismakovirus 1, Bovine-associated cosmakovirus 1, Dragonfly-associated dragosmakovirus 1, Bovine-associated drosmakovirus 1, Camel-associated drosmakovirus 1, Camel-associated drosmakovirus 2, Bovine-associated rhizomakovirus 1, Bovine-associated rhizomakovirus 2, Chicken-associated rhizomakovirus 1, Chicken-associated rhizomakovirus 2, Human-associated rhizomakovirus 1, Human-associated rhizomakovirus 2, Human-associated rhizomakovirus 3, Bovine-associated porpurismakovirus 1, Camel-associated porpurismakovirus 1, Camel-associated porpurismako virus 2, camel-associated porpuris makovirus 3, camel-associated porpuris makovirus 4, chimpanzee-associated porpuris makovirus 1, chimpanzee-associated porpuris makovirus 2, gorilla-associated porpuris makovirus 1, howler monkey-associated porpuris makovirus 1, human-associated porpuris makovirus 1, human-associated porpuris makovirus 2, lemur-associated porpuris makovirus 1, porcine-associated porpuris makovirus 1, porcine-associated porpuris makovirus 2, porcine-associated porpuris makovirus 3, porcine-associated porpuris makovirus 4, porcine-associated porpuris makovirus Smacovirus 5, Pig-associated Porpurismacovirus 6, Pig-associated Porpurismacovirus 7, Pig-associated Porpurismacovirus 8, Pig-associated Porpurismacovirus 9, Pig-associated Porpurismacovirus 10, Rat-associated Porpurismacovirus 1, Sheep-associated Porpurismacovirus 1, Sheep-associated Porpurismacovirus 2, Sheep-associated Porpurismacovirus 3, Turkey-associated Porpurismacovirus 1, Abaca bunchy top virus, Banana bunchy top virus, Cardamom bushy dwarf virus, Medicago leaf curl virus,Broad bean necrotic stunt virus, Broad bean necrotic yellows virus, Broad bean yellow leaf virus, Milk vetch dwarf virus, Pea necrotic yellow dwarf virus, Pea yellow stunt virus, Subterranean clover stunt virus, Coconut leaf decay virus, Brisa virus, Biento virus, Beet curl top Iran virus, Exomis Microphylla latent virus, Spinach curl-top Arizona virus, Abutilon golden mosaic virus, Abutilon mosaic Bolivia virus, Abutilon mosaic Brazil virus, Abutilon mosaic virus, African cassava mosaic Burkina Faso virus, African cassava mosaic virus, Ageratum enation virus, Ageratum leaf curl Sichuan virus, Ageratum leaf curl virus, Ageratum yellow vein Hualia virus, Ageratum yellow vein Sri Lanka virus, Ageratum yellow vein virus, Allamanda leaf curl virus, Allamanda leaf spot distortion virus, Cranberry yellow vein virus, Andrographis yellow vein curl virus, Asistasia mosaic Madagascar virus, Bean calico mosaic virus, Bean yellows dwarf virus, Bean dwarf mosaic virus, Bean golden mosaic virus, Bean golden yellow mosaic virus, Bean leaf wrinkle virus, Bean white yellows dwarf mosaic virus, Bean yellow mosaic virus Xicovirus, Bhendi yellow vein Bhubaneswar virus, Bhendi yellow vein Haryana virus, Bhendi yellow vein mosaic Delhi virus, Bhendi yellow vein mosaic virus, Bitter gourd yellow mosaic virus, Blainvillea yellow spot virus, Blechum intraveinal yellowing virus, Blechum yellow vein virus, Boerhavia yellow spot virus, Cabbage leaf cigar Jamaica virus, Cabbage leaf cigar virus, Capraria yellow spot virus, Cassava mosaic Madagascar virus, Catharanthus yellow mosaic virus, Centrosema yellow spot virus, Chayote yellow mosaic virus, Chenopodium leaf cigar virus, Chilean leaf cigar Ahmedabad virus, Chilean leaf cigar Bhavanisagar virus, Chilean leaf cigar Gonda virus, Chilean leaf cigar India virus, Chilean leaf cigar Kanpur virus, Chilean leaf cigar Sri Lanka virus, Chilean leaf cigar Veranado virus, Chilean leaf cigar virus, Chinodeltmate Amazonas virus,Chinodeltoma tomato virus, Cleome golden mosaic virus, Cleome leaf wrinkle virus, Clerodendrum golden mosaic virus, Clerodendrum yellow mosaic virus, Clerodendrum golden mosaic China virus, Clerodendrum golden mosaic Jiangsu virus, Cnidoscolus mosaic leaf deformation virus, Coccinia mosaic Tamil Nadu virus, Common bean mottle virus, Common bean severe mosaic virus, Tunaso golden mosaic virus, Tunaso yellow spot virus, Tunaso yellow vein mosaic virus, Tunaso yellow vein virus, Cotton yellow spot virus, Cotton leaf wrinkle virus, Cotton leaf cigar Arabad virus, Cotton leaf cigar Bangalore virus, Cotton leaf cigar Barasat virus, Cotton leaf cigar Gezira virus, Cotton leaf cigar Cochrane virus, Cotton leaf cigar Multan virus, Cotton yellow mosaic virus, Cowpea golden mosaic virus, Safflower borome yellow vein virus, Croton golden mosaic virus, Croton yellow vein mosaic virus, Cucurbit leaf wrinkle virus, Dalechampia yellow mosaic virus, Datura leaf cigar virus Datura leaf distortion virus, Datura mosaic virus, Desmodium leaf distortion virus, Desmodium mottle virus, Dicliptera yellow mottle Cuba virus, Dicliptera yellow mottle virus, Dolichos yellow mosaic virus, Duranta leaf cigar virus, East African cassava mosaic Cameroon virus, East African cassava mosaic Kenya virus, East African cassava mosaic Malawi virus, East African cassava mosaic Zanzibar virus, Eclipta yellow vein virus, Emilia yellow vein Fujian virus, Emilia yellow vein Thailand virus, Emilia yellow vein virus, Erectite yellow mosaic virus, Euphorbia yellow vein mosaic virus, Euphorbia yellow vein virus, Euphorbia leaf cigar virus (Guangxi), Euphorbia leaf cigar virus, Euphorbia mosaic Peru virus, Euphorbia mosaic virus, Euphorbia yellow cigar virus, Euphorbia yellow mosaic virus, French bean leaf cigar virus, Hedyotis Uncinella yellow mosaic virus, Hemidesmus yellow mosaic virus, Hibiscus golden mosaic virus,Hollyhock leaf cigar virus, Hollyhock yellow vein mosaic virus, Hollyhock yellow vein virus, Honeysuckle yellow vein virus, Horsegram yellow mosaic virus, Indian cassava mosaic virus, Jackmontia mosaic Yucatan virus, Jackmontia yellow mosaic virus, , Jackmontia yellow vein virus, Jatropha leaf cigar Gujarat virus, Jatropha leaf cigar virus, Jatropha leaf yellow mosaic virus, Jatropha mosaic India virus, Jatropha mosaic Nigeria virus, Jatropha mosaic virus, Jatropha yellow mosaic virus, Kudzu mosaic virus, Leonurus mosaic virus, Lindernia anagallis yellow vein virus, Lisianthus leaf cirrus virus, Polygonum vita yellow vein virus, Polygonum vita yellow vein virus, Luffa yellow mosaic virus, Lisianthes yellow mosaic virus, Macroptilium bright mosaic virus, Macroptilium common mosaic virus, Macroptilium golden mosaic virus, Macroptilium mosaic Puerto Rico virus, Macroptilium yellow mosaic Florida virus, Macroptilium yellow mosaic virus, Macroptilium yellow spot virus, Macroptilium yellow vein virus, Malvastrum golden mosaic virus, Malvastrum leaf cirrus Philippines virus, Malvastrum leaf cirrus virus, Malvastrum yellow mosaic Herscher virus, Malvastrum yellow mosaic Jamaica virus, Malvastrum yellow mosaic virus, Malvastrum yellow vein Kambo Zia virus, Malvastrum yellow vein Honghe virus, Malvastrum yellow vein Lahore virus, Malvastrum yellow vein virus, Malvastrum yellow vein Yunnan virus, Melochia mosaic virus, Melochia yellow mosaic virus, Melon yellow leaf curl virus, Melon yellow leaf curl virus, Melon yellow mosaic virus, Melomya mosaic Puerto Rico virus, Melomya mosaic virus, Mesta yellow vein mosaic Bahreich virus, Mimosa yellow leaf curl virus, Mirabilis leaf curl virus, Mung bean yellow mosaic India virus, Mung bean yellow mottle mosaic virus, Okra yellow leaf curl virus, Okra mottle virus, Okra yellow crinkle virus, Okra yellow mosaic Mexico virus, Oxalis yellow vein virus, Papaya leaf wrinkle virus, Papaya leaf curl China virus, Papaya leaf curl Gandong virus, Papaya leaf curl virus,Passion fruit leaf cigar virus, Passion fruit leaf distortion virus, Passion fruit severe leaf distortion virus, Pavonia mosaic virus, Pavonia yellow mosaic virus, Pea leaf distortion virus, Pedilanthus leaf cigar virus, Pepper golden mosaic virus, Pepper huasteco yellow vein virus, Pepper leaf cigar Bangladesh virus, Pepper leaf cigar Lahore virus, Pepper leaf cigar virus, Pepper leaf cigar Yunnan virus, Pepper leaf cigar virus, Pepper yellow leaf cigar Ase virus, Pepper yellow leaf cigar Indonesia virus, Pepper yellow leaf cigar Indonesia virus 2, Kosi Yellow leaf cigar virus, Pepper yellow leaf cigar virus, Pepper yellow leaf vein virus, Potato yellow mosaic Panama virus, Potato yellow mosaic virus, Pouzolzia golden mosaic virus, Pouzolzia mosaic Canton virus, Pouzolzia yellow mosaic virus, Premna leaf cigar virus, Pumpkin yellow mosaic virus, Radish leaf cigar virus, Ramie mosaic Yunnan virus, Rhynchosia golden mosaic Havana virus, Rhynchosia golden mosaic Sinaloa virus, Rhynchosia golden mosaic virus, Rhynchosia mild mosaic virus, Rhynchosia Rugose golden mosaic virus, Tankirimame yellow mosaic Indian virus, Tankirimame yellow mosaic virus, Rose leaf cigar virus, Sauropus leaf cigar virus, Senecio yellow mosaic virus, Senna leaf cigar virus, Fern horn mosaic virus, Fern golden mosaic virus, Fern yellow mottle virus, Fern yellow vein virus, Fern ciliaris golden mosaic virus, Fern common mosaic virus, Fern golden mosaic bracovirus, Fern golden mosaic Brazil virus, Fern golden mosaic backup virus, Fern golden mosaic Costa Rica virus, Fern golden mosaic Florida virus, Fern golden mosaic Lala virus, Fern golden mosaic virus, Fern golden mottle virus, Fern golden spot virus, Fern golden vein virus, Fern leaf cigar virus, Sidamicrantha mosaic virus, Fern mosaic Alagoas virus, Fern mosaic Bolivia virus 1, Fern mosaic Bolivia virus 2, Fern mosaic Sinaloa virus, Fern spot Alagoas virus, Fern spot virus, Fern yellow blotch virusFern yellow leaf cigar virus, Fern yellow mosaic Alagoas virus, Fern yellow mosaic China virus, Fern yellow mosaic virus, Fern yellow mosaic Yucatan virus, Fern yellow mottle virus, Fern yellow net virus, Fern yellow vein Vietnam virus, Fern yellow vein virus, Sidastrum yellow leaf spot virus, Siegesbeckia yellow vein Guangxi virus, Siegesbeckia yellow vein virus, Eggplant mosaic Bolivia virus, South African cassava mosaic virus, Soybean blister mosaic virus, Soybean yellow blight virus Rotch virus, Soybean mild mottle virus, Spilanthes yellow vein virus, Spinach yellow vein virus, Squash leaf cigar China virus, Squash leaf cigar Philippines virus, Pumpkin leaf cigar virus, Squash leaf cigar Yunnan virus, Squash mild leaf cigar virus, Sri Lankan cassava mosaic virus, Stachytarpheta leaf cigar virus, Sunhemp leaf distortion virus, Sweet potato golden vein Korea virus, Sweet potato leaf cigar Canary virus, Sweet potato leaf cigar China virus, Sweet potato leaf cigar Georgia virus , sweet potato cigar Guangxi virus, sweet potato cigar Henan virus, sweet potato cigar Hubei virus, sweet potato cigar Sao Paulo virus, sweet potato cigar Shandong virus, sweet potato cigar Sichuan virus 1, sweet potato cigar Sichuan virus 2, sweet potato cigar South Carolina virus, sweet potato cigar virus, sweet potato mosaic virus, Synedrella yellow vein removal virus, Terfilia golden mosaic virus, tobacco cigar shoot virus, tobacco cigar Comoros virus, tobacco cigar Cuban virus, tobacco cigar Dominican Republic virus, Tobacco cigar Pusa virus, Tobacco cigar Thailand virus, Tobacco cigar Yunnan virus, Tobacco cigar Zimbabwe virus, Tobacco leaf wrinkle virus, Tobacco mottle cigar virus, Tobacco yellow crinkle virus, Tomato golden yellow mosaic virus, Tomato golden yellow mottle virus, Tomato tinola pass virus, Tomato yellow leaf cigar virus, Tomato yellow leaf distortion virus, Tomato yellow mottle Gaian virus, Tomato yellow mottle virus, Tomato common mosaic virus, Tomato stunt cigar virus, Tomato dwarf leaf virus, Tomato enation leaf cigar virus,Tomato golden leaf distortion virus, Tomato sooty mold virus, Tomato golden mosaic virus, Tomato golden mottle virus, Tomato golden vein virus, Tomato intravein yellowing virus, Tomato latent virus, Tomato leaf cigar Anjouan virus, Tomato leaf cigar Arusha virus, Tomato yellow leaf cigar Bangalore virus, Tomato leaf cigar Bangladesh virus, Tomato leaf cigar Burkina Faso virus, Tomato leaf cigar Cebu virus, Tomato leaf cigar China virus, Tomato leaf cigar Comoros virus, Tomato cigar Diana virus, Tomato cigar Ghana virus, Tomato cigar Guangdong virus , Tomato leaf cigar Guangxi virus, Tomato yellow leaf cigar Gujarat virus, Tomato leaf cigar Hainan virus, Tomato leaf cigar Hanoi virus, Tomato leaf cigar Shinchu virus, Tomato leaf cigar Iran virus, Tomato cigar Japan virus, Tomato leaf cigar Java virus, Tomato yellow leaf cigar Joydebpur virus, Tomato yellow leaf cigar Karnataka virus, Tomato cigar Karnataka virus 2, Tomato cigar Karnataka virus 3, Tomato cigar Kerala virus, Tomato yellow leaf cigar Laos virus, Tomato cigar Riwa virus, Tomato cigar Madagascar virus, Tomato cigar Mahe virus, Tomato cigar Male Tomato cigar virus, Tomato leaf cigar Mali virus, Tomato leaf cigar Mindanao virus, Tomato leaf cigar Moheli virus, Tomato leaf cigar Namakeri virus, Tomato yellow leaf cigar New Delhi virus, Tomato cigar New Delhi virus 2, Tomato cigar New Delhi virus 4, Tomato cigar New Delhi virus 5, Tomato cigar Nigeria virus, Tomato yellow leaf cigar Palampur virus, Tomato cigar Patna virus, Tomato yellow leaf cigar Philippines virus, Tomato yellow leaf cigar Pune virus, Tomato cigar purple vein virus, Tomato cigar Rajasthan virus, Tomato cigar Seychelles virus Virus, Tomato leaf cigar Sinaloa virus, Tomato yellow leaf cigar Sri Lanka virus, Tomato yellow leaf cigar Sudan virus, Tomato leaf cigar Sulawesi virus, Tomato yellow leaf cigar Taiwan virus, Tomato leaf cigar Tanzania virus, Tomato leaf cigar Toliara virus, Tomato leaf cigar Uganda virus, Tomato yellow leaf cigar Vietnam virus, Tomato yellow leaf cigar virus, Tomato leaf deformation virus, Tomato leaf distortion virus, Tomato mild mosaic virus, Tomato mild yellow leaf cigar Aragua virus, Tomato mosaic Havana virus, Tomato mottle hairy leaf cigar virus, Tomato mottle Taino virus,Tomato spot virus, Tomato mottle wrinkle virus, Tomato wrinkle mosaic virus, Tomato wrinkled yellow leaf cigar virus, Tomato severe leaf cigar Karacada virus, Tomato yellow leaf cigar virus, Tomato severe wrinkle virus, Tomato twisted leaf virus, Tomato wrinkle mosaic virus, Tomato yellow leaf cigar Axarchia virus, Tomato yellow leaf cigar China virus, Tomato yellow leaf cigar Guangdong virus, Tomato yellow leaf cigar Indonesia virus, Tomato yellow leaf cigar Kanchanaburi virus, Tomato yellow leaf cigar Malaga virus, Tomato yellow leaf cigar Mali virus, Tomato yellow leaf cigar Sardinia virus, Tomato yellow leaf cigar Shuangbai virus, Tomato yellow leaf cigar Thailand virus, Tomato yellow leaf cigar Vietnam virus, Tomato yellow leaf cigar virus, Tomato yellow leaf cigar Yunnan virus, Tomato yellow leaf distortion virus, Tomato yellow margin leaf cigar virus, Tomato yellow mottle virus, Tomato yellow spot virus Virus, Tomato yellow vein streak virus, Spiral grass yellow mosaic virus, Velvet bean golden mosaic virus, Mucuna pruriens severe mosaic virus, Vernonia crinkle virus, Vernonia yellow vein Fujian virus, Vernonia yellow vein virus, Vigna yellow mosaic virus, Vinca leaf cirrus virus, Watermelon yellow stunt virus, West African assistasia virus 1, West African assistasia virus 2, West African assistasia virus 3, Whitefly-associated begomovirus 1, Whitefly-associated begomovirus 2, Whitefly-associated begomovirus 3, Whitefly-associated begomovirus 4, Whitefly-associated begomovirus 6, Whitefly-associated begomovirus 7, Wissadula golden mosaic virus, Wissadula yellow mosaic virus, Alfalfa leaf cirrus virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus, plantain lanceolata latent virus, beet curl top virus, horseradish curl top virus, spinach severe curl top virus, Eragrostis curvula streak virus, grapevine red blotch virus, cherry blossom latent virus, Wild Vitis latent virus, Axonopus compressus streak virus, barley streak mosaic virus, chickpea yellows Australian virus, chickpea yellows virus, chickpea yellows dwarf virus,Chickpea red leaf virus, Chickpea yellow dwarf virus, Chickpea yellows virus, Chloris striate mosaic virus, Digitaria ciliaris striate mosaic virus, Digitaria didactyla striate mosaic virus, Crabgrass streak virus, Dragonfly-associated trout, Torevirus, Eragrostis minor streak virus, Eragrostis streak virus, Maize streak dwarfing virus, Maize streak reunion virus, Maize streak virus, Maize streak mosaic virus, Miscanthus streak virus, Auto dwarf virus, Panicum streak virus, Paspalum dilatatum streak mosaic virus, Paspalum mosaic virus, Rice latent virus 1, Rice latent virus 2, Saccharum streak virus, Sporobolus striate mosaic virus 1, Sporobolus Striate mosaic virus 2, Sugarcane yellow streak virus, Sugarcane streak Egyptian virus, Sugarcane streak reunion virus, Sugarcane streak virus, Sugarcane streak virus, Sugarcane white streak virus, Sweet potato asymptomatic virus 1, Switchgrass mosaic associated virus, Tobacco yellow dwarf virus, Urochloa streak virus, Wheat dwarf Indian virus, Wheat dwarf virus, Tomato pseudo-rolled top virus, Sesame rolled top virus, Turnip rolled top virus, Turnip leaf cigar virus, Citrus yellow dwarf associated virus, Mulberry mosaic dwarf associated virus, Blackbird-associated gemmicircular virus 1, Cattle-associated gemmicircular virus 1, Bromus-associated gemmicircular virus 1, Cassava-associated gemmicircular virus Lar virus 1, chickadee-associated gemmicircular virus 1, chicken-associated gemmicircular virus 1, chicken-associated gemmicircular virus 2, dragonfly-associated gemmicircular virus 1, horse-associated gemmicircular virus 1, fur seal-associated gemmicircular virus 1, Gerygone-associated gemmicircular virus 1, Gerygone-associated gemmicircular virus 2, Gerygone-associated gemmicircular virus 3, St. John's wort-associated gemmicircular virus 1, llama-associated gemmicircular virus 1, mallard-associated gemmicircular virus 1, Miniopterus-associated gemmicircular virus 1, mongoose-associated gemmicircular virus 1, mosquito-associated gemmicircular virus 1, Odonata-associated gemmicircular virus 1,Odonata-associated gemicircular virus 2, Poaceae-associated gemicircular virus 1, Pig-associated gemicircular virus 1, Pig-associated gemicircular virus 2, Fruit bat-associated gemicircular virus 1, Fruit bat-associated gemicircular virus 2, Fruit bat-associated gemicircular virus 3, Fruit bat-associated gemicircular virus 4, Fruit bat-associated gemicircular virus 5, Fruit bat-associated gemicircular virus 6, Fruit bat-associated gemicircular virus 7, Fruit bat-associated Gemicircular virus 8, fruit bat-associated gemicircular virus 9, fruit bat-associated gemicircular virus 10, rat-associated gemicircular virus 1, sclerotial gemicircular virus 1, sewage-borne gemicircular virus 1, sewage-borne gemicircular virus 2, sewage-borne gemicircular virus 3, sewage-borne gemicircular virus 4, sewage-borne gemicircular virus 5, sheep-associated gemicircular virus 1, soybean-associated gemicircular virus 1, dragonfly-associated gemicircular virus Dugivirus 1, Dog-associated gemigolvirus 1, Mallard-associated gemigolvirus 1, Fruit bat-associated gemigolvirus 1, Sewage-borne gemigolvirus 1, Starling-associated gemigolvirus 1, Badger-associated gemigolvirus 1, Black robin-associated gemigolvirus 1, Blackbird-associated gemigolvirus 1, Cattle-associated gemigolvirus 1, Dragonfly-associated gemigolvirus 1, Human-associated gemigolvirus 1, Human-associated gemigolvirus 2, Human-associated gemigolvirus 3, Human-associated gemigolvirus 4, Human-associated gemigolvirus Mikivivirus 5, mongoose-associated gemikivivirus 1, flying fox-associated gemikivivirus 1, horseshoe bat-associated g...

Claims

1. a) a CRISPR-associated (Cas) protein; and b) an RNase protein, The Cas protein is a catalytically inactive Cas13 (dCas13) comprising a sequence selected from SEQ ID NOs: 47 to 48; A fusion protein, wherein the RNase protein is RNase T1 comprising a sequence selected from SEQ ID NOs: 63-66 and 68.

2. The fusion protein of claim 1 , wherein the fusion protein further comprises a nuclear localization signal (NLS).

3. 3. The fusion protein of claim 2, wherein the NLS comprises a sequence selected from SEQ ID NOs: 110-730.

4. A fusion protein according to any one of claims 1 to 3, comprising a sequence selected from SEQ ID NOs: 732 and 733 and a sequence selected from SEQ ID NOs: 49 to 89.

5. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein of any one of claims 1 to 4.

6. A composition comprising the fusion protein of any one of claims 1 to 4 or the nucleic acid molecule of claim 5.

7. The composition of claim 6, further comprising one or more targeting nucleic acid molecules.

8. 8. The composition of claim 7, wherein the targeting nucleic acid molecule is a CRISPR guide RNA (crRNA).

9. The composition of claim 8 further comprising a targeting DNA oligo.

10. 10. The composition of any of claims 6 to 9 for use in a method for reducing the number of or cleaving an RNA transcript in a subject, the method comprising administering to the subject the composition and a guide nucleic acid comprising a sequence complementary to a target RNA sequence within the RNA transcript.

11. The composition described in claim 10, wherein the method is an in vitro method or an in vivo method.

12. 10. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 4 or the nucleic acid molecule of claim 5 for use in a method for treating an RNA-related disease or disorder in a subject, the method comprising administering to the subject the pharmaceutical composition and a guide nucleic acid comprising a sequence complementary to a target RNA sequence within the RNA transcript; The pharmaceutical composition, wherein the disease or disorder is myotonic dystrophy or RNA virus infection.

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