Patent Publication Number: US-2022220473-A1

Title: Protein translational control

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Patent Application Ser. No. 62/834,582, filed Apr. 16, 2019, which is incorporated herein by reference in its entirety. 
    
    
     STATEMENT OF GOVERNMENT SUPPORT 
     This invention was made with government support under EY029166 and NS103172, awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 16, 2020, is named Sequence_Listing.txt. 
     BACKGROUND 
     Existing RNA-targeting CRISPR-Cas applications provide tools to degrade RNA or modulate RNA structure but do not, on their own enhance gene expression or increase translational protein control. Likewise, existing methods of enhancing and/or increasing gene expression, such as the delivery of messenger RNAs, prove to be technically challenging. Indeed, there are few known or well characterized methods to increase mRNA translation. 
     The vast majority of gene regulatory drugs have been designed to knockdown gene expression (i.e. siRNAs, miRNAs, anti-sense, etc.). Some methods exist to enhance gene expression, such as the delivery of mRNAs; however, therapeutic delivery of such large and charged RNA molecules is technically challenging, inefficient, and not particularly practical. Classical gene therapy approaches involve delivery of a gene product as viral-encoded products (e.g. AAV or lentivirus-packaged products); however, these methods suffer from not being able to accurately reproduce the correct alternatively spliced isoforms in the correct ratios. In addition, gene delivery can exclude important non-coding regulatory sequences. Other methods of regulating protein translation involve engineered RNA binding proteins which are not easily delivered to cells and can be highly immunogenic. Often, engineered RNA binding proteins require extensive engineering for each target RNA sequence and certain of these possess limits to their applicability. More problematically, the act of expression of certain of these types of engineered RNA binding proteins, when combined with translation initiation factor functions in a fusion protein context, could disrupt the stoichiometry of translation machinery maintained by the cell. 
     As such, there is a need to provide compositions and methods for recruiting translational pre-initiation complexes in a manner which overcomes gene therapy barriers and protein engineering challenges, thereby controlling translation in cells and in gene therapy techniques. 
     SUMMARY 
     In one aspect, provided herein are complexes comprising: a Cas polypeptide; and a capped-sgRNA comprising (i) an m7G cap or an analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. In some embodiments of any of the complexes described herein, the RNA molecule is a messenger RNA (mRNA). In some embodiments of any of the complexes described herein, the mRNA has an endogenous m7G cap. In some embodiments of any of the complexes described herein, the target sequence is downstream of the endogenous m7G cap of the mRNA. In some embodiments of any of the complexes described herein, the mRNA comprises a start codon, and wherein the 5′ end of the target sequence is upstream of the start codon. In some embodiments of any of the complexes described herein, the 5′ end of the target sequence is between 1 to 50 nucleotides upstream of the first nucleotide of the start codon. In some embodiments of any of the complexes described herein, the 5′ end of the target sequence is between 1 to 15 nucleotides upstream of the first nucleotide of the start codon. In some embodiments of any of the complexes described herein, the target sequence comprises the start codon. In some embodiments of any of the complexes described herein, the mRNA comprises a start codon, and wherein the 5′ end of the target sequence is downstream of the start codon. In some embodiments of any of the complexes described herein, the 5′ end of the target sequence is between 1 to 50 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the complexes described herein, 5′ end of the target sequence is between 1 to 15 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the complexes described herein, the 5′ end of the target sequence is between 1 to 5 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the complexes described herein, the spacer is at least 80% complementary to the target sequence. In some embodiments of any of the complexes described herein, the spacer is at least 90% complementary to the target sequence. In some embodiments of any of the complexes described herein, the spacer comprises about 10 to about 40 nucleotides. In some embodiments of any of the complexes described herein, the spacer comprises about 15 to about 25 nucleotides. In some embodiments of any of the complexes described herein, the spacer comprises about 20 nucleotides. In some embodiments of any of the complexes described herein, the spacer is connected to the m7G cap or analog there of via a linker. In some embodiments of any of the complexes described herein, the linker comprises about 5 to about 25 nucleotides. In some embodiments of any of the complexes described herein, the linker comprises about 8 to about 15 nucleotides. In some embodiments of any of the complexes described herein, the linker is not complementary to any sequence in the mRNA. In some embodiments of any of the complexes described herein, the linker is conjugated to the m7G cap or analog thereof via polyethylene glycol. In some embodiments of any of the complexes described herein, the Cas polypeptide is a nuclease-deficient Cas (dCas) polypeptide, wherein the dCas comprises an inactivated target cleavage domain and a retained guide cleavage domain. In some embodiments of any of the complexes described herein, the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. In some embodiments of any of the complexes described herein, the direct repeat is capable of binding to a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. In some embodiments of any of the complexes described herein, the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas9 (dCas9) polypeptide. In some embodiments of any of the complexes described herein, the direct repeat is capable of binding to a nuclease-deficient Cas9 (dCas9) polypeptide. In some aspects, provided herein is a nucleic acid comprising a sequence encoding the capped-sgRNA in any of the complexes described herein. In some embodiments, the nucleic acid further comprises a sequence encoding the Cas polypeptide in any of the complexes described herein. 
     In another aspect, provided herein are nucleic acids comprising a sequence encoding a capped-sgRNA, wherein the capped-sgRNA comprises: (i) an m7G cap or analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to a Cas polypeptide. In some embodiments of any of the nucleic acids described herein, the RNA molecule is an mRNA. In some embodiments of any of the nucleic acids described herein, the mRNA has an endogenous m7G cap. In some embodiments of any of the nucleic acids described herein, the target sequence is downstream of the endogenous m7G cap of the mRNA. In some embodiments of any of the nucleic acids described herein, the mRNA comprises a start codon, and wherein the 5′ end of the target sequence is upstream of the start codon. In some embodiments of any of the nucleic acids described herein, the 5′ end of the target sequence is between 1 and 50 nucleotides upstream of the first nucleotide of the start codon. In some embodiments of any of the nucleic acids described herein, the 5′ end of the target sequence is between 1 to 15 nucleotides upstream of the first nucleotide of the start codon. In some embodiments of any of the nucleic acids described herein, the target sequence comprises the start codon. In some embodiments of any of the nucleic acids described herein, the mRNA comprises a start codon, and wherein the 5′ end of the target sequence is downstream of the start codon. In some embodiments of any of the nucleic acids described herein, the 5′ end of the target sequence is between 1 to 50 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the nucleic acids described herein, the 5′ end of the target sequence is between 1 to 15 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the nucleic acids described herein, the 5′ end of the target sequence is between 1 to 5 nucleotides downstream of the last nucleotide of the start codon. In some embodiments of any of the nucleic acids described herein, the spacer is at least 80% complementary to the target sequence. In some embodiments of any of the nucleic acids described herein, the spacer is at least 90% complementary to the target sequence. In some embodiments of any of the nucleic acids described herein, the spacer comprises about 10 to about 40 nucleotides. In some embodiments of any of the nucleic acids described herein, the spacer comprises about 15 to about 25 nucleotides. In some embodiments of any of the nucleic acids described herein, the spacer comprises about 20 nucleotides. In some embodiments of any of the nucleic acids described herein, the spacer is connected to the m7G cap or analog thereof via a linker. In some embodiments of any of the nucleic acids described herein, the linker comprises about 5 to about 25 nucleotides. In some embodiments of any of the nucleic acids described herein, the linker comprises about 8 to about 20 nucleotides. In some embodiments of any of the nucleic acids described herein, the linker is not complementary to any sequence in the mRNA. In some embodiments of any of the nucleic acids described herein, the linker is conjugated to the m7G cap or analog thereof via polyethylene glycol. In some embodiments of any of the nucleic acids described herein, the nucleic acids further comprise a sequence encoding a RNase P processing site. In some embodiments of any of the nucleic acids described herein, the nucleics further comprise a poly-T sequence. In some embodiments of any of the nucleic acids described herein, the nucleic acids further comprise a poly-T sequence downstream of the sequence encoding a RNase P processing site. In some embodiments of any of the nucleic acids described herein, the nucleic acids further comprise a sequence encoding the Cas polypeptide. In some embodiments of any of the nucleic acids described herein, the Cas polypeptide is a nuclease-deficient Cas polypeptide. In some embodiments of any of the nucleic acids described herein, the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. In some embodiments of any of the nucleic acids described herein, the direct repeat is capable of binding to a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d. In some embodiments of any of the nucleic acids described herein, the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas9 (dCas9) polypeptide. In some embodiments of any of the nucleic acids described herein, the nucleic acids further comprise one or more RNA polymerase II promoters. In some embodiments of any of the nucleic acids described herein, the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from the same promoter. In some embodiments of any of the nucleic acids described herein, the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from different promoters. In some aspects, provided herein are vectors comprising the nucleic acids of any of the above embodiments. In some embodiments, the vector is an AAV vector. In some embodiments, provided herein are cells comprising the nucleic acid of any of the above embodiments. 
     In another aspect, provided herein are methods of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising (a) a sequence encoding a Cas polypeptide; and (b) a sequence encoding a capped-sgRNA comprising (i) an m7G cap or analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. In some embodiments of any of the methods of regulating translation of an mRNA in a cell described herein, the Cas polypeptide is a nuclease-deficient Cas13 (dCas13) polypeptide. In some embodiments of any of the methods of regulating translation of an mRNA in a cell described herein, the dCas13 polypeptide is dCas13b or dCas13d. In some embodiments of any of the methods of regulating translation of an mRNA in a cell described herein, the dCas13b comprises an inactivated target cleavage domain and a retained guide cleavage domain. In some embodiments of any of the methods of regulating translation of an mRNA in a cell described herein, the dCas13d comprises an inactivated target cleavage domain and a retained guide cleavage domain. 
     INCORPORATION BY REFERENCE 
     All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       A better understanding of the features and advantages can be obtained by reference to the following detailed description that sets forth illustrative embodiments and accompanying drawings (“Figure” and “Fig.” herein), of which: 
         FIGS. 1A-1G  show modulation of translation using dCas and capped-sgRNA.  FIG. 1A  shows exemplary constructs for generating dCas and Capped-sgRNA.  FIG. 1B  shows an exemplary structure of unprocessed capped-sgRNA containing an RNase P processing site.  FIG. 1C  shows an exemplary chemical composition of a capped-sgRNA.  FIG. 1D  shows capped-sgRNA processing by RNase P.  FIG. 1E  shows a schematic of localized capped-sgRNA recruitment and binding of dCas to the capped-sgRNA.  FIG. 1F  shows an exemplary two construct system for expressing dCas and capped-sgRNA.  FIG. 1G  shows the sgRNA targeting window in the target ATF4 ORF.  FIG. 1H  shows results of capped-sgRNA modulation on the translation of the target ATF4 ORF.  FIG. 1I  shows results of using the control uncapped-sgRNAs on the translation of the target ATF4 ORF. 
     
    
    
     DETAILED DESCRIPTION 
     Embodiments according to the invention disclosed herein will be described more fully hereinafter. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure. 
     Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/−15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. 
     Unless otherwise defined, all terms (including 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. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. While not explicitly defined below, such terms should be interpreted according to their common meaning. 
     Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof. 
     As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. 
     The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. 
     The terms “acceptable,” “effective,” “efficient” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose. 
     Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). 
     As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the recited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.” “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present disclosure. 
     As used herein, the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect. 
     Translational Enhancement Via M7G Cap Recruitment 
     Translation initiation in mammalian cells starts with the binding of the 5′ methyl-7 guanosine (m7G) cap structure by Eukaryotic Initiation Factor 4E (EIF4E), which results in the nucleation of translational pre-initiation complexes on the adjacent 5′ untranslated region (5′UTR) of mRNA. The bound pre-initiation complexes then scan the 5′UTR unidirectionally (5′ to 3′) for suitable start codons (e.g., “AUG”) to prime and initiate translation. The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA, and serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export, and cap-dependent protein synthesis. 
     Provided herein are compositions and methods for enhancing protein production by recruiting an m7G cap to an mRNA using a capped-sgRNA and a Cas polypeptide. In the compositions and methods provided herein, the bound pre-initiation complexes do not necessarily scan the 5′UTR unidirectionally 5′ to 3′. 
     In some aspects, provided herein is a complex comprising at least one Cas polypeptide or nucleic acid encoding the at least one Cas polypeptide, and at least one m7G capped single guide RNA (capped-sgRNA) or nucleic acid encoding the at least one capped-sgRNA, where the at least one capped-sgRNA is capable of targeting the at least one Cas polypeptide to a target sequence in an RNA molecule (e.g., an mRNA). In some embodiments, upon hybridization between the sgRNA and the target sequence, the m7G cap of the sgRNA is brought closer to a desired start codon in the target mRNA as compared to the endogenous m7G cap of the target mRNA. Also provided are methods of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising (a) a sequence encoding a at least one Cas polypeptide; and (b) a sequence encoding at least one capped-sgRNA comprising (i) an m7G cap; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. 
     In some aspects, provided herein is a complex comprising a Cas polypeptide or nucleic acid encoding the Cas polypeptide, and an m7G capped single guide RNA (capped-sgRNA) or nucleic acid encoding the capped-sgRNA, where the capped-sgRNA is capable of targeting the Cas polypeptide to a target sequence in an RNA molecule (e.g., an mRNA). In some embodiments, upon hybridization between the sgRNA and the target sequence, the m7G cap of the sgRNA is brought closer to a desired start codon in the target mRNA as compared to the endogenous m7G cap of the target mRNA. Also provided are methods of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising (a) a sequence encoding a Cas polypeptide; and (b) a sequence encoding a capped-sgRNA comprising (i) an m7G cap; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. 
     Each strand of DNA or RNA has a 5′ end and a 3′ end, corresponding to the carbon position on the deoxyribose (or ribose) ring. “Upstream” as described herein can mean toward the 5′ end of an RNA molecule and “downstream” as described herein can mean towards the 3′ end of an RNA molecule. A “start codon” as described herein can refer to the first codon of a messenger RNA transcript translated by a ribosome. The most common start codon is AUG. Alternative start codons from both prokaryotes and eukaryotes include, but not limited to, GUG, UUG, AUU, and CUG. 
     The term “cell” as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. 
     The term “encode” as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide, an mRNA, or an effector RNA if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the effector RNA, the mRNA, or an mRNA that can for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. 
     As used herein, the term “expression” or “gene expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample. 
     The term “target sequence” can refer to a nucleic acid sequence present in an RNA molecule to which a spacer of a guide RNA (e.g, a capped-sgRNA as disclosed herein) can hybridize, provided sufficient conditions for hybridization exist. Hybridization between the spacer and the target sequence can, for example, be based on Watson-Crick base pairing rules, which enables programmability in the spacer sequence. The spacer sequence can be designed, for instance, to hybridize with any target sequence. 
     The “spacer” or “spacer sequence” is comprised within a single guide RNA can include a nucleotide sequence that is complementary to a specific sequence within a target RNA. 
     “Binding” as used herein can refer to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it means that the molecule X binds to molecule Y in a non-covalent manner). Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10 −6  M, less than 10 −7  M, less than 10 −8  M, less than 10 −9 M, less than 10 −10  M, less than 10 −11  M, less than 10 −12  M, less than 10 −13  M, less than 10 −14  M, or less than 10 −15 M. Kd is dependent on environmental conditions, e.g., pH and temperature, as is known by those in the art. “Affinity” can refer to the strength of binding, and increased binding affinity is correlated with a lower Kd. 
     The terms “hybridizing” or “hybridize” can refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a partially, substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences or segments of sequences are “substantially complementary” if at least 80% of their individual bases are complementary to one another. Two nucleic acid sequences or segments of sequences are “partially complementary” if at least 50% of their individual bases are complementary to one another. 
     As used herein, “complementary” can mean that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of sequence identity. “Complementary” also means that two nucleic acid sequences can hybridize under low, middle, and/or high stringency condition(s). 
     As used herein, “substantially complementary” means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99%, or 100% of sequence identity. “Substantially complementary” can also mean that two nucleic acid sequences can hybridize under high stringency condition(s). 
     Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5×Denhardt&#39;s solution, 6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt&#39;s solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art. 
     As used herein, “operably linked” refers to the situation in which part of a linear DNA sequence can influence the other parts of the same DNA molecule. For example, when a promoter controls the transcription of the coding sequence, it is operatively linked to the coding sequence. 
     As used herein, a “polypeptide” refers to, without limitation, proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized. A polypeptide may have one or more modifications, such as a post-translational modification (such as glycosylation, etc.) or any other modification (such as PEGylation, etc.). The polypeptide may contain one or more non-naturally-occurring amino acids (such as an amino acid with a side chain modification). Polypeptides described herein typically comprise at least about 10 amino acids. 
     As used herein, “contacting” a cell with a nucleic acid molecule can include allowing the nucleic acid molecule to be in sufficient proximity with the cell such that the nucleic acid molecule can be introduced into the cell. 
     A “promoter” can be a region of DNA that leads to initiation of transcription of a gene. 
     As used herein, “nuclease-deficient” may refer to a polypeptide with reduced nuclease activity, reduced endo- or exo-DNAse activity or RNAse activity, reduced nickase activity, or reduced ability to cleave DNA and/or RNA. “Reduced nuclease activity” means a decline in nuclease, nickase, DNAse, or RNAse activity of at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 35%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more or any value between any of the listed values. Alternatively, “reduced nuclease activity” may refer to a decline of at least about 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 2000-fold or more or any value between any of the listed values. 
     “Nucleic acids” may be naturally occurring nucleic acids such as DNA and RNA, or artificial nucleic acids including peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). Nucleic acids disclosed herein can be single-stranded or double-stranded nucleic acids. 
     I. m7G Cap 
     The m7G cap, or 7-methylguanosine cap, is a guanine nucleotide methylated on the 7 position and can be linked to an RNA molecule (e.g., a sgRNA or an mRNA) via a 5′ to 5′ triphosphate linkage. In one embodiment, capped RNA molecules (e.g., capped-sgRNAs) disclosed herein include a single methyl group on the terminal G residue at the N-7 position. In vivo, the m7G cap structure is added enzymatically to mRNA produced by RNA polymerase II. In one embodiment, the adjacent nucleotides can be 2′-O-methylated to different extents. For example, the m7G cap can be a m7GpppN, or Cap 0, an m7GpppNm, or Cap 1, and an m7GpppNmpNm, or Cap 2. Exemplary structures of Cap 0 and Cap 1, are shown below: 
     
       
         
         
             
             
         
       
     
     The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA, and serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export, and cap-dependent protein synthesis. The capped-sgRNA disclosed herein exploits these biological functions to recruit pre-initiation complexes for enhancement of protein translation. 
     The capped-sgRNA disclosed herein includes a nucleic acid sequence which is transcribed by, e.g. an RNA polymerase II, and the sgRNA thereby becomes 5′ capped upon transcription with an m7G cap. The transcription of capped-sgRNA nucleic acid sequence is catalyzed by bacteriophage RNA polymerases, such as, without limitation, RNA polymerase T7, T3, and SP6. When bacteriophage RNA polymerases are used to generate the sgRNAs of the present disclosure, cap analogs initiate transcription. In one embodiment, the m7G(5′)pppG cap analog is incorporated into the sgRNA and simulates the m7G cap structure. In another embodiment, standard cap analogs are incorporated into the sgRNA in the forward (e.g., [m7G(5′)pppG(pN)]) or the reverse orientation (e.g., [G(5′)pppm7G(pN)]) resulting in two forms of isomeric RNAs. In another embodiment, chemical modifications at either the 2′ or 3′ OH group results in the cap being incorporated solely in the forward orientation. In some embodiments, the m7G cap is an anti-reverse cap analog (ARCA), wherein one of the 3′ OH groups (closer m7G) is eliminated from the cap analog and is substituted with —OCH 3 . This modification forces RNA polymerases to initiate transcription with the remaining —OH group in G and thus synthesize RNA transcripts capped exclusively in the correct orientation. An exemplary structure of ARCA (m7(3′-O-methyl)-G(5′)ppp(5′)G) is shown below: 
     
       
         
         
             
             
         
       
     
     Additional cap analogs contemplated herein also include unmethylated cap analogs (e.g., GpppG), trimethylated cap analogs (e.g., m 3   2.2.7 GP 3 G), and m 2   7,3′-O GP 3 (2′OMe)ApG. 
     In one embodiment, the m7G cap disclosed herein includes chemical modifications relative to the naturally occurring m7G cap. For example, chemical modifications that can reduce the sensitivity of the m7G cap to cellular decapping enzymes are useful for the capped-RNAs disclosed herein. Suitable chemical modifications include, without limitation, those with 1,2-dithiodiphosphate. See those described in e.g., Strenkowska et al., Nucleic Acids Res. 44(20):9578-9590 (2016), phosphate-modified cap analogues described in e.g., Walczak et al., Chem Sci. 8(1):260-267 (2017)), as well as those described in Basolo et al., Eur J Endocrinol., 145(5):599-604 (2001), and Borghardt et al., Can Respir J. 2018 Jun. 19; 2018:2732017, all of which are incorporated herein by reference in their entirety. 
     II. Capped-sgRNA 
     Provided herein are capped-sgRNAs, nucleic acids comprising and/or encoding the capped-sgRNAs, and methods of using the same for regulating protein translation. The capped-sgRNA can include an m7G cap or an analog thereof, a spacer capable of specifically hybridizing with a target sequence in an RNA molecule, and a direct repeat capable of binding to a Cas polypeptide. In some embodiments, the capped-sgRNA includes from 5′ to 3′, an m7G cap or an analog thereof, a spacer sequence, and a direct repeat sequence. In one embodiment, the 5′ cap is linked to the spacer sequence via a linker. In one embodiment, the capped-sgRNA is derived from an unprocessed capped-sgRNA that further includes a Ribonuclease P (RNase P) processing site, and a polyadenylated (poly-A) tail at the 3′ end. 
     In some embodiments of the capped-sgRNAs disclosed herein, the spacer sequence and the scaffolding sequence or direct repeat sequence are not contiguous. In some embodiments, a scaffold sequence comprises a direct repeat sequence. 
     In some embodiments, the capped-sgRNA sequence is synthetic or comprises non-naturally occurring nucleotides. In some embodiments, a capped-guideRNA of the disclosure or a sequence encoding the guide RNA comprises or consists of modified or synthetic RNA nucleotides. Exemplary modified RNA nucleotides include, but are not limited to, pseudouridine (Y), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G), hypoxanthine, xanthine, xanthosine, 7-methylguanine, 5, 6-Dihydrouracil, 5-methylcytosine, 5-methylcytidine, 5-hydroxymethylcytosine, isoguanine, and isocytosine. Capped-sgRNAs of the disclosure may bind modified RNA within a target sequence. Within a target sequence, capped-guide RNAs of the disclosure may bind modified RNA. Exemplary epigenetically or post-transcriptionally modified RNA include, but are not limited to, 2′-0-Methylation (2′-OMe) (2′-0-methylation occurs on the oxygen of the free T-OH of the ribose moiety), N6-methyladenosine (m6A), and 5-methylcytosine (m5C). In some embodiments of the compositions of the disclosure, a capped-guide RNA of the disclosure comprises at least one sequence encoding a non-coding C/D box small nucleolar RNA (snoRNA) sequence. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the target sequence of the RNA molecule comprises at least one 2′-OMe. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the at least one sequence that is complementary to the target RNA comprises a box C motif (RETGAETGA) and a box D motif (CUGA). 
     In some embodiments, a sequence encoding a capped-guide RNA of the disclosure comprises or consists essentially of a spacer sequence and a scaffold or direct repeat sequence, wherein the spacer and the scaffold or direct repeat are operably linked. In one embodiment, the spacer and scaffold and/or direct repeat are separated by a linker sequence. In some embodiments, the linker sequence may include or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, nucleotides or any number of nucleotides in between. In some embodiments, the linker sequence may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, nucleotides or any number of nucleotides in between. 
     In some embodiments, therapeutic or pharmaceutical compositions including the capped-sgRNAs or methods of gene therapy using the capped-sgRNAs of the disclosure do not include a PAMmer oligonucleotide. In other embodiments, optionally, non-therapeutic or non-pharmaceutical compositions may include a PAMmer oligonucleotide. In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof includes a sequence complementary to a protospacer flanking sequence (PFS). In some embodiments, including those wherein a guide RNA or a portion thereof includes a sequence complementary to a PFS, the RNA binding protein may include a sequence isolated or derived from a Cas protein, such as, without limitation, a Cas9, Cas13b, or Cas13d protein. In some embodiments, including those wherein a guide RNA or a portion thereof includes a sequence complementary to a PFS, the RNA binding protein may include a sequence encoding a Cas protein, such as, without limitation, a Cas9, Cas 13b, or Cas13d protein, or an RNA-binding portion thereof. In some embodiments, the guide RNA or a portion thereof does not include a sequence complementary to a PFS. 
     Spacer 
     The capped-sgRNA disclosed herein comprises a “spacer” or “spacer sequence” that is complementary to a specific sequence within a target RNA. The spacer sequence can be designed to hybridize with any target sequence of interest. 
     In one embodiment, the spacer sequence comprised within the capped-sgRNA is about 10 to about 30 nucleotides (e.g., about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides). In another embodiment, the spacer sequence is about 15 to about 25 nucleotides (e.g., about 18 to about 22 nucleotides, or about 20 nucleotides). In another embodiment, the spacer sequence is at least 50% complementary, at least 60% complementary, or at least 70% complementary to a target sequence in an RNA molecule (e.g., an mRNA). In another embodiment, the spacer sequence is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) complementary to a target sequence in an RNA molecule (e.g., an mRNA). In another embodiment, the spacer sequence is 100% complementary to the target sequence. Exemplary spacer sequences for the capped-sgRNAs disclosed herein are: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 278) 
               
               
                   
                 TTTGCTGGAATCGAGGAATGTGCTT 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 279) 
               
               
                   
                 GTTGCGGTGCTTTGCTGGAATCGAG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 280) 
               
               
                   
                 TTTCGGTCATGTTGCGGTGCTTTGC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 281) 
               
               
                   
                 AGGAAGCTCATTTCGGTCATGTTGC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 282) 
               
               
                   
                 CTCGCTGCTCAGGAAGCTCATTTCG 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 283) 
               
               
                   
                 CCACCAACACCTCGCTGCTCAGGAA 
               
            
           
         
       
     
     In another embodiment of the capped-sgRNAs disclosed herein, spacer sequences may comprise a CRISPR RNA (crRNA). In another embodiment, spacer sequences of the disclosure comprise or consist of a sequence having sufficient complementarity to a target sequence of an RNA molecule to bind selectively to the target sequence. Upon binding to a target sequence of an RNA molecule, the spacer sequence may guide one or more of a scaffold or direct repeat sequence and a Cas polypeptide or fusion protein to the RNA molecule. In some embodiments, a spacer sequence having sufficient complementarity to a target sequence of an RNA molecule to bind selectively (partially or substantially) to the target sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in between to the target sequence. In some embodiments, a spacer sequence having sufficient complementarity to a target sequence of an RNA molecule to bind selectively to the target sequence has 100% identity the target sequence. 
     In some embodiments of the compositions of the disclosure, a capped-guide RNA or a portion thereof comprises or consists of between 10 and 100 nucleotides, inclusive of the endpoints. In some embodiments, a spacer sequence of the disclosure comprises or consists of between 10 and 30 nucleotides, inclusive of the endpoints. In some embodiments, a spacer sequence of the disclosure comprises or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence of the disclosure comprises or consists of 20 nucleotides. In some embodiments, the spacer sequence of the disclosure comprises or consists of 21 nucleotides. In some embodiments, a scaffold or direct repeat sequence of the disclosure comprises or consists of between 10 and 100 nucleotides, inclusive of the endpoints. In some embodiments, a scaffold or direct repeat sequence of the disclosure comprises or consists of 30, 35, 40, 45, 50, 55, 60, 65, 70, 76, 80, 87, 90, 95, 100, or any number of nucleotides in between. In some embodiments, the scaffold or direct repeat sequence of the disclosure comprises or consists of between 85 and 95 nucleotides, inclusive of the endpoints. In some embodiments, the scaffold or direct repeat sequence of the disclosure comprises or consists of 85 nucleotides. In some embodiments, the scaffold or direct repeat sequence of the disclosure comprises or consists of 90 nucleotides. In some embodiments, the scaffold or direct repeat sequence of the disclosure comprises or consists of 93 nucleotides. 
     In some embodiments of the compositions of the disclosure, a capped-guide RNA or a portion thereof does not comprise a nuclear localization sequence (NLS). 
     In some embodiments of the compositions of the disclosure, a capped-guide RNA, or a portion thereof does not comprise a sequence complementary to a protospacer adjacent motif (PAM). 
     In some embodiments, therapeutic or pharmaceutical compositions of the disclosure do not include a PAMmer oligonucleotide. In other embodiments, optionally, non-therapeutic or non-pharmaceutical compositions may include a PAMmer oligonucleotide. In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof includes a sequence complementary to a protospacer flanking sequence (PFS). In some embodiments, including those wherein a guide RNA or a portion thereof includes a sequence complementary to a PFS, the RNA binding protein may include a sequence isolated or derived from a Cas protein, such as, without limitation, a Cas9, Cas13b, or Cas13d protein. In some embodiments, including those wherein a guide RNA or a portion thereof includes a sequence complementary to a PFS, the RNA binding protein may include a sequence encoding a Cas protein, such as, without limitation, a Cas9, Cas 13b, or Cas13d protein, or an RNA-binding portion thereof. In some embodiments, the guide RNA or a portion thereof does not comprise a sequence complementary to a PFS. 
     Target RNA 
     The “target sequence” can be a stretch of nucleic acid sequences or a sequence motif present in an RNA molecule (e.g., mRNA) of interest to which a spacer sequence of the capped-sgRNA hybridizes, provided sufficient conditions for hybridization exist. Hybridization between the spacer and the target sequence is, for example, based on Watson-Crick base pairing rules, which enables programmability of the spacer sequence. 
     In one embodiment, the mRNA including the target sequence additionally includes one or more start codons and/or an endogenous m7G cap. In another embodiment, the target sequence is located downstream of an endogenous m7G cap, with its 5′ end located either upstream or downstream of a desired start codon. Any start codon in the target mRNA can be selected as the desired start codon. Upon hybridization between the spacer sequence of the capped-sgRNA and the target sequence, the m7G cap of the capped-sgRNA can be recruited to the vicinity of the desired start codon, and closer in proximity to the desired start codon than the endogenous m7G cap of the mRNA. In one embodiment, the m7G cap of the bound capped-sgRNA recruits translation initiation factors (e.g., EIF4E) and initiates protein translation from the desired start codon. In another embodiment, recruitment of the translation initiation factors occurs subsequent to the binding of the m7G cap of the capped-sgRNA. Without wishing to be bound by theory, the recruitment of an m7G cap via a capped-sgRNA to the vicinity of a desired start codon in a transcript allows for enhanced protein translation, as compared to protein translation initiated by the endogenous m7G cap of the transcript. 
     In one embodiment, the target sequence includes the desired start codon of the target mRNA. For example, the 5′ end of the target sequence can be upstream of the desired start codon and the 3′ end of the target sequence can be downstream of the desired start codon. In one embodiment, the 5′ end of the target sequence is located between 1 and 50 nucleotides (e.g., about 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides) upstream of the first nucleotide of the desired start codon (e.g., an “A”). In another embodiment, the 5′ end of the target sequence is located between 1 and 50 nucleotides (e.g., about 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides) downstream of the last nucleotide of the desired start codon. In some embodiments, the target sequence does not overlap with the desired start codon. The location ranges of the target sequence between about 1 and 50 nucleotides upstream or downstream of the desired start codon accounts for the differing structural properties of the various Cas proteins capable of being used with the capped-sgRNAs disclosed herein. In another embodiment, the target sequence is not required to be located between 1 and 50 nucleotides upstream or downstream from a desired start codon, rather the target sequence is located anywhere on the transcript. This is particularly relevant if the 5′UTR is large. 
     Direct Repeat/Scaffold 
     The capped-sgRNA disclosed herein includes both a “spacer sequence” and a “direct repeat” (or “DR” or “direct repeat sequence” or “DR sequence”). In one embodiment, DR is comprised within a scaffold sequence which is capable of binding to a corresponding (or cognate) Cas polypeptide. In another embodiment, a DR is capable of binding to a corresponding (or cognate) Cas polypeptide. A direct repeat sequence disclosed herein is a repetitive sequence found within a CRISPR locus (naturally-occurring in a bacterial genome or plasmid). It is well known that one would be able to infer the DR sequence of a corresponding Cas protein if the sequence of the associated CRISPR locus is known. 
     Generally, a DR is a nucleic acid sequence that consists of two or more repeats of a specific sequence, i.e., nucleotide sequences present in multiple copies in the genome. A DR sequence may or may not have intervening nucleotides. In one embodiment, a DR sequence disclosed herein includes about 10 to about 100 nucleotides (e.g. about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 nucleotides). In another embodiment, the DR sequence is orientated either 5′ or 3′ to a spacer within the sgRNA. In one embodiment, the direct repeat sequence is located 5′ to the spacer. In another embodiment, the DR sequence is located 3′ to the spacer. 
     Exemplary DR sequences for the capped-sgRNAs disclosed herein are shown below. Exemplary direct repeat sequences for Cas13a are SEQ ID Nos 284-298. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 Cas13a 
                   
                   
               
               
                 Cas13a 
                 abbreviation 
                   
                   
               
               
                 number 
                 name 
                 Organism 
                 Direct Repeat sequence 
               
               
                   
               
             
            
               
                 Cas13a1 
                 LshCas13a 
                 
                   Leptotrichia 
                 
                 CCACCCCAATATCGAAGGGGACTAAAAC (SEQ ID 
               
               
                   
                   
                 
                   shahii 
                 
                 NO: 284) 
               
               
                   
               
               
                 Cas13a2 
                 LwaCas13a 
                 
                   Leptotrichia 
                 
                 GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC (SEQ 
               
               
                   
                   
                 
                   wadei 
                 
                 ID NO: 285) 
               
               
                   
               
               
                 Cas13a3 
                 LseCas13a 
                 
                   Listeria 
                 
                 GTAAGAGACTACCTCTATATGAAAGAGGACTAAAAC (SEQ 
               
               
                   
                   
                 
                   seeligeri 
                 
                 ID NO: 286) 
               
               
                   
               
               
                 Cas13a4 
                 LbmCas13a 
                 
                   Lachnospiraceae 
                 
                 GTATTGAGAAAAGCCAGATATAGTTGGCAATAGAC (SEQ ID 
               
               
                   
                   
                 
                   bacterium 
                 
                 NO: 287) 
               
               
                   
                   
                 MA2020 
                   
               
               
                   
               
               
                 Cas13a5 
                 LbnCas13a 
                 Lachnospiraceae 
                 GTTGATGAGAAGAGCCCAAGATAGAGGGCAATAAC (SEQ 
               
               
                   
                   
                 bacterium 
                 ID NO: 288) 
               
               
                   
                   
                 NK4A179 
                   
               
               
                   
               
               
                 Cas13a6 
                 CamCas13a 
                 [ Clostridium ] 
                 GTCTATTGCCCTCTATATCGGGCTGTTCTCCAAAC (SEQ ID 
               
               
                   
                   
                 
                   aminophilum 
                 
                 NO: 289) 
               
               
                   
                   
                 DSM 10710 
                   
               
               
                   
               
               
                 Cas13a7 
                 CgaCas13a 
                 
                   Carnobacterium 
                 
                 ATTAAAGACTACCTCTAAATGTAAGAGGACTATAAC (SEQ 
               
               
                   
                   
                 
                   gallinarum 
                 
                 ID NO: 290) 
               
               
                   
                   
                 DSM 4847 
                   
               
               
                   
               
               
                 Cas13a8 
                 Cga2Cas13a 
                 
                   Carnobacterium 
                 
                 AATATAAACTACCTCTAAATGTAAGAGGACTATAAC (SEQ 
               
               
                   
                   
                 
                   gallinarum 
                 
                 ID NO: 291) 
               
               
                   
                   
                 DSM 4847 
                   
               
               
                   
               
               
                 Cas13a9 
                 Pprcas13a 
                 
                   Paludibacter 
                 
                 CTTGTGGATTATCCCAAAATTGAAGGGAACTACAAC (SEQ 
               
               
                   
                   
                 
                   propionicigenes 
                 
                 ID NO: 292) 
               
               
                   
                   
                 WB4 
                   
               
               
                   
               
               
                 Cas13a10 
                 LweCas13a 
                 
                   Listeria 
                 
                 GATTTAGAGTACCTCAAAATAGAAGAGGTCTAAAAC (SEQ 
               
               
                   
                   
                   weihen - 
                 ID NO: 293) 
               
               
                   
                   
                 
                   stephanensis  
                 
                   
               
               
                   
                   
                 FSL R9-0317 
                   
               
               
                   
               
               
                 Cas13a11 
                 LbfCas13a 
                 
                   Listeriaceae 
                 
                 GATTTAGAGTACCTCAAAACAAAAGAGGACTAAAAC (SEQ 
               
               
                   
                   
                   bacterium  FSL 
                 ID NO: 294) 
               
               
                   
                   
                 M6-0635 
                   
               
               
                   
                   
                 ( Listeria   
                   
               
               
                   
                   
                   newyorkensis ) 
                   
               
               
                   
               
               
                 Cas13a12 
                 Lwa2cas13a 
                 
                   Leptotrichia 
                 
                 GATATAGATAACCCCAAAAACGAAGGGATCTAAAAC (SEQ 
               
               
                   
                   
                   wadei  F0279 
                 ID NO: 295) 
               
               
                   
               
               
                 Cas13a13 
                 RcsCas13a 
                 
                   Rhodobacter 
                 
                 GCCTCACATCACCGCCAAGACGACGGCGGACTGAAC (SEQ 
               
               
                   
                   
                   capsulatus  SB 
                 ID NO: 296) 
               
               
                   
                   
                 1003 
                   
               
               
                   
               
               
                 Cas13a14 
                 RcrCas13a 
                 
                   Rhodobacter 
                 
                 GCCTCACATCACCGCCAAGACGACGGCGGACTGAAC (SEQ 
               
               
                   
                   
                 
                   capsulatus 
                 
                 ID NO: 297) 
               
               
                   
                   
                 R121 
                   
               
               
                   
               
               
                 Cas13a15 
                 RcdCas13a 
                 
                   Rhodobacter 
                 
                 GCCTCACATCACCGCCAAGACGACGGCGGACTGAAC (SEQ 
               
               
                   
                   
                 
                   capsulatus 
                 
                 ID NO: 298) 
               
               
                   
                   
                 DE442 
               
               
                   
               
            
           
         
       
     
     An exemplary Cas13b direct repeat sequence is: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 299) 
               
               
                   
                 GTTGTGGAAGGTCCAGTTTTGAGGGGCTATTACAAC 
               
            
           
         
       
     
     An exemplary Cas13d (contig e-k87_11092736) Direct Repeat Sequence is: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 300) 
               
               
                   
                 GTGAGAAGTCTCCTTATGGGGAGATGCTAC 
               
            
           
         
       
     
     An exemplary Cas13d (160582958_gene49834) Direct Repeat Sequence is: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 301) 
               
               
                   
                 GAACTACACCCCTCTGTTCTTGTAGGGGTCTAACAC 
               
            
           
         
       
     
     Additional exemplary Cas13d Direct Repeat sequences are: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 302) 
               
               
                   
                 CACCCGTGCAAAATTGCAGGGGTCTAAAAC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 303) 
               
               
                   
                 GACCAACACCTCTGCAAAACTGCAGGGGTCTAAAAC 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 304) 
               
               
                   
                 AACCCCTACCAACTGGTCGGGGTTTGAAAC 
               
            
           
         
       
     
     An exemplary scaffold sequence for Cas9 is: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 305) 
               
               
                 TTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCC 
               
               
                 GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC 
               
            
           
         
       
     
     Scaffold/DR sequences of the disclosure bind the CRISPR/Cas RNA-binding protein of the disclosure. Scaffold/DR sequences of the disclosure may include a trans acting RNA (tracrRNA). Upon binding to a target sequence of an RNA molecule, the scaffold/DR sequence guides a fusion protein to the RNA molecule. In some embodiments, a scaffold/DR sequence having sufficient complementarity to a target sequence of an RNA molecule to bind selectively (partially or substantially) to the target sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in between to the target sequence. In some embodiments, a scaffold/DR sequence having sufficient complementarity to a target sequence of an RNA molecule to bind selectively to the target sequence has 100% identity the target sequence. In some embodiments, scaffold/DR sequences of the disclosure comprise a secondary structure or a tertiary structure. Exemplary secondary structures include, but are not limited to, a helix, a stem loop, a bulge, a tetraloop and a pseudo not. Exemplary tertiary structures include, but are not limited to, an A-form of a helix, a B-form of a helix, and a Z-form of a helix. Exemplary tertiary structures include, but are not limited to, a twisted or helicized stem loop. Exemplary tertiary structures include, but are not limited to, a twisted or helicized pseudoknot. In some embodiments, scaffold/DR sequences of the disclosure comprise at least one secondary structure or at least one tertiary structure. In some embodiments, scaffold/DR sequences of the disclosure include one or more secondary structure(s) or one or more tertiary structure(s). 
     Linker 
     The capped-sgRNA disclosed herein can include a “linker” or “linker sequence” between the m7G cap or analog thereof and the spacer and/or DR sequences. In one embodiment, the linker sequence includes about 5 to about 25 nucleotides (e.g., about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides). In another embodiment, the linker sequence is non-complementary to any sequence within the RNA molecule comprising the target sequence. Exemplary sequences for such a linker include, without limitation: GTCAGATCG (SEQ ID NO: 306), GTCAGATCGCCT (SEQ ID NO: 307), GTCAGATCGCCTGGA (SEQ ID NO: 308), and GTCAGATCGCCTGGAATT (SEQ ID NO: 309). Any suitable linker sequences known in the art are also contemplated herein. In some embodiments, the linker sequence is modified to adjust the editing window. 
     RNase P Processing Site 
     Some embodiments provide a nucleic acid encoding the capped-sgRNA described herein, where an unprocessed capped-sgRNA is first generated upon transcription of the nucleic acid. In one embodiment, an unprocessed capped-sgRNA includes an RNase P processing site, which is downstream of the spacer and/or direct repeat and upstream of a poly-A tail. In this manner, an RNase P binds to the processing site and removes the downstream sequence (e.g., the poly-A tail) to generate a capped-sgRNA disclosed herein. Exemplary RNase P processing sites can be found at Esakova and Krasilnikov, RNA 16:1725-1747, 2010 (e.g., See FIG. 1 of Esakova and Krasilnikov), incorporated herein by reference in its entirety. The RNase P processing site is known to include elements recognizable by RNase P, such as those described in Kirseborn et al. Biochimie 89: 1183-1194, 2007 and Lai et al. FEBS Left 584: 287-296, 2010, both of which are incorporated herein by reference in their entirety. For example, the RNase P processing site can include all or a portion of a bacterial (e.g.,  E. coli ) pre-tRNA, 4.5S rRNA precursor, or yeast pre-rRNA that includes an RNase cleavage site. Structures that resemble tmRNA, operon mRNAs, phage RNAs, OLE RNA from extremophilic bacteria are also contemplated herein as RNase P processing sites. All or a portion of a viral non-tRNA such as TYMV RNA are also useful as RNase P processing sites. In some embodiments, an RNase P processing site includes a tRNA-like small RNA (e.g., GenBank Accession No. FJ209302). 
     An exemplary structure of an unprocessed capped-sgRNA is shown in  FIG. 1B . In this embodiment, the unprocessed capped-sgRNA includes from 5′ to 3′: an m7G cap, a linker, a spacer, a direct repeat, an RNase P processing site, and a poly-A tail. In another embodiment, the RNase P processing site and poly-A tail is removed upon RNase P processing, thereby generating the capped-sgRNA with the structure of  FIG. 1C , wherein a′ is a guanosine or adenine, b′ is a spacer sequence and c′ is a direct repeat sequence. 
     III. CRISPR/Cas Polypeptides 
     The capped-sgRNAs disclosed herein are capable of binding with their cognate or corresponding RNA-binding CRISPR/Cas polypeptides (e.g., via a direct repeat sequence in the capped-sgRNA). In one embodiment, the capped-sgRNA includes a spacer sequence that confers target specificity to the Cas/sgRNA complex. CRISPR/Cas polypeptides are well known in the art and any particular Cas polypeptide can be adapted for use in the capped-sgRNA systems disclosed herein. In some embodiments, the Cas polypeptides for use as disclosed herein have altered activity compared to its corresponding wild type Cas polypeptide. In some embodiments, the Cas polypeptides are nuclease-deficient Cas (dCas) polypeptides. Nuclease-deficient Cas polypeptides have altered (e.g., diminished or abolished) nuclease activity without substantially diminished binding affinity to the sgRNA. These Cas polypeptides are useful, for example, in mediating the direct association between the capped-sgRNA and the target mRNA, and in protecting the 3′ end of the capped-sgRNA from degradation. In some embodiments, the dCas for use with the capped-sgRNA disclosed herein is devoid of cleavage activity that is applicable to the target RNA. In some embodiments, the dCas polypeptide for use with the capped-sgRNA disclosed herein retains cleavage activity that is applicable to the capped-sgRNA. In some embodiments, the dCas13 comprises an inactivated target cleavage domain and a retained or partially retained (i.e., activated or partially activated) guide cleavage domain. 
     In one embodiment, the Cas polypeptide is Cas13b. In another embodiment, the Cas polypeptide is dead or nuclease deficient Cas13b (dCas13b). In another embodiment, the dCas13b comprises an inactivated target cleavage domain and a retained (i.e., activated) guide cleavage domain as exemplified in  FIGS. 1B and 1D . In one embodiment, the Cas polypeptide disclosed herein is a Type II CRISPR Cas protein. In some embodiments, the Type II CRISPR Cas protein includes a Cas9 protein. Exemplary Cas9 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, bacteria or archaea. Exemplary Cas9 proteins of the disclosure may be isolated or derived from any species, including, but not limited to,  Streptococcus pyogenes, Haloferax mediteranii, Mycobacterium tuberculosis, Francisella tularensis  subsp.  novicida, Pasteurella multocida, Neisseria meningitidis, Campylobacter jejune, Streptococcus thermophilus, Campylobacter lari  CF89-12,  Mycoplasma gallisepticum  str. F,  Nitratifractor salsuginis  str. DSM 16511 , Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea , a  Gluconacetobacter diazotrophicus , an  Azospirillum  B510, a  Sphaerochaeta globus  str. Buddy,  Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactor alocis, Treponema denticola, Legionella pneumophila  str. Paris,  Sutterella wadsworthensis, Corynebacter diphtherias, Streptococcus aureus , and  Francisella novicida.    
     Some embodiments of the capped-sgRNA compositions or methods disclosed herein provide a Cas9 polypeptide. In certain embodiments, the Cas9 polypeptide lacks part or all of the nuclease domains (e.g., the RuvC and/or HNH domains) of a wild type Cas9 polypeptide, and therefore are nuclease-deficient. These truncated Cas9 polypeptides have a smaller size as compared to a wild type Cas9. The RuvC and HNH nuclease domains can also be inactivated, for example, as a result of point mutations within these domains. For instance, D10A and H840A mutations in  Streptococcus pyogenes  Cas9 (SpCas9) results in nuclease-deficient SpCas9. An exemplary sequence of a nuclease-deficient SpCas9 is SEQ ID NO: 310. The RuvC domain is distributed among 3 non-contiguous portions of the nuclease-deficient Cas9 primary structure (residues 1-60, 719-775, and 910-1099). The HNH domain is composed of residues 776-909. 
     
       
         
           
               
               
            
               
                 SEQ ID NO: 310 
                   
               
               
                 (SEQ ID NO: 310) 
               
               
                 Arg Thr Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn 
                   
               
               
                 1               5                   10                  15 
               
               
                   
               
               
                 Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys 
               
               
                             20                  25                  30 
               
               
                   
               
               
                 Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn 
               
               
                         35                  40                  45 
               
               
                   
               
               
                 Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr 
               
               
                     50                  55                  60 
               
               
                   
               
               
                 Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg 
               
               
                 65                  70                  75                  80 
               
               
                   
               
               
                 Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp 
               
               
                                 85                  90                  95 
               
               
                   
               
               
                 Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp 
               
               
                             100                 105                 110 
               
               
                   
               
               
                 Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val 
               
               
                         115                 120                 125 
               
               
                   
               
               
                 Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu 
               
               
                     130                 135                 140 
               
               
                   
               
               
                 Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu 
               
               
                 145                 150                 155                 160 
               
               
                   
               
               
                 Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu 
               
               
                                 165                 170                 175 
               
               
                   
               
               
                 Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln 
               
               
                             180                 185                 190 
               
               
                   
               
               
                 Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val 
               
               
                         195                 200                 205 
               
               
                   
               
               
                 Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu 
               
               
                     210                 215                 220 
               
               
                   
               
               
                 Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe 
               
               
                 225                 230                 235                 240 
               
               
                   
               
               
                 Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser 
               
               
                                 245                 250                 255 
               
               
                   
               
               
                 Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr 
               
               
                             260                 265                 270 
               
               
                   
               
               
                 Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr 
               
               
                         275                 280                 285 
               
               
                   
               
               
                 Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu 
               
               
                     290                 295                 300 
               
               
                   
               
               
                 Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser 
               
               
                 305                 310                 315                 320 
               
               
                   
               
               
                 Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu 
               
               
                                 325                 330                 335 
               
               
                   
               
               
                 Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile 
               
               
                             340                 345                 350 
               
               
                   
               
               
                 Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly 
               
               
                         355                 360                 365 
               
               
                   
               
               
                 Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys 
               
               
                     370                 375                 380 
               
               
                   
               
               
                 Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu 
               
               
                 385                 390                 395                 400 
               
               
                   
               
               
                 Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile 
               
               
                                 405                 410                 415 
               
               
                   
               
               
                 His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr 
               
               
                             420                 425                 430 
               
               
                   
               
               
                 Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe 
               
               
                         435                 440                 445 
               
               
                   
               
               
                 Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe 
               
               
                     450                 455                 460 
               
               
                   
               
               
                 Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe 
               
               
                 465                 470                 475                 480 
               
               
                   
               
               
                 Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg 
               
               
                                 485                 490                 495 
               
               
                   
               
               
                 Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys 
               
               
                             500                 505                 510 
               
               
                   
               
               
                 His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys 
               
               
                         515                 520                 525 
               
               
                   
               
               
                 Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly 
               
               
                     530                 535                 540 
               
               
                   
               
               
                 Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys 
               
               
                 545                 550                 555                 560 
               
               
                   
               
               
                 Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys 
               
               
                                 565                 570                 575 
               
               
                   
               
               
                 Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser 
               
               
                             580                 585                 590 
               
               
                   
               
               
                 Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe 
               
               
                         595                 600                 605 
               
               
                   
               
               
                 Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr 
               
               
                     610                 615                 620 
               
               
                   
               
               
                 Leu Thr Leu Phe Glu Asp Arg Glu Net Ile Glu Glu Arg Leu Lys Thr 
               
               
                 625                 630                 635                 640 
               
               
                   
               
               
                 Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg 
               
               
                                 645                 650                 655 
               
               
                   
               
               
                 Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile 
               
               
                             660                 665                 670 
               
               
                   
               
               
                 Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp 
               
               
                         675                 680                 685 
               
               
                   
               
               
                 Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu 
               
               
                     690                 695                 700 
               
               
                   
               
               
                 Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp 
               
               
                 705                 710                 715                 720 
               
               
                   
               
               
                 Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys 
               
               
                                 725                 730                 735 
               
               
                   
               
               
                 Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val 
               
               
                             740                 745                 750 
               
               
                   
               
               
                 Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu 
               
               
                         755                 760                 765 
               
               
                   
               
               
                 Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys 
               
               
                     770                 775                 780 
               
               
                   
               
               
                 Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu 
               
               
                 785                 790                 795                 800 
               
               
                   
               
               
                 His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr 
               
               
                                 805                 810                 815 
               
               
                   
               
               
                 Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile 
               
               
                             820                 825                 830 
               
               
                   
               
               
                 Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe 
               
               
                         835                 840                 845 
               
               
                   
               
               
                 Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys 
               
               
                     850                 855                 860 
               
               
                   
               
               
                 Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys 
               
               
                 865                 870                 875                 880 
               
               
                   
               
               
                 Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln 
               
               
                                 885                 890                 895 
               
               
                   
               
               
                 Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu 
               
               
                             900                 905                 910 
               
               
                   
               
               
                 Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln 
               
               
                         915                  920                925 
               
               
                   
               
               
                 Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys 
               
               
                     930                 935                 940 
               
               
                   
               
               
                 Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu 
               
               
                 945                 950                 955                 960 
               
               
                   
               
               
                 Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys 
               
               
                                 965                 970                 975 
               
               
                   
               
               
                 Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn 
               
               
                             980                 985                 990 
               
               
                   
               
               
                 Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser 
               
               
                         995                 1000                1005 
               
               
                   
               
               
                 Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile 
               
               
                     1010                1015                1020 
               
               
                   
               
               
                 Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 
               
               
                 1025                1030                1035               1040 
               
               
                   
               
               
                 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn 
               
               
                                 1045                1050                1055 
               
               
                   
               
               
                 Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly 
               
               
                             1060                1065                1070 
               
               
                   
               
               
                 Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val 
               
               
                         1075                1080                1085 
               
               
                   
               
               
                 Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr 
               
               
                     1090                1095                1100 
               
               
                   
               
               
                 Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys 
               
               
                 1105                1110                1115               1120 
               
               
                   
               
               
                 Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe 
               
               
                                 1125                1130                1135 
               
               
                   
               
               
                 Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu 
               
               
                             1140                1145                1150 
               
               
                   
               
               
                 Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile 
               
               
                         1155                1160                1165 
               
               
                   
               
               
                 Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu 
               
               
                     1170                1175                1180 
               
               
                   
               
               
                 Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu 
               
               
                 1185                1190                1195               1200 
               
               
                   
               
               
                 Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu 
               
               
                                 1205                1210                1215 
               
               
                   
               
               
                 Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser 
               
               
                             1220                1225                1230 
               
               
                   
               
               
                 Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys 
               
               
                         1235                1240                1245 
               
               
                   
               
               
                 Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His 
               
               
                     1250                1255                1260 
               
               
                   
               
               
                 Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 
               
               
                 1265                1270                1275               1280 
               
               
                   
               
               
                 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr 
               
               
                                 1285                1290                1295 
               
               
                   
               
               
                 Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile 
               
               
                             1300                1305                1310 
               
               
                   
               
               
                 His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr 
               
               
                         1315                1320                1325 
               
               
                   
               
               
                 Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val 
               
               
                     1330                1335                1340 
               
               
                   
               
               
                 Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr 
               
               
                 1345                1350                1355               1360 
               
               
                   
               
               
                 Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ala Tyr Pro Tyr Asp Val 
               
               
                                 1365                1370                1375 
               
               
                   
               
               
                 Pro Asp Tyr Ala Ser Leu 
               
            
           
         
       
     
     Some embodiments provide a Cas polypeptide that is a Cas9 polypeptide that lacks all or a part of (1) an HNH domain, (2) at least one RuvC nuclease domain, (3) a Cas9 polypeptide DNase active site, (4) a ββα-metal fold comprising a Cas9 polypeptide active site, or (5) a Cas9 polypeptide that lacks all or part of one or more of the HNH domain, at least one RuvC nuclease domain, a Cas9 polypeptide DNase active site, and/or a ββα-metal fold comprising a Cas9 polypeptide active site as compared to a corresponding wild type Cas9 polypeptide. 
     The Cas9 polypeptides described herein can be archaeal or bacterial Cas9 polypeptides. Exemplary Cas9 polypeptide include those derived from  Haloferax mediteranii, Mycobacterium tuberculosis, Francisella tularensis  subsp.  novicida, Pasteurella multocida, Neisseria meningitidis, Campylobacter jejune, Streptococcus thermophilus  LMD-9 CRISPR 3,  Campylobacter lari  CF89-12,  Mycoplasma gallisepticum  str. F,  Nitratifractor salsuginis  str. DSM 1651 1 , Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea, Gluconacetobacter diazotrophicus, Azospirillum  B510 , Sphaerochaeta globus  str. Buddy,  Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filif actor alocis, Treponema denticola, Legionella pneumophila  str. Paris,  Sutterella wadsworthensis, Corynebacter diphtheriae, Streptococcus aureus , and  Francisella novicida.    
     Any Cas polypeptides with altered nuclease activity as compared to a naturally occurring Cas polypeptide is contemplated herein. Additional types of nuclease-deficient Cas polypeptides are described in e.g., Brezgin et al. Int J Mol Sci 20(23):6041, 2019 and Xu and Lei, J Mol Biol 431:34-47, 2019, incorporated by reference in its entirety. 
     Exemplary Cas polypeptide sequences disclosed in the methods for translational enhancement WO2019/204828 are incorporated herein by reference in their entirety and can be used in conjunction with the corresponding capped-sgRNAs disclosed herein. 
     In some embodiments of the compositions of the disclosure, the CRISPR Cas protein comprises a Type V CRISPR Cas protein. In some embodiments, the Type V CRISPR Cas protein comprises a Cpf1 protein. Exemplary Cpf1 proteins of the disclosure may be isolated or derived from any species, including but not limited to, bacteria or archaea. Exemplary Cpf1 proteins of the disclosure may be isolated or derived from any species, including but not limited to,  Francisella tularensis  subsp.  novicida, Acidaminococcus  sp. BV3L6 and  Lachnospiraceae bacterium  sp. ND2006. Exemplary Cpf1 proteins of the disclosure may be nuclease inactivated. 
     In some embodiments, the CRISPR Cas protein comprises a Type VI CRISPR Cas protein or portion thereof. In some embodiments, the Type VI CRISPR Cas protein comprises a Cas13 protein or portion thereof. Exemplary Cas13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, bacteria or archaea. Exemplary Cas13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to,  Leptotrichia wadei, Listeria seeligeri  serovar 1/2b (strain ATCC 35967/DSM 20751/CIP 100100/SLCC 3954),  Lachnospiraceae bacterium, Clostridium aminophilum  DSM 10710 , Carnobacterium gallinarum  DSM 4847 , Paludibacter propionicigenes  WB4,  Listeria weihenstephanensis  FSL R9-0317,  Listeria weihenstephanensis  FSL R9-0317, bacterium FSL M6-0635 ( Listeria newyorkensis ),  Leptotrichia wadei  F0279,  Rhodobacter capsulatus  SB 1003,  Rhodobacter capsulatus  R121,  Rhodobacter capsulatus  DE442 and  Corynebacterium ulcerans . Exemplary Cas13 proteins of the disclosure may be DNA nuclease inactivated. Exemplary Cas13 proteins of the disclosure include, but are not limited to, Cas13a, Cas13b, Cas13c, Cas13d, and orthologs thereof. Exemplary Cas13b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively. 
     In some embodiments of the compositions of the disclosure, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a Cas13d protein or also called CasRx/Cas13d proteins. CasRX/Cas13d is an effector of the type VI-D CRISPR-Cas systems. In some embodiments, the CasRX/Cas13d protein is an RNA-guided RNA endonuclease enzyme that can cut or bind RNA. In some embodiments, the CasRX/Cas13d protein can include one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains. In some embodiments, the CasRX/Cas13d protein can include either a wild-type or mutated HEPN domain. In some embodiments, the CasRX/Cas13d protein includes a mutated HEPN domain that cannot cut RNA but can process guide RNA. In some embodiments, the CasRX/Cas13d protein does not require a protospacer flanking sequence. Also see WO Publication No. WO2019/040664 &amp; US2019/0062724, which is incorporated herein by reference in its entirety, for further examples and sequences of CasRX/Cas13d protein. 
     In some instances, the Cas polypeptide has a sequence that is at least 80% identical (e.g. at least 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% identical) to any of the Cas polypeptides (e.g., any of the wild type or nuclease-deficient Cas polypeptides) of the present disclosure. 
     Exemplary Cas9 sequences include, but are not limited to: 
     
       
         
           
               
               
             
               
                   
               
               
                 Name 
                 Protein Sequence 
               
               
                   
               
             
            
               
                   S .  pyogenes  Cas9 
                 MDKKYSIGLDIGINSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
               
               
                   
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI 
               
               
                   
                 FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD 
               
               
                   
                 NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG 
               
               
                   
                 LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK 
               
               
                   
                 NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ 
               
               
                   
                 SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQ 
               
               
                   
                 IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT 
               
               
                   
                 PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL 
               
               
                   
                 GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ 
               
               
                   
                 KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN 
               
               
                   
                 QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV 
               
               
                   
                 DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN 
               
               
                   
                 TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI 
               
               
                   
                 KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI 
               
               
                   
                 RKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS 
               
               
                   
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF 
               
               
                   
                 EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV 
               
               
                   
                 NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS 
               
               
                   
                 AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI 
               
               
                   
                 TGLYETRIDLSQLGGD (SEQ ID NO: 311) 
               
               
                   
               
               
                 
                   Staphylococcus 
                 
                 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRR 
               
               
                   aureus  Cas9 
                 RRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGV 
               
               
                   
                 HNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDY 
               
               
                   
                 VKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEML 
               
               
                   
                 MGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQK 
               
               
                   
                 KKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIA 
               
               
                   
                 KILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDN 
               
               
                   
                 QIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII 
               
               
                   
                 ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKC 
               
               
                   
                 LYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSS 
               
               
                   
                 SDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATR 
               
               
                   
                 GLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANA 
               
               
                   
                 DFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK 
               
               
                   
                 YSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLL 
               
               
                   
                 MYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN 
               
               
                   
                 KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEV 
               
               
                   
                 NSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYR 
               
               
                   
                 EYLENIVINDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 
               
               
                   
                 312) 
               
               
                   
               
               
                   S .  thermophilus   
                 MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRK 
               
               
                 CRISPR 1 Cas9 
                 KHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGIS 
               
               
                   
                 YLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGK 
               
               
                   
                 KHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRT 
               
               
                   
                 DYGRYRTSGETLDNIEGILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKK 
               
               
                   
                 LSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYR 
               
               
                   
                 KMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQF 
               
               
                   
                 RKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEK 
               
               
                   
                 LLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKAN 
               
               
                   
                 KDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHD 
               
               
                   
                 LINNSNQEENDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSERE 
               
               
                   
                 LKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRA 
               
               
                   
                 HKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTL 
               
               
                   
                 VSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRK 
               
               
                   
                 ISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHD 
               
               
                   
                 PQTFEKVIEPILENYPNKQINDKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYY 
               
               
                   
                 DSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFDKGTGT 
               
               
                   
                 YKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKH 
               
               
                   
                 YVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKN 
               
               
                   
                 EGDKPKLDF (SEQ ID NO: 313) 
               
               
                   
               
               
                   N .  meningitidis  Cas9 
                 MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLA 
               
               
                   
                 MARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAA 
               
               
                   
                 ALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGD 
               
               
                   
                 FRIPAELALNKEEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLK 
               
               
                   
                 EGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILE 
               
               
                   
                 QGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTL 
               
               
                   
                 MEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEI 
               
               
                   
                 LEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPI 
               
               
                   
                 PADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEEN 
               
               
                   
                 RKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYV 
               
               
                   
                 EIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVET 
               
               
                   
                 SRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASN 
               
               
                   
                 GQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDG 
               
               
                   
                 KTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKL 
               
               
                   
                 SSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD 
               
               
                   
                 LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQ 
               
               
                   
                 VQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQG 
               
               
                   
                 KDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKI 
               
               
                   
                 GKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR (SEQ ID NO: 314) 
               
               
                   
               
               
                 
                   Parvibaculum 
                 
                 MERIFGEDIGTTSIGFSVIDYSSTQSAGNIQRLGVRIFPEARDPDGTPLNQQRRQKRMMR 
               
               
                 
                   lavamentivorans 
                 
                 RQLRRRRIRRKALNETLHEAGFLPAYGSADWPVVMADEPYELRRRGLEEGLSAYEFGR 
               
               
                 Cas9 
                 AIYHLAQHRHFKGRELEESDTPDPDVDDEKEAANERAATLKALKNEQTTLGAWLARR 
               
               
                   
                 PPSDRKRGIHAHRNVVAEEFERLWEVQSKFHPALKSEEMRARISDTIFAQRPVFWRKN 
               
               
                   
                 TLGECRFMPGEPLCPKGSWLSQQRRMLEKLNNLAIAGGNARPLDAEERDAILSKLQQQ 
               
               
                   
                 ASMSWPGVRSALKALYKQRGEPGAEKSLKFNLELGGESKLLGNALEAKLADMFGPD 
               
               
                   
                 WPAHPRKQEIRHAVHERLWAADYGETPDKKRVIILSEKDRKAHREAAANSFVADFGIT 
               
               
                   
                 GEQAAQLQALKLPTGWEPYSIPALNLFLAELEKGERFGALVNGPDWEGWRRTNEPHR 
               
               
                   
                 NQPTGEILDKLPSPASKEERERISQLRNPTVVRTQNELRKVVNNLIGLYGKPDRIRIEVG 
               
               
                   
                 RDVGKSKREREEIQSGIRRNEKQRKKATEDLIKNGIANPSRDDVEKWILWKEGQERCP 
               
               
                   
                 YTGDQIGFNALFREGRYEVEHIWPRSRSFDNSPRNKTLCRKDVNIEKGNRMPFEAFGH 
               
               
                   
                 DEDRWSAIQIRLQGMVSAKGGTGMSPGKVKRFLAKTMPEDFAARQLNDTRYAAKQIL 
               
               
                   
                 AQLKRLWPDMGPEAPVKVEAVTGQVTAQLRKLWTLNNILADDGEKTRADHRHHAID 
               
               
                   
                 ALTVACTHPGMTNKLSRYWQLRDDPRAEKPALTPPWDTIRADAEKAVSEIVVSHRVR 
               
               
                   
                 KKVSGPLHKETTYGDTGTDIKTKSGTYRQFVTRKKIESLSKGELDEIRDPRIKEIVAAHV 
               
               
                   
                 AGRGGDPKKAFPPYPCVSPGGPEIRKVRLTSKQQLNLMAQTGNGYADLGSNHHIAIYR 
               
               
                   
                 LPDGKADFEIVSLFDASRRLAQRNPIVQRTRADGASFVMSLAAGEAIMIPEGSKKGIWI 
               
               
                   
                 VQGVWASGQVVLERDTDADHSTTTRPMPNPILKDDAKKVSIDPIGRVRPSND (SEQ ID 
               
               
                   
                 NO: 315) 
               
               
                   
               
               
                 
                   Corynebacter 
                 
                 MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDSGLDPDEIKSAVTRLASSGI 
               
               
                   diphtheria  Cas9 
                 ARRTRRLYRRKRRRLQQLDKFIQRQGWPVIELEDYSDPLYPWKVRAELAASYIADEKE 
               
               
                   
                 RGEKLSVALRHIARHRGWRNPYAKVSSLYLPDGPSDAFKAIREEIKRASGQPVPETATV 
               
               
                   
                 GQMVTLCELGTLKLRGEGGVLSARLQQSDYAREIQEICRMQEIGQELYRKIIDVVFAAE 
               
               
                   
                 SPKGSASSRVGKDPLQPGKNRALKASDAFQRYRIAALIGNLRVRVDGEKRILSVEEKNL 
               
               
                   
                 VFDHLVNLTPKKEPEWVTIAEILGIDRGQLIGTATMTDDGERAGARPPTHDTNRSIVNS 
               
               
                   
                 RIAPLVDWWKTASALEQHAMVKALSNAEVDDFDSPEGAKVQAFFADLDDDVHAKLD 
               
               
                   
                 SLHLPVGRAAYSEDTLVRLTRRMLSDGVDLYTARLQEFGIEPSWTPPTPRIGEPVGNPA 
               
               
                   
                 VDRVLKTVSRWLESATKTWGAPERVIIEHVREGFVTEKRAREMDGDMRRRAARNAK 
               
               
                   
                 LFQEMQEKLNVQGKPSRADLWRYQSVQRQNCQCAYCGSPITFSNSEMDHIVPRAGQG 
               
               
                   
                 STNTRENLVAVCHRCNQSKGNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRST 
               
               
                   
                 DFKKFTKAVVERFQRATMDEEIDARSMESVAWMANELRSRVAQHFASHGTTVRVYR 
               
               
                   
                 GSLTAEARRASGISGKLKFFDGVGKSRLDRRHHAIDAAVIAFTSDYVAETLAVRSNLK 
               
               
                   
                 QSQAHRQEAPQWREFTGKDAEHRAAWRVWCQKMEKLSALLTEDLRDDRVVVMSNV 
               
               
                   
                 RLRLGNGSAHKETIGKLSKVKLSSQLSVSDIDKASSEALWCALTREPGFDPKEGLPANP 
               
               
                   
                 ERHIRVNGTHVYAGDNIGLEPVSAGSIALRGGYAELGSSEHHARVYKITSGKKPAFAML 
               
               
                   
                 RVYTIDLLPYRNQDLFSVELKPQTMSMRQAEKKLRDALATGNAEYLGWLVVDDELV 
               
               
                   
                 VDTSKIATDQVKAVEAELGTIRRWRVDGFFSPSKLRLRPLQMSKEGIKKESAPELSKIID 
               
               
                   
                 RPGWLPAVNKLFSDGNVTVVRRDSLGRVRLESTAHLPVTWKVQ (SEQ ID NO: 316) 
               
               
                   
               
               
                 
                   Streptococcus 
                 
                 MTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNAERRGFRGSRRLNRR 
               
               
                   pasteurianus  Cas9 
                 KKHRVKRVRDLFEKYGIVTDFRNLNLNPYELRVKGLTEQLKNEELFAALRTISKRRGIS 
               
               
                   
                 YLDDAEDDSTGSTDYAKSIDENRRLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEA 
               
               
                   
                 HRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRT 
               
               
                   
                 DYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNLKVSTETGK 
               
               
                   
                 LSIEQKESLVEFAKNTATLGPAKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYR 
               
               
                   
                 KLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQS 
               
               
                   
                 TAFNKGWHSFSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTD 
               
               
                   
                 EIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEK 
               
               
                   
                 DDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQGERCLYSGKPISIQELVHNS 
               
               
                   
                 NNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDY 
               
               
                   
                 VLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQSALRELGK 
               
               
                   
                 DTKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNPIVWVD 
               
               
                   
                 YGKNQVVDKQTGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNR 
               
               
                   
                 KVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQNGFDTFIKKYNKDKTQFLMYQKD 
               
               
                   
                 SLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLK 
               
               
                   
                 YYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTG 
               
               
                   
                 NYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDIASGEQEIYRFLSRTMPNVNHY 
               
               
                   
                 VELKPYDKEKFDNVQELVEALGEADKVGRCIKGLNKPNISIYKVRTDVLGNKYFVKK 
               
               
                   
                 KGDKPKLDFKNNKK (SEQ ID NO: 317) 
               
               
                   
               
               
                 
                   Neisseria cinerea 
                 
                 MAAFKPNPMNYILGLDIGIASVGWAIVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAA 
               
               
                 Cas9 
                 ARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAAL 
               
               
                   
                 DRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNTHALQTGDFR 
               
               
                   
                 TPAELALNKFEKESGHIRNQRGDYSHTFNRKDLQAELNLLFEKQKEFGNPHVSDGLKE 
               
               
                   
                 GIETLLMTQRPALSGDAVQKMLGHCIFEPTEPKAAKNTYTAERFVWLTKLNNLRILEQ 
               
               
                   
                 GSERPLTDTERATLMDEPYRKSKLTYAQARKLLDLDDTAFFKGLRYGKDNAEASTLM 
               
               
                   
                 EMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRVQPEIL 
               
               
                   
                 EALLKHISFDKFVQISLKALRRIVPLMEQGNRYDEACTEIYGDHYGKKNTEEKIYLPPIP 
               
               
                   
                 ADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENR 
               
               
                   
                 KDREKSAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEI 
               
               
                   
                 DHALPFSRTWDDSFNNKVLALGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSR 
               
               
                   
                 FPRSKKQRILLQKFDEDGFKERNLNDTRYINRFLCQFVADHMLLTGKGKRRVFASNGQ 
               
               
                   
                 ITNLLRGFWGLRKVRAENDRHHALDAVVVACSTIAMQQKITRFVRYKEMNAFDGKTI 
               
               
                   
                 DKETGEVLHQKAHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSS 
               
               
                   
                 RPEAVHKYVTPLFISRAPNRKMSGQGHMETVKSAKRLDEGISVLRVPLTQLKLKDLEK 
               
               
                   
                 MVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK 
               
               
                   
                 TGVWVHNHNGIADNATIVRVDVFEKGGKYYLVPIYSWQVAKGILPDRAVVQGKDEE 
               
               
                   
                 DWTVMDDSFEFKFVLYANDLIKLTAKKNEFLGYFVSLNRATGAIDIRTHDTDSTKGKN 
               
               
                   
                 GIFQSVGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR (SEQ ID NO: 318) 
               
               
                   
               
               
                 
                   Campylobacter lari 
                 
                 MRILGEDIGINSIGWAFVENDELKDCGVRIFTKAENPKNKESLALPRRNARSSRRRLKR 
               
               
                 Cas9 
                 RKARLIAIKRILAKELKLNYKDYVAADGELPKAYEGSLASVYELRYKALTQNLETKDL 
               
               
                   
                 ARVILHIAKHRGYMNKNEKKSNDAKKGKILSALKNNALKLENYQSVGEYFYKEFFQK 
               
               
                   
                 YKKNTKNFIKIRNTKDNYNNCVLSSDLEKELKLILEKQKEFGYNYSEDFINEILKVAFFQ 
               
               
                   
                 RPLKDFSHLVGACTFFEEEKRACKNSYSAWEEVALTKIINEIKSLEKISGEIVPTQTINEV 
               
               
                   
                 LNLILDKGSITYKKFRSCINLHESISFKSLKYDKENAENAKLIDFRKLVEFKKALGVHSL 
               
               
                   
                 SRQELDQISTHITLIKDNVKLKTVLEKYNLSNEQINNLLEIEFNDYINLSFKALGMILPLM 
               
               
                   
                 REGKRYDEACEIANLKPKTVDEKKDFLPAFCDSIFAHELSNPVVNRAISEYRKVLNALL 
               
               
                   
                 KKYGKVHKIHLELARDVGLSKKAREKIEKEQKENQAVNAWALKECENIGLKASAKNI 
               
               
                   
                 LKLKLWKEQKEICIYSGNKISIEHLKDEKALEVDHIYPYSRSFDDSFINKVLVFTKENQE 
               
               
                   
                 KLNKTPFEAFGKNIEKWSKIQTLAQNLPYKKKNKILDENFKDKQQEDFISRNLNDTRYI 
               
               
                   
                 ATLIAKYTKEYLNFLLLSENENANLKSGEKGSKIHVQTISGMLTSVLRHTWGFDKKDR 
               
               
                   
                 NNHLHHALDAIIVAYSINSIIKAFSDERKNQELLKARFYAKELTSDNYKHQVKFFEPFK 
               
               
                   
                 SFREKILSKIDEIFVSKPPRKRARRALHKDTFHSENKIIDKCSYNSKEGLQIALSCGRVRK 
               
               
                   
                 IGTKYVENDTIVRVDIFKKQNKFYAIPIYAMDFALGILPNKIVITGKDKNNNPKQWQTID 
               
               
                   
                 ESYEFCFSLYKNDLILLQKKNMQEPEFAYYNDFSISTSSICVEKHDNKFENLTSNQKLLF 
               
               
                   
                 SNAKEGSVKVESLGIQNLKVFEKYIITPLGDKIKADFQPRENISLKTSKKYGLR (SEQ ID 
               
               
                   
                 NO: 319) 
               
               
                   
               
               
                   T .  denticola  Cas9 
                 MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDLWGMRCFETAETAEVRRLH 
               
               
                   
                 RGARRRIERRKKRIKLLQELFSQEIAKTDEGFFQRMKESPFYAEDKTILQENTLENDKDF 
               
               
                   
                 ADKTYHKAYPTINHLIKAWIENKVKPDPRLLYLACHNIIKKRGHFLFEGDFDSENQFDT 
               
               
                   
                 SIQALFEYLREDMEVDIDADSQKVKEILKDSSLKNSEKQSRLNKILGLKPSDKQKKAIT 
               
               
                   
                 NLISGNKINFADLYDNPDLKDAEKNSISFSKDDFDALSDDLASILGDSFELLLKAKAVY 
               
               
                   
                 NCSVLSKVIGDEQYLSFAKVKIYEKHKTDLTKLKNVIKKHFPKDYKKVEGYNKNEKN 
               
               
                   
                 NNNYSGYVGVCKTKSKKLIINNSVNQEDFYKFLKTILSAKSEIKEVNDILTEIETGTFLP 
               
               
                   
                 KQISKSNAEIPYQLRKMELEKILSNAEKHFSFLKQKDEKGLSHSEKIIMLLTFKIPYYIGPI 
               
               
                   
                 NDNHKKFFPDRCWVVKKEKSPSGKTTPWNEFDHIDKEKTAEAFITSRINFCTYLVGES 
               
               
                   
                 VLPKSSLLYSEYTVLNEINNLQIIIDGKNICDIKLKQKIYEDLFKKYKKITQKQISTFIKHE 
               
               
                   
                 GICNKTDEVIILGIDKECTSSLKSYIELKNIFGKQVDEISTKNMLEEIIRWATIYDEGEGKT 
               
               
                   
                 ILKTKIKAEYGKYCSDEQIKKILNLKFSGWGRLSRKFLETVTSEMPGFSEPVNIITAMRE 
               
               
                   
                 TQNNLMELLSSEFTFTENIKKINSGEEDAEKQFSYDGLVKPLFLSPSVIUMLWQTLKLV 
               
               
                   
                 KEISHITQAPPKKIFIEMAKGAELEPARTKTRLKILQDLYNNCKNDADAFSSEIKDLSGKI 
               
               
                   
                 ENEDNLRLRSDKLYLYYTQLGKCMYCGKPIEIGHVFDTSNYDIDHIYPQSKIKDDSISNR 
               
               
                   
                 VLVCSSCNKNKEDKYPLKSEIQSKQRGEWNFLQRNNFISLEKLNRLTRATPISDDETAK 
               
               
                   
                 FIARQLVETRQATKVAAKVLEKMFPETKIVYSKAETVSMFRNKFDIVKCREINDFHHA 
               
               
                   
                 HDAYLNIVVGNVYNIKFTNNPWNFIKEKRDNPKIADTYNYYKVEDYDVKRNNITAWE 
               
               
                   
                 KGKTIITVKDMLKRNTPIYTRQAACKKGELFNQTIMKKGLGQHPLKKEGPFSNISKYGG 
               
               
                   
                 YNKVSAAYYTLIEYEEKGNKIRSLETIPLYLVKDIQKDQDVLKSYLTDLLGKKEFKILVP 
               
               
                   
                 KIKINSLLKINGFPCHITGKTNDSFLLRPAVQFCCSNNEVLYFKKIIRFSEIRSQREKIGKTI 
               
               
                   
                 SPYEDLSFRSYIKENLWKKTKNDEIGEKEFYDLLQKKNLEIYDMLLTKHKDTIYKKRPN 
               
               
                   
                 SATIDILVKGKEKFKSLIIENQFEVILEILKLFSATRNVSDLQHIGGSKYSGVAKIGNKISS 
               
               
                   
                 LDNCILIYQSITGIFEKRIDLLKV (SEQ ID NO: 320) 
               
               
                   
               
               
                   S .  mutans  Cas9 
                 MKKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGALLFDSGN 
               
               
                   
                 TAEDRRLKRTARRRYTRRRNRILYLQEIFSEEMGKVDDSFFHRLEDSFLVTEDKRGERH 
               
               
                   
                 PIEGNLEEEVKYHENEPTIYHLRQYLADNPEKVDLRLVYLALAHIIKERGHFLIEGKEDT 
               
               
                   
                 RNNDVQRLFQEFLAVYDNTFENSSLQEQNVQVEEILTDKISKSAKKDRVLKLFPNEKSN 
               
               
                   
                 GRFAEFLKLIVGNQADFKKHFELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAK 
               
               
                   
                 KLYDSILLSGILTVTDVGTKAPLSASMIQRYNEHQMDLAQLKQFIRQKLSDKYNEVFSD 
               
               
                   
                 VSKDGYAGYIDGKTNQEAFYKYLKGLLNKIEGSGYFLDKIEREDFLRKQRTFDNGSIPH 
               
               
                   
                 QIHLQEMRAIIRRQAEFYPFLADNQDRIEKLLTFRIPYYVGPLARGKSDFAWLSRKSAD 
               
               
                   
                 KITPWNFDEIVDKESSAEAFINRMTNYDLYLPNQKVLPKHSLLYEKFTVYNELTKVKY 
               
               
                   
                 KTEQGKTAFFDANMKQEIFDGVFKVYRKVTKDKLMDFLEKEFDEFRIVDLTGLDKEN 
               
               
                   
                 KVFNASYGTYHDLCKILDKDELDNSKNEKILEDIVLTLTLFEDREMIRKRLENYSDLLT 
               
               
                   
                 KEQVKKLERRHYTGWGRLSAELIHGIRNKESRKTILDYLIDDGNSNRNFMQLINDDALS 
               
               
                   
                 FKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDELVKIMGHQPENIVVEMA 
               
               
                   
                 RENQFTNQGRRNSQQRLKGLTDSIKEFGSQILKEHPVENSQLQNDRLFLYYLQNGRDM 
               
               
                   
                 YTGEELDIDYLSQYDIDHIIPQAFIKDNSIDNRVLTSSKENRGKSDDVPSKDVVRKMKSY 
               
               
                   
                 WSKLLSAKLITQRKFDNLTKAERGGLTDDDKAGFIKRQLVETRQITKHVARILDERFNT 
               
               
                   
                 ETDENNKKIRQVKIVILKSNLVSNERKEFELYKVREINDYHHAHDAYLNAVIGKALLG 
               
               
                   
                 VYPQLEPEFVYGDYPHFHGHKENKATAKKFFYSNIMNFFKKDDVRTDKNGEIIWKKD 
               
               
                   
                 EHISNIKKVLSYPQVNIVKKVEEQTGGFSKESILPKGNSDKLIPRKTKKFYWDTKKYGG 
               
               
                   
                 FDSPIVAYSILVIADIEKGKSKKLKTVKALVGVTIMEKMTFERDPVAFLERKGYRNVQE 
               
               
                   
                 ENIIKLPKYSLFKLENGRKRLLASARELQKGNEIVLPNHLGTLLYHAKNIHKVDEPKHL 
               
               
                   
                 DYVDKHKDEFKELLDVVSNFSKKYTLAEGNLEKIKELYAQNNGEDLKELASSFINLLT 
               
               
                   
                 FTAIGAPATFKFFDKNIDRKRYTSTTEILNATLIHQSITGLYETRIDLNKL GGD (SEQ ID 
               
               
                   
                 NO: 321) 
               
               
                   
               
               
                   S .  thermophilus   
                 MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGIT 
               
               
                 CRISPR 3 Cas9 
                 AEGRRLKRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYP 
               
               
                   
                 IFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNS 
               
               
                   
                 KNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGI 
               
               
                   
                 FSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKL 
               
               
                   
                 YDAILLSGELTVTDNETEAPLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVEKDDT 
               
               
                   
                 KNGYAGYIDGKINQEDFYVYLKKLLAEFEGADYFLEKIDREDFLRKQRTEDNGSIPYQI 
               
               
                   
                 HLQEMRAILDKQAKFYPFLAKNKERIEKILITRIPYYVGPLARGNSDFAWSIRKRNEKIT 
               
               
                   
                 PWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAES 
               
               
                   
                 MRDYQFLDSKQKKDIVRLYEKDKRKVTDKDHEYLHAIYGYDGIELKGIEKQENSSLST 
               
               
                   
                 YHDLLNIINDKEFLDDSSNEAHEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRH 
               
               
                   
                 YTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQKAQIIG 
               
               
                   
                 DEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMARENQYTNQ 
               
               
                   
                 GKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYT 
               
               
                   
                 GDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDVPSLEVVKKRKTFW 
               
               
                   
                 YQLLKSKLISQRKFDNLTKAERGGLSPEDKAGFIQRQLVETRQIIKHVARLLDEKENNK 
               
               
                   
                 KDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVVASALLKK 
               
               
                   
                 YPKLEPEFVYGDYPKYNSFRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNE 
               
               
                   
                 ETGESVWNKESDLATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKP 
               
               
                   
                 NSNENLVGAKEYLDPKKYGGYAGISNSFTVLVKGTIEKGAKKKITNVLEFQGISILDRIN 
               
               
                   
                 YRKDKLNELLEKGYKDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLS 
               
               
                   
                 QKFVKLLYHAKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNS 
               
               
                   
                 AFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLK 
               
               
                   
                 DATLIHQSVTGLYETRIDLAKLGEG (SEQ ID NO: 322) 
               
               
                   
               
               
                   C .  jejuni  Cas9 
                 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLAR 
               
               
                   
                 RKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFA 
               
               
                   
                 RVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKEN 
               
               
                   
                 SKEFTNVRNKKESYERCIAQSELKDELKLIFKKQREFGESFSKKFEEEVLSVAFYKRALK 
               
               
                   
                 DFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNIEGILYTKDDLNALLN 
               
               
                   
                 EVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEI 
               
               
                   
                 AKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKY 
               
               
                   
                 DEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYG 
               
               
                   
                 KVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLF 
               
               
                   
                 KEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQT 
               
               
                   
                 PFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARL 
               
               
                   
                 VLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRN 
               
               
                   
                 NHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGF 
               
               
                   
                 RQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVN 
               
               
                   
                 GKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILM 
               
               
                   
                 DENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKIL 
               
               
                   
                 FKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK (SEQ ID NO: 323) 
               
               
                   
               
               
                   P .  multocida  Cas9 
                 MQTTNLSYILGLDLGIASVGWAVVEINENEDPIGLIDVGVRIFERAEVPKTGESLALSRR 
               
               
                   
                 LARSTRRLIRRRAHRLLLAKRFLKREGILSTIDLEKGLPNQAWELRVAGLERRLSAIEW 
               
               
                   
                 GAVLLHLIKHRGYLSKRKNESQTNNKELGALLSGVAQNHQLLQSDDYRTPAELALKK 
               
               
                   
                 FAKEEGHIRNQRGAYTHTFNRLDLLAELNLLFAQQHQFGNPHCKEHIQQYMTELLMW 
               
               
                   
                 QKPALSGEAILKMLGKCTHEKNEFKAAKHTYSAERFVWLTKLNNLRILEDGAERALNE 
               
               
                   
                 EERQLLINHPYEKSKLTYAQVRKLLGLSEQAIFKHLRYSKENAESATFMELKAWHAIR 
               
               
                   
                 KALENQGLKDTWQDLAKKPDLLDEIGTAFSLYKTDEDIQQYLTNKVPNSVINALLVSL 
               
               
                   
                 NFDKFIELSLKSLRKILPLMEQGKRYDQACREIYGHHYGEANQKTSQLLPAIPAQEIRNP 
               
               
                   
                 VVLRTLSQARKVINAIIRQYGSPARVHIETGRELGKSFKERREIQKQQEDNRTKRESAV 
               
               
                   
                 QKFKELFSDFSSEPKSKDILKFRLYEQQHGKCLYSGKEINIHRLNEKGYVEIDHALPFSR 
               
               
                   
                 TWDDSFNNKVLVLASENQNKGNQTPYEWLQGKINSERWKNFVALVLGSQCSAAKKQ 
               
               
                   
                 RLLTQVIDDNKFIDRNLNDTRYIARFLSNYIQENLLLVGKNKKNVFTPNGQITALLRSR 
               
               
                   
                 WGLIKARENNNRHHALDAIVVACATPSMQQKITRFIRFKEVHPYKIENRYEMVDQESG 
               
               
                   
                 EIISPHFPEPWAYFRQEVNIRVFDNHPDTVLKEMLPDRPQANHQFVQPLFVSRAPTRKM 
               
               
                   
                 SGQGHMETIKSAKRLAEGISVLRIPLTQLKPNLLENMVNKEREPALYAGLKARLAEFN 
               
               
                   
                 QDPAKAFATPFYKQGGQQVKAIRVEQVQKSGVLVRENNGVADNASIVRTDVFIKNNK 
               
               
                   
                 FFLVPIYTWQVAKGILPNKAIVAHKNEDEWEEMDEGAKFKFSLFPNDLVELKTKKEYF 
               
               
                   
                 FGYYIGLDRATGNISLKEHDGEISKGKDGVYRVGVKLALSFEKYQVDELGKNRQICRP 
               
               
                   
                 QQRQPVR (SEQ ID NO: 324) 
               
               
                   
               
               
                   F .  novicida  Cas9 
                 MNFKILPIAIDLGVKNTGVFSAFYQKGTSLERLDNKNGKVYELSKDSYTLLMNNRTAR 
               
               
                   
                 RHQRRGIDRKQLVKRLFKLIWTEQLNLEWDKDTQQAISFLFNRRGFSFITDGYSPEYLN 
               
               
                   
                 IVPEQVKAILMDIFDDYNGEDDLDSYLKLATEQESKISEIYNKLMQKILEFKLMKLCTDI 
               
               
                   
                 KDDKVSTKTLKEITSYEFELLADYLANYSESLKTQKFSYTDKQGNLKELSYYHHDKYN 
               
               
                   
                 IQEFLKRHATINDRILDILLTDDLDIWNFNFEKFDFDKNEEKLQNQEDKDHIQAHLHHF 
               
               
                   
                 VFAVNKIKSEMASGGRHRSQYFQEITNVLDENNHQEGYLKNFCENLHNKKYSNLSVK 
               
               
                   
                 NLVNLIGNLSNLELKPLRKYFNDKIHAKADHWDEQKFTETYCHWILGEWRVGVKDQD 
               
               
                   
                 KKDGAKYSYKDLCNELKQKVTKAGLVDFLLELDPCRTIPPYLDNICNRKPPKCQSLILN 
               
               
                   
                 PKFLDNQYPNWQQYLQELKKLQSIQNYLDSFETDLKVLKSSKDQPYFVEYKSSNQQIA 
               
               
                   
                 SGQRDYKDLDARILQFIFDRVKASDELLLNEIYFQAKKLKQKASSELEKLESSKKLDEVI 
               
               
                   
                 ANSQLSQILKSQHTNGIFEQGTFLHLVCKYYKQRQRARDSRLYIMPEYRYDKKLHKYN 
               
               
                   
                 NTGRFDDDNQLLTYCNHKPRQKRYQLLNDLAGVLQVSPNFLKDKIGSDDDLFISKWL 
               
               
                   
                 WHIRGFKKACEDSLKIQKDNRGLLNHKINIARNTKGKCEKEIFNLICKIEGSEDKKGNY 
               
               
                   
                 KHGLAYELGVLLFGEPNEASKPEFDRKIKKFNSIYSFAQIQQIAFAERKGNANTCAVCS 
               
               
                   
                 ADNAHRMQQIKITEPVEDNKDKIILSAKAQRLPAIPTRIVDGAVKKMATILAKNIVDDN 
               
               
                   
                 WQNIKQVLSAKHQLHIPIITESNAFEFEPALADVKGKSLKDRRKKALERISPENIFKDKN 
               
               
                   
                 NRIKEFAKGISAYSGANLTDGDFDGAKEELDHIIPRSHKKYGTLNDEANLICVTRGDNK 
               
               
                   
                 NKGNRIFCLRDLADNYKLKQFETTDDLEIEKKIADTIWDANKKDFKFGNYRSFINLTPQ 
               
               
                   
                 EQKAFRHALFLADENPIKQAVIRAINNRNRTFVNGTQRYFAEVLANNIYLRAKKENLN 
               
               
                   
                 TDKISFDYFGIPTIGNGRGIAEIRQLYEKVDSDIQAYAKGDKPQASYSHLIDAMLAFCIA 
               
               
                   
                 ADEHRNDGSIGLEIDKNYSLYPLDKNTGEVFTKDIFSQIKITDNEFSDKKLVRKKAIEGF 
               
               
                   
                 NTHRQMTRDGIYAENYLPILIHKELNEVRKGYTWKNSEEIKIFKGKKYDIQQLNNLVY 
               
               
                   
                 CLKFVDKPISIDIQISTLEELRNILTINNIAATAEYYYINLKTQKLHEYYIENYNTALGYK 
               
               
                   
                 KYSKEMEFLRSLAYRSERVKIKSIDDVKQVLDKDSNFIIGKITLPFKKEWQRLYREWQN 
               
               
                   
                 TTIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEGKFLVKRKTWDNNFIYQILNDS 
               
               
                   
                 DSRADGTKPFIPAFDISKNEIVEAIIDSFTSKNIFWLPKNIELQKVDNKNIFAIDTSKWFEV 
               
               
                   
                 ETPSDLRDIGIATIQYKIDNNSRPKVRVKLDYVIDDDSKINYFMNHSLLKSRYPDKVLEI 
               
               
                   
                 LKQSTIIEFESSGFNKTIKEMLGMKLAGIYNETSNN (SEQ ID NO: 325) 
               
               
                   
               
               
                 
                   Lactobacillus 
                 
                 MKVNNYHIGLDIGTSSIGWVAIGKDGKPLRVKGKTAIGARLFQEGNPAADRRMFRTTR 
               
               
                   buchneri  Cas9 
                 RRLSRRKWRLKLLEEIFDPYITPVDSTFFARLKQSNLSPKDSRKEFKGSMLFPDLTDMQ 
               
               
                   
                 YHKNYPTIYHLRHALMTQDKKFDIRMVYLAIHHIVKYRGNFLNSTPVDSFKASKVDFV 
               
               
                   
                 DQFKKLNELYAAINPEESFKINLANSEDIGHQFLDPSIRKFDKKKQIPKIVPVMMNDKVT 
               
               
                   
                 DRLNGKIASEIIHAILGYKAKLDVVLQCTPVDSKPWALKFDDEDIDAKLEKILPEMDEN 
               
               
                   
                 QQSIVAILQNLYSQVTLNQIVPNGMSLSESMIEKYNDHHDHLKLYKKLIDQLADPKKK 
               
               
                   
                 AVLKKAYSQYVGDDGKVIEQAEFWSSVKKNLDDSELSKQIMDLIDAEKFMPKQRTSQ 
               
               
                   
                 NGVIPHQLHQRELDEIIEHQSKYYPWLVEINPNKHDLHLAKYKIEQLVAFRVPYYVGP 
               
               
                   
                 MITPKDQAESAETVFSWMERKGTETGQITPWNFDEKVDRKASANRFIKRMTTKDTYLI 
               
               
                   
                 GEDVLPDESLLYEKFKVLNELNMVRVNGKLLKVADKQAIFQDLFENYKHVSVKKLQN 
               
               
                   
                 YIKAKTGLPSDPEISGLSDPEHFNNSLGTYNDFKKLFGSKVDEPDLQDDFEKIVEWSTVF 
               
               
                   
                 EDKKILREKLNEITWLSDQQKDVLESSRYQGWGRLSKKLLTGIVNDQGERIIDKLWNT 
               
               
                   
                 NKNFMQIQSDDDFAKRIHEANADQMQAVDVEDVLADAYTSPQNKKAIRQVVKVVDD 
               
               
                   
                 IQKAMGGVAPKYISIEFTRSEDRNPRRTISRQRQLENTLKDTAKSLAKSINPELLSELDN 
               
               
                   
                 AAKSKKGLTDRLYLYFTQLGKDIYTGEPINIDELNKYDIDHILPQAFIKDNSLDNRVLVL 
               
               
                   
                 TAVNNGKSDNVPLRMFGAKMGHFWKQLAEAGLISKRKLKNLQTDPDTISKYAMEGFI 
               
               
                   
                 RRQLVETSQVIKLVANILGDKYRNDDTKIIEITARMNHQMRDEFGFIKNREINDYHHAF 
               
               
                   
                 DAYLTAFLGRYLYHRYIKLRPYFVYGDFKKFREDKVTMRNFNFLHDLTDDTQEKIAD 
               
               
                   
                 AETGEVIWDRENSIQQLKDVYHYKFMLISHEVYTLRGAMFNQTVYPASDAGKRKLIPV 
               
               
                   
                 KADRPVNVYGGYSGSADAYMAIVRIHNKKGDKYRVVGVPMRALDRLDAAKNVSDA 
               
               
                   
                 DFDRALKDVLAPQLTKTKKSRKTGEITQVIEDIEIVLGKVMYRQLMIDGDKKFMLGSS 
               
               
                   
                 TYQYNAKQLVLSDQSVKTLASKGRLDPLQESMDYNNVYTEILDKVNQYFSLYDMNKF 
               
               
                   
                 RHKLNLGFSKFISFPNHNVLDGNTKVSSGKREILQEILNGLHANPTFGNLKDVGITTPFG 
               
               
                   
                 QLQQPNGILLSDETKIRYQSPTGLFERTVSLKDL (SEQ ID NO: 326) 
               
               
                   
               
               
                 
                   Listeria innocua 
                 
                 MKKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKIAGDSEKKQIKKNFWGVRLFDEGQ 
               
               
                 Cas9 
                 TAADRRMARTARRRIERRRNRISYLQGIFAEEMSKTDANFFCRLSDSFYVDNEKRNSR 
               
               
                   
                 HPFFATIEEEVEYHKNYPTIYHLREELVNSSEKADLRLVYLALAHIIKYRGNFLIEGALD 
               
               
                   
                 TQNTSVDGIYKQFIQTYNQVFASGIEDGSLKKLEDNKDVAKILVEKVTRKEKLERILKL 
               
               
                   
                 YPGEKSAGMFAQFISLIVGSKGNFQKPFDLIEKSDIECAKDSYEEDLESLLALIGDEYAE 
               
               
                   
                 LFVAAKNAYSAVVLSSIITVAETETNAKLSASMIERFDTHEEDLGELKAFIKLHLPKHYE 
               
               
                   
                 EIFSNTEKHGYAGYIDGKTKQADFYKYMKMTLENIEGADYFIAKIEKENFLRKQRTFD 
               
               
                   
                 NGAIPHQLHLEELEAILHQQAKYYPFLKENYDKIKSLVTFRIPYFVGPLANGQSEFAWL 
               
               
                   
                 TRKADGEIRPWNIEEKVDFGKSAVDFIEKMTNKDTYLPKENVLPKHSLCYQKYLVYNE 
               
               
                   
                 LTKVRYINDQGKTSYFSGQEKEQIFNDLFKQKRKVKKKDLELFLRNMSHVESPTIEGLE 
               
               
                   
                 DSFNSSYSTYHDLLKVGIKQEILDNPVNTEMLENIVKILTVFEDKRMIKEQLQQFSDVL 
               
               
                   
                 DGVVLKKLERRHYTGWGRLSAKLLMGIRDKQSHLTILDYLMNDDGLNRNLMQLINDS 
               
               
                   
                 NLSFKSIIEKEQVTTADKDIQSIVADLAGSPAIKKGILQSLKIVDELVSVMGYPPQTIVVE 
               
               
                   
                 MARENQTTGKGKNNSRPRYKSLEKAIKEFGSQILKEHPTDNQELRNNRLYLYYLQNGK 
               
               
                   
                 DMYTGQDLDIHNLSNYDIDHIVPQSFITDNSIDNLVLTSSAGNREKGDDVPPLEIVRKRK 
               
               
                   
                 VFWEKLYQGNLMSKRKFDYLTKAERGGLTEADKARFIHRQLVETRQITKNVANILHQ 
               
               
                   
                 RFNYEKDDHGNTMKQVRIVTLKSALVSQFRKQFQLYKVRDVNDYHHAHDAYLNGVV 
               
               
                   
                 ANTLLKVYPQLEPEFVYGDYHQFDWFKANKATAKKQFYTNIMLFFAQKDRIIDENGEI 
               
               
                   
                 LWDKKYLDTVKKVMSYRQMNIVKKTEIQKGEFSKATIKPKGNSSKLIPRKTNWDPMK 
               
               
                   
                 YGGLDSPNMAYAVVIEYAKGKNKLVFEKKIIRVTIMERKAFEKDEKAFLEEQGYRQPK 
               
               
                   
                 VLAKLPKYTLYECEEGRRRMLASANEAQKGNQQVLPNHLVTLLHHAANCEVSDGKSL 
               
               
                   
                 DYIESNREMFAELLAHVSEFAKRYTLAEANLNKINQLFEQNKEGDIKAIAQSFVDLMAF 
               
               
                   
                 NAMGAPASFKFFETTIERKRYNNLKELLNSTIIYQSITGLYESRKRLDD (SEQ ID NO: 
               
               
                   
                 327) 
               
               
                   
               
               
                   L .  pneumophilia   
                 MESSQILSPIGIDLGGKFTGVCLSHLEAFAELPNHANTKYSVILIDHNNFQLSQAQRRAT 
               
               
                 Cas9 
                 RHRVRNKKRNQFVKRVALQLFQHILSRDLNAKEETALCHYLNNRGYTYVDTDLDEYI 
               
               
                   
                 KDETTINLLKELLPSESEHNFIDWFLQKMQSSEFRKILVSKVEEKKDDKELKNAVKNIK 
               
               
                   
                 NFITGFEKNSVEGHRIIRKVYFENIKSDITKDNQLDSIKKKIPSVCLSNLLGHLSNLQWK 
               
               
                   
                 NLHRYLAKNPKQFDEQTFGNEFLRMLKNFRHLKGSQESLAVRNLIQQLEQSQDYISILE 
               
               
                   
                 KTPPEITIPPYEARTNTGMEKDQSLLLNPEKLNNLYPNWRNLIPGIIDAHPFLEKDLEHT 
               
               
                   
                 KLRDRKRIISPSKQDEKRDSYILQRYLDLNKKIDKFKIKKQLSFLGQGKQLPANLIETQK 
               
               
                   
                 EMETHFNSSLVSVLIQIASAYNKEREDAAQGIWFDNAFSLCELSNINPPRKQKILPLLVG 
               
               
                   
                 AILSEDFINNKDKWAKFKIFWNTHKIGRTSLKSKCKEIEEARKNSGNAFKIDYEEALNH 
               
               
                   
                 PEHSNNKALIKIIQTIPDIIQAIQSHLGHNDSQALIYHNPFSLSQLYTILETKRDGFHKNCV 
               
               
                   
                 AVTCENYWRSQKTEIDPEISYASRLPADSVRPFDGVLARMMQRLAYEIAMAKWEQIK 
               
               
                   
                 HIPDNSSLLIPIYLEQNRFEFEESFKKIKGSSSDKTLEQAIEKQNIQWEEKFQRIINASMNI 
               
               
                   
                 CPYKGASIGGQGEIDHIYPRSLSKKHFGVIFNSEVNLIYCSSQGNREKKEEHYLLEHLSP 
               
               
                   
                 LYLKHQFGTDNVSDIKNFISQNVANIKKYISFHLLTPEQQKAARHALFLDYDDEAFKTI 
               
               
                   
                 TKFLMSQQKARVNGTQKFLGKQIMEFLSTLADSKQLQLEFSIKQITAEEVHDHRELLSK 
               
               
                   
                 QEPKLVKSRQQSFPSHAIDATLTMSIGLKEFPQFSQELDNSWFINHLMPDEVHLNPVRS 
               
               
                   
                 KEKYNKPNISSTPLFKDSLYAERFIPVWVKGETFAIGFSEKDLFEIKPSNKEKLFTLLKTY 
               
               
                   
                 STKNPGESLQELQAKSKAKWLYFPINKTLALEFLHHYFHKEIVTPDDTTVCHFINSLRY 
               
               
                   
                 YTKKESITVKILKEPMPVLSVKFESSKKNVLGSFKHTIALPATKDWERLFNHPNFLALK 
               
               
                   
                 ANPAPNPKEFNEFIRKYFLSDNNPNSDIPNNGHNIKPQKHKAVRKVFSLPVIPGNAGTM 
               
               
                   
                 MRIRRKDNKGQPLYQLQTIDDTPSMGIQINEDRLVKQEVLMDAYKTRNLSTIDGINNSE 
               
               
                   
                 GQAYATFDNWLTLPVSTFKPEIIKLEMKPHSKTRRYIRITQSLADFIKTIDEALMIKPSDS 
               
               
                   
                 IDDPLNMPNEIVCKNKLFGNELKPRDGKMKIVSTGKIVTYEFESDSTPQWIQTLYVTQL 
               
               
                   
                 KKQP (SEQ ID NO: 328) 
               
               
                   
               
               
                   N .  lactamica  Cas9 
                 MAAFKPNPMNYILGLDIGIASVGWAMVEVDEEENPIRLIDLGVRVFERAEVPKTGDSL 
               
               
                   
                 AMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQDADFDENGLVKSLPNTPWQLRA 
               
               
                   
                 AALDRKLTCLEWSAVLLHLVKIIRGYLSQRKNEGETADKELGALLKGVADNAHALQT 
               
               
                   
                 GDFRIPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELNLLFEKQKEFGNPHVSD 
               
               
                   
                 GLKEDIETLLMAQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLR 
               
               
                   
                 ILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEAS 
               
               
                   
                 TLMEMKAYHAISRALEKEGLKDKKSPLNLSTELQDEIGTAFSLFKTDKDITGRLKDRVQ 
               
               
                   
                 PEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYCKKNAEEKIYL 
               
               
                   
                 PPIPADEIRNPVVLRALSQARKVINCVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQE 
               
               
                   
                 ENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKG 
               
               
                   
                 YVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARV 
               
               
                   
                 ETSRFPRSKKQRILLQKFDEEGFKERNLNDTRYVNRFLCQFVADHILLTGKGKRRVFAS 
               
               
                   
                 NGQITNLLRGFWGLRKVRTENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD 
               
               
                   
                 GKTIDKETGEVLHQKAHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAE 
               
               
                   
                 KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGISVLRVPLTQLKLK 
               
               
                   
                 GLEKMVNREREPKLYDALKAQLETHKDDPAKAFAEPFYKYDKAGSRTQQVKAVRIEQ 
               
               
                   
                 VQKTGVWVRNHNGIADNATMVRVDVFEKGGKYYLVPIYSWQVAKGILPDRAVVAFK 
               
               
                   
                 DEEDWTVMDDSFEFRFVLYANDLIKLTAKKNEFLGYFVSLNRATGAIDIRTHDTDSTK 
               
               
                   
                 GKNGIFQSVGVKTALSFQKNQIDELGKEIRPCRLKKRPPVR (SEQ ID NO: 329) 
               
               
                   
               
               
                   N .  meningitides   
                 MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLA 
               
               
                 Cas9 
                 MARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAA 
               
               
                   
                 ALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGD 
               
               
                   
                 FRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLK 
               
               
                   
                 EGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILE 
               
               
                   
                 QGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTL 
               
               
                   
                 MEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQPEI 
               
               
                   
                 LEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPI 
               
               
                   
                 PADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEEN 
               
               
                   
                 RKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYV 
               
               
                   
                 EIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVET 
               
               
                   
                 SRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASN 
               
               
                   
                 GQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDG 
               
               
                   
                 KTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKL 
               
               
                   
                 SSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD 
               
               
                   
                 LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQ 
               
               
                   
                 VQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQG 
               
               
                   
                 KDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKI 
               
               
                   
                 GKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR (SEQ ID NO: 330) 
               
               
                   
               
               
                   B .  longum  Cas9 
                 MLSRQLLGASHLARPVSYSYNVQDNDVHCSYGERCFMRGKRYRIGIDVGLNSVGLAA 
               
               
                   
                 VEVSDENSPVRLLNAQSVIHDGGVDPQKNKEAITRKNMSGVARRTRRMRRRKRERLH 
               
               
                   
                 KLDMLLGKFGYPVIEPESLDKPFEEWHVRAELATRYIEDDELRRESISIALRHMARHRG 
               
               
                   
                 WRNPYRQVDSLISDNPYSKQYGELKEKAKAYNDDATAAEEESTPAQLVVAMLDAGY 
               
               
                   
                 AEAPRLRWRTGSKKPDAEGYLPVRLMQEDNANELKQIFRVQRVPADEWKPLFRSVFY 
               
               
                   
                 AVSPKGSAEQRVGQDPLAPEQARALKASLAFQEYRIANVITNLRIKDASAELRKLTVDE 
               
               
                   
                 KQSIYDQLVSPSSEDITWSDLCDFLGFKRSQLKGVGSLTEDGEERISSRPPRLTSVQRIYE 
               
               
                   
                 SDNKIRKPLVAWWKSASDNEHEAMIRLLSNTVDIDKVREDVAYASAIEFIDGLDDDAL 
               
               
                   
                 TKLDSVDLPSGRAAYSVETLQKLTRQMLTTDDDLHEARKTLFNVTDSWRPPADPIGEP 
               
               
                   
                 LGNPSVDRVLKNVNRYLMNCQQRWGNPVSVNIEHVRSSFSSVAFARKDKREYEKNNE 
               
               
                   
                 KRSIFRSSLSEQLRADEQMEKVRESDLRRLEAIQRQNGQCLYCGRTITFRTCEMDHIVP 
               
               
                   
                 RKGVGSTNTRTNFAAVCAECNRMKSNTPFAIWARSEDAQTRGVSLAEAKKRVTMFTF 
               
               
                   
                 NPKSYAPREVKAFKQAVIARLQQTEDDAAIDNRSIESVAWMADELHRRIDWYFNAKQ 
               
               
                   
                 YVNSASIDDAEAETMKTTVSVFQGRVTASARRAAGIEGKIHFIGQQSKTRLDRRHHAV 
               
               
                   
                 DASVIAMMNTAAAQTLMERESLRESQRLIGLMPGERSWKEYPYEGTSRYESFHLWLD 
               
               
                   
                 NMDVLLELLNDALDNDRIAVMQSQRYVLGNSIAHDATIHPLEKVPLGSAMSADLIRRA 
               
               
                   
                 STPALWCALTRLPDYDEKEGLPEDSHREIRVHDTRYSADDEMGFFASQAAQIAVQEGS 
               
               
                   
                 ADIGSAIHHARVYRCWKTNAKGVRKYFYGMIRVFQTDLLRACHDDLFTVPLPPQSISM 
               
               
                   
                 RYGEPRVVQALQSGNAQYLGSLVVGDEIEMDFSSLDVDGQIGEYLQFFSQFSGGNLAW 
               
               
                   
                 KHWVVDGFFNQTQLRIRPRYLAAEGLAKAFSDDVVPDGVQKIVTKQGWLPPVNTASK 
               
               
                   
                 TAVRIVRRNAFGEPRLSSAHHMPCSWQWRHE (SEQ ID NO: 331) 
               
               
                   
               
               
                   A .  muciniphila  Cas9 
                 MSRSLTFSFDIGYASIGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFKRREYRRLRR 
               
               
                   
                 NIRSRRVRIERIGRLLVQAQIITPEMKETSGHPAPFYLASEALKGHRTLAPIELWHVLRW 
               
               
                   
                 YAHNRGYDNNASWSNSLSEDGGNGEDTERVKHAQDLMDKHGTATMAETICRELKLE 
               
               
                   
                 EGKADAPMEVSTPAYKNLNTAFPRLIVEKEVRRILELSAPLIPGLTAEIIELIAQHHPLTT 
               
               
                   
                 EQRGVLLQHGIKLARRYRGSLLFGQLIPRFDNRIISRCPVTWAQVYEAELKKGNSEQSA 
               
               
                   
                 RERAEKLSKVPTANCPEFYEYRMARILCNIRADGEPLSAEIRRELMNQARQEGKLTKAS 
               
               
                   
                 LEKAISSRLGKETETNVSNYFTLHPDSEEALYLNPAVEVLQRSGIGQILSPSVYRIAANR 
               
               
                   
                 LRRGKSVTPNYLLNLLKSRGESGEALEKKIEKESKKKEADYADTPLKPKYATGRAPYA 
               
               
                   
                 RTVLKKVVEEILDGEDPTRPARGEAHPDGELKAHDGCLYCLLDTDSSVNQHQKERRL 
               
               
                   
                 DTMTNNHLVRHRMLILDRLLKDLIQDFADGQKDRISRVCVEVGKELTTFSAMDSKKIQ 
               
               
                   
                 RELTLRQKSHTDAVNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGATYGDHELE 
               
               
                   
                 NLELEHIVPHSFRQSNALSSLVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLHIC 
               
               
                   
                 SLNNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQNHEAMKEIGMTE 
               
               
                   
                 GMMTQSSHLMKLACKSIKTSLPDAHIDMIPGAVTAEVRKAWDVFGVFKELCPEAADP 
               
               
                   
                 DSGKILKENLRSLTHLHHALDACVLGLIPYIIPAHHNGLLRRVLAMRRIPEKLIPQVRPV 
               
               
                   
                 ANQRHYVLNDDGRMMLRDLSASLKENIREQLMEQRVIQHVPADMGGALLKETMQRV 
               
               
                   
                 LSVDGSGEDAMVSLSKKKDGKKEKNQVKASKLVGVFPEGPSKLKALKAAIEIDGNYG 
               
               
                   
                 VALDPKPVVIRHIKVFKRIMALKEQNGGKPVRILKKGMLIHLTSSKDPKHAGVWRIESI 
               
               
                   
                 QDSKGGVKLDLQRAHCAVPKNKTHECNWREVDLISLLKKYQMKRYPTSYTGTPR 
               
               
                   
                 (SEQ ID NO: 332) 
               
               
                   
               
               
                   O .  laneus  Cas9 
                 METTLGIDLGTNSIGLALVDQEEHQILYSGVRIFPEGINKDTIGLGEKEESRNATRRAKR 
               
               
                   
                 QMRRQYFRKKLRKAKLLELLIAYDMCPLKPEDVRRWKNWDKQQKSTVRQFPDTPAF 
               
               
                   
                 REWLKQNPYELRKQAVTEDVTRPELGRILYQMIQRRGFLSSRKGKEEGKIFTGKDRMV 
               
               
                   
                 GIDETRKNLQKQTLGAYLYDIAPKNGEKYRFRTERVRARYTLRDMYIREFEIIWQRQA 
               
               
                   
                 GHLGLAHEQATRKKNIFLEGSATNVRNSKLITHLQAKYGRGHVLIEDTRITVITQLPLK 
               
               
                   
                 EVLGGKIEIEEEQLKFKSNESVLFWQRPLRSQKSLLSKCVFEGRNFYDPVHQKWIIAGPT 
               
               
                   
                 PAPLSHPEFEEFRAYQFINNITYGKNEHLTAIQREAVFELMCTESKDFINTEKIPKHLKLFE 
               
               
                   
                 KFNFDDTTKVPACTTISQLRKLFPHPVWEEKREEIWHCFYFYDDNTLLFEKLQKDYAL 
               
               
                   
                 QTNDLEKIKKIRLSESYGNVSLKAIRRINPYLKKGYAYSTAVLLGGIRNSFGKREENFKE 
               
               
                   
                 YEPEIEKAVCRILKEKNAEGEVIRKIKDYLVHNRFGFAKNDRAFQKLYHHSQAITTQAQ 
               
               
                   
                 KERLPETGNLRNPIVQQGLNELRRTVNKLLATCREKYGPSFKFDHIHVEMGRELRSSKT 
               
               
                   
                 EREKQSRQIRENEKKNEAAKVKLAEYGLKAYRDNIQKYLLYKEIEEKGGTVCCPYTGK 
               
               
                   
                 TLNISHTLGSDNSVQIEHIIPYSISLDDSLANKTLCDATFNREKGELTPYDFYQKDPSPEK 
               
               
                   
                 WGASSWEEIEDRAFRLLPYAKAQRFIRRKPQESNEFISRQLNDTRYISKKAVEYLSAICS 
               
               
                   
                 DVKAFPGQLTAELRHLWGLNNILQSAPDITFPLPVSATENHREYYVITNEQNEVIRLFPK 
               
               
                   
                 QGETPRIIKGELLLTGEVERKVFRCKGMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAP 
               
               
                   
                 KPISADGQIVLKGRIEKGVFVCNQLKQKLKTGLPDGSYWISLPVISQTFKEGESVNNSKL 
               
               
                   
                 TSQQVQLFGRVREGIFRCHNYQCPASGADGNFWCTLDTDTAQPAFTPIKNAPPGVGGG 
               
               
                   
                 QIILTGDVDDKGIFHADDDLHYELPASLPKGKYYGIFTVESCDPTLIPIELSAPKTSKGEN 
               
               
                   
                 LIEGNIWVDEHTGEVRFDPKKNREDQRHHAIDAIVIALSSQSLFQRLSTYNARRENKKR 
               
               
                   
                 GLDSTEHFPSPWPGFAQDVRQSVVPLLVSYKQNPKTLCKISKTLYKDGKKIHSCGNAV 
               
               
                   
                 RGQLHKETVYGQRTAPGAIIKSYHIRKDIRELKTSKHIGKVVDITIRQMLLKHLQENY 
               
               
                   
                 HIDITQEFNIPSNAFFKEGVYRIFLPNKHGEPVPIKKIRMKEELGNAERLKDNINQYVNP 
               
               
                   
                 RNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPREGRNIVSILQINDTFLIGL 
               
               
                   
                 KEEEPEVYRNDLSTLSKHLYRVQKLSGMYYTFRHHLASTLNNEREEFRIQSLEAWKRA 
               
               
                   
                 NPVKVQIDEIGRITFLNGPLC (SEQ ID NO: 333) 
               
               
                   
               
            
           
         
       
     
     Nuclease deficient  S. pyogenes  Cas9 proteins may comprise a substitution of an Alanine (A) for an Aspartic Acid (D) at position 10 and an alanine (A) for a Histidine (H) at position 840. Exemplary nuclease deficient  S. pyogenes  Cas9 proteins of the disclosure may comprise or consist of the amino acid sequence (D10A and H840A bolded and underlined): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 334) 
               
            
           
           
               
               
            
               
                 1 
                 MDKKYSIGL   A    IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR 
               
               
                   
                 HSIKKNLIGA LLFDSGETAE 
               
               
                   
               
               
                 61 
                 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR 
               
               
                   
                 LEESFLVEED KKHERHPIFG 
               
               
                   
               
               
                 121 
                 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH 
               
               
                   
                 MIKFRGHFLI EGDLNPDNSD 
               
               
                   
               
               
                 181 
                 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR 
               
               
                   
                 RLENLIAQLP GEKKNGLFGN 
               
               
                   
               
               
                 241 
                 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA 
               
               
                   
                 QIGDQYADLF LAAKNLSDAI 
               
               
                   
               
               
                 301 
                 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR 
               
               
                   
                 QQLPEKYKEI FFDQSKNGYA 
               
               
                   
               
               
                 361 
                 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR 
               
               
                   
                 KQRTFDNGSI PHQIHLGELH 
               
               
                   
               
               
                 421 
                 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS 
               
               
                   
                 RFAWMTRKSE ETITPWNFEE 
               
               
                   
               
               
                 481 
                 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV 
               
               
                   
                 YNELTKVKYV TEGMRKPAFL 
               
               
                   
               
               
                 541 
                 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI 
               
               
                   
                 SGVEDRFNAS LGTYHDLLKI 
               
               
                   
               
               
                 601 
                 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA 
               
               
                   
                 HLFDDKVMKQ LKRRRYTGWG 
               
               
                   
               
               
                 661 
                 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD 
               
               
                   
                 SLTFKEDIQK AQVSGQGDSL 
               
               
                   
               
               
                 721 
                 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV 
               
               
                   
                 IEMARENQTT QKGQKNSRER 
               
               
                   
               
               
                 781 
                 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR 
               
               
                   
                 DMYVDQELDI NRLSDYDVD   A     
               
               
                   
               
               
                 841 
                 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK 
               
               
                   
                 NYWRQLLNAK LITQRKFDNL 
               
               
                   
               
               
                 901 
                 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN 
               
               
                   
                 TKYDENDKLI REVKVITLKS 
               
               
                   
               
               
                 961 
                 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK 
               
               
                   
                 YPKLESEFVY GDYKVYDVRK 
               
               
                   
               
               
                 1021 
                 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR 
               
               
                   
                 PLIETNGETG EIVWDKGRDF 
               
               
                   
               
               
                 1081 
                 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI 
               
               
                   
                 ARKKDWDPKK YGGFDSPTVA 
               
               
                   
               
               
                 1141 
                 YSVLVVAKVE KGKDKKLKSV KELLGITIME RSSFEKNPID 
               
               
                   
                 FLEAKGYKEV KKDLIIKLPK 
               
               
                   
               
               
                 1201 
                 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS 
               
               
                   
                 HYEKLKGSPE DNEQKQLFVE 
               
               
                   
               
               
                 1261 
                 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK 
               
               
                   
                 PIREQAENII HLFTLTNLGA 
               
               
                   
               
               
                   
                 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI 
               
               
                   
                 DLSQLGGD. 
               
            
           
         
       
     
     Exemplary wild type  Francisella tularensis  subsp.  Novicida  Cpf1 (FnCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 335) 
               
            
           
           
               
               
            
               
                 1 
                 MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN IKARGLILDD 
               
               
                   
                 EKRAKDYKKA KQIIDKYHQF 
               
               
                   
               
               
                 61 
                 FIEEILSSVC ISEDLLQNYS DVYFKLKKSD DDNLQKDFKS 
               
               
                   
                 AKDTIKKQIS EYIKDSEKFK 
               
               
                   
               
               
                 121 
                 NLFNQNLIDA KKGQESDLIL WLKQSKDNGI ELFKANSDIT 
               
               
                   
                 DIDEALEIIK SFKGWTTYFK 
               
               
                   
               
               
                 181 
                 GFHENRKNVY SSNDIPTSII YRIVDDNLPK FLENKAKYES 
               
               
                   
                 LKDKAPEAIN YEQIKKDLAE 
               
               
                   
               
               
                 241 
                 ELTFDIDYKT SEVNQRVFSL DEVFEIANFN NYLNQSGITK 
               
               
                   
                 FNTIIGGKFV NGENTKRKGI 
               
               
                   
               
               
                 301 
                 NEYINLYSQQ INDKTLKKYK MSVLFKQILS DTESKSFVID 
               
               
                   
                 KLEDDSDVVT TMQSFYEQIA 
               
               
                   
               
               
                 361 
                 AFKTVEEKSI KETLSLLFDD LKAQKLDLSK IYFKNDKSLT 
               
               
                   
                 DLSQQVFDDY SVIGTAVLEY 
               
               
                   
               
               
                 421 
                 ITQQIAPKNL DNPSKKEQEL IAKKTEKAKY LSLETIKLAL 
               
               
                   
                 EEFNKHRDID KQCRFEEILA 
               
               
                   
               
               
                 481 
                 NFAAIPMIFD EIAQNKDNLA QISIKYQNQG KKDLLQASAE 
               
               
                   
                 DDVKAIKDLL DQTNNLLHKL 
               
               
                   
               
               
                 541 
                 KIFHISQSED KANILDKDEH FYLVFEECYF ELANIVPLYN 
               
               
                   
                 KIRNYITQKP YSDEKFKLNF 
               
               
                   
               
               
                 601 
                 ENSTLANGWD KNKEPDNTAI LFIKDDKYYL GVMNKKNNKI 
               
               
                   
                 FDDKAIKENK GEGYKKIVYK 
               
               
                   
               
               
                 661 
                 LLPGANKMLP KVFFSAKSIK FYNPSEDILR IRNHSTHTKN 
               
               
                   
                 GSPQKGYEKF EFNIEDCRKF 
               
               
                   
               
               
                 721 
                 IDFYKQSISK HPEWKDFGFR FSDTQRYNSI DEFYREVENQ 
               
               
                   
                 GYKLTFENIS ESYIDSVVNQ 
               
               
                   
               
               
                 781 
                 GKLYLFQIYN KDFSAYSKGR PNLHTLYWKA LFDERNLQDV 
               
               
                   
                 VYKLNGEAEL FYRKQSIPKK 
               
               
                   
               
               
                 841 
                 ITHPAKEAIA NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF 
               
               
                   
                 HCPITINFKS SGANKFNDEI 
               
               
                   
               
               
                 901 
                 NLLLKEKAND VHILSIDRGE RHLAYYTLVD GKGNIIKQDT 
               
               
                   
                 FNIIGNDRMK TNYHDKLAAI 
               
               
                   
               
               
                 961 
                 EKDRDSARKD WKKINNIKEM KEGYLSQVVH EIAKLVIEYN 
               
               
                   
                 AIVVFEDLNF GFKRGRFKVE 
               
               
                   
               
               
                 1021 
                 KQVYQKLEKM LIEKLNYLVF KDNEFDKTGG VLRAYQLTAP 
               
               
                   
                 FETFKKMGKQ TGIIYYVPAG 
               
               
                   
               
               
                 1081 
                 FTSKICPVTG FVNQLYPKYE SVSKSQEFFS KFDKICYNLD 
               
               
                   
                 KGYFEFSFDY KNFGDKAAKG 
               
               
                   
               
               
                 1141 
                 KWTIASFGSR LINFRNSDKN HNWDTREVYP TKELEKLLKD 
               
               
                   
                 YSIEYGHGEC IKAAICGESD 
               
               
                   
               
               
                 1201 
                 KKFFAKLTSV LNTILQMRNS KTGTELDYLI SPVADVNGNF 
               
               
                   
                 FDSRQAPKNM PQDADANGAY 
               
               
                   
               
               
                 1261 
                 HIGLKGLMLL GRIKNNQEGK KLNLVIKNEE YFEFVQNRNN 
               
            
           
         
       
     
     Exemplary wild type  Lachnospiraceae bacterium  sp. ND2006 Cpf1 (LbCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 336) 
               
            
           
           
               
               
            
               
                 1 
                 AASKLEKFTN CYSLSKTLRF KAIPVGKTQE NIDNKRLLVE 
               
               
                   
                 DEKRAEDYKG VKKLLDRYYL 
               
               
                   
               
               
                 61 
                 SFINDVLHSI KLKNLNNYIS LFRKKTRTEK ENKELENLEI 
               
               
                   
                 NLRKEIAKAF KGAAGYKSLF 
               
               
                   
               
               
                 121 
                 KKDIIETILP EAADDKDEIA LVNSFNGFTT AFTGFFDNRE 
               
               
                   
                 NMFSEEAKST SIAFRCINEN 
               
               
                   
               
               
                 181 
                 LTRYISNMDI FEKVDAIFDK HEVQEIKEKI LNSDYDVEDF 
               
               
                   
                 FEGEFFNFVL TQEGIDVYNA 
               
               
                   
               
               
                 241 
                 IIGGFVTESG EKIKGLNEYI NLYNAKTKQA LPKFKPLYKQ 
               
               
                   
                 VLSDRESLSF YGEGYTSDEE 
               
               
                   
               
               
                 301 
                 VLEVFRNTLN KNSEIFSSIK KLEKLFKNFD EYSSAGIFVK 
               
               
                   
                 NGPAISTISK DIFGEWNLIR 
               
               
                   
               
               
                 361 
                 DKWNAEYDDI HLKKKAVVTE KYEDDRRKSF KKIGSFSLEQ 
               
               
                   
                 LQEYADADLS VVEKLKEIII 
               
               
                   
               
               
                 421 
                 QKVDEIYKVY GSSEKLFDAD FVLEKSLKKN DAVVAIMKDL 
               
               
                   
                 LDSVKSFENY IKAFFGEGKE 
               
               
                   
               
               
                 481 
                 TNRDESFYGD FVLAYDILLK VDHIYDAIRN YVTQKPYSKD 
               
               
                   
                 KFKLYFQNPQ FMGGWDKDKE 
               
               
                   
               
               
                 541 
                 TDYRATILRY GSKYYLAIMD KKYAKCLQKI DKDDVNGNYE 
               
               
                   
                 KINYKLLPGP NKMLPKVFFS 
               
               
                   
               
               
                 601 
                 KKWMAYYNPS EDIQKIYKNG TFKKGDMFNL NDCHKLIDFF 
               
               
                   
                 KDSISRYPKW SNAYDFNFSE 
               
               
                   
               
               
                 661 
                 TEKYKDIAGF YREVEEQGYK VSFESASKKE VDKLVEEGKL 
               
               
                   
                 YMFQIYNKDF SDKSHGTPNL 
               
               
                   
               
               
                 721 
                 HTMYFKLLFD ENNHGQIRLS GGAELFMRRA SLKKEELVVH 
               
               
                   
                 PANSPIANKN PDNPKKTTTL 
               
               
                   
               
               
                 781 
                 SYDVYKDKRF SEDQYELHIP IAINKCPKNI FKINTEVRVL 
               
               
                   
                 LKHDDNPYVI GIDRGERNLL 
               
               
                   
               
               
                 841 
                 YIVVVDGKGN IVEQYSLNEI INNFNGIRIK TDYHSLLDKK 
               
               
                   
                 EKERFEARQN WTSIENIKEL 
               
               
                   
               
               
                 901 
                 KAGYISQVVH KICELVEKYD AVIALEDLNS GFKNSRVKVE 
               
               
                   
                 KQVYQKFEKM LIDKLNYMVD 
               
               
                   
               
               
                 961 
                 KKSNPCATGG ALKGYQITNK FESFKSMSTQ NGFIFYIPAW 
               
               
                   
                 LTSKIDPSTG FVNLLKTKYT 
               
               
                   
               
               
                 1021 
                 SIADSKKFIS SFDRIMYVPE EDLFEFALDY KNFSRTDADY 
               
               
                   
                 IKKWKLYSYG NRIRIFAAAK 
               
               
                   
               
               
                 1081 
                 KNNVFAWEEV CLTSAYKELF NKYGINYQQG DIRALLCEQS 
               
               
                   
                 DKAFYSSFMA LMSLMLQMRN 
               
               
                   
               
               
                 1141 
                 SITGRTDVDF LISPVKNSDG IFYDSRNYEA QENAILPKNA 
               
               
                   
                 DANGAYNIAR KVLWAIGQFK 
               
               
                   
               
               
                 1201 
                 KAEDEKLDKV KIAISNKEWL EYAQTSVK 
               
            
           
         
       
     
     Exemplary wild type  Acidaminococcus  sp. BV3L6 Cpf1 (AsCpf1) proteins of the disclosure may comprise or consist of the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 337) 
               
            
           
           
               
               
            
               
                 1 
                 MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED 
               
               
                   
                 KARNDHYKEL KPIIDRIYKT 
               
               
                   
               
               
                 61 
                 YADQCLQLVQ LDWENLSAAI DSYRKEKTEE TRNALIEEQA 
               
               
                   
                 TYRNAIHDYF IGRTDNLTDA 
               
               
                   
               
               
                 121 
                 INKRHAEIYK GLFKAELFNG KVLKQLGTVT TTEHENALLR 
               
               
                   
                 SFDKFTTYFS GFYENRKNVF 
               
               
                   
               
               
                 181 
                 SAEDISTAIP HRIVQDNFPK FKENCHIFTR LITAVPSLRE 
               
               
                   
                 HFENVKKAIG IFVSTSIEEV 
               
               
                   
               
               
                 241 
                 FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV 
               
               
                   
                 LNLAIQKNDE TAHIIASLPH 
               
               
                   
               
               
                 301 
                 RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL 
               
               
                   
                 RNENVLETAE ALFNELNSID 
               
               
                   
               
               
                 361 
                 LTHIFISHKK LETISSALCD HWDTLRNALY ERRISELTGK 
               
               
                   
                 ITKSAKEKVQ RSLKHEDINL 
               
               
                   
               
               
                 421 
                 QEIISAAGKE LSEAFKQKTS EILSHAHAAL DQPLPTTLKK 
               
               
                   
                 QEEKEILKSQ LDSLLGLYHL 
               
               
                   
               
               
                 481 
                 LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY 
               
               
                   
                 ATKKPYSVEK FKLNFQMPTL 
               
               
                   
               
               
                 541 
                 ASGWDVNKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF 
               
               
                   
                 EPTEKTSEGF DKMYYDYFPD 
               
               
                   
               
               
                 601 
                 AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLEITK 
               
               
                   
                 EIYDLNNPEK EPKKFQTAYA 
               
               
                   
               
               
                 661 
                 KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP 
               
               
                   
                 SSQYKDLGEY YAELNPLLYH 
               
               
                   
               
               
                 721 
                 ISFQRIAEKE IMDAVETGKL YLFQIYNKDF AKGHHGKPNL 
               
               
                   
                 HTLYWTGLFS PENLAKTSIK 
               
               
                   
               
               
                 781 
                 LNGQAELFYR PKSRMKRMAH RLGEKMLNKK LKDQKTPIPD 
               
               
                   
                 TLYQELYDYV NHRLSHDLSD 
               
               
                   
               
               
                 841 
                 EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ 
               
               
                   
                 AANSPSKFNQ RVNAYLKEHP 
               
               
                   
               
               
                 901 
                 ETPIIGIDRG ERNLIYITVI DSTGKILEQR SLNTIQQFDY 
               
               
                   
                 QKKLDNREKE RVAARQAWSV 
               
               
                   
               
               
                 961 
                 VGTIKDLKQG YLSQVIHEIV DLMIHYQAVV VLENLNFGFK 
               
               
                   
                 SKRTGIAEKA VYQQFEKMLI 
               
               
                   
               
               
                 1021 
                 DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG 
               
               
                   
                 FLFYVPAPYT SKIDPLTGFV 
               
               
                   
               
               
                 1081 
                 DPFVWKTIKN HESRKHFLEG FDFLHYDVKT GDFILHFKMN 
               
               
                   
                 RNLSFQRGLP GFMPAWDIVF 
               
               
                   
               
               
                 1141 
                 EKNETQFDAK GTPFIAGKRI VPVIENHRFT GRYRDLYPAN 
               
               
                   
                 ELIALLEEKG IVFRDGSNIL 
               
               
                   
               
               
                 1201 
                 PKLLENDDSH AIDTMVALIR SVLQMRNSNA ATGEDYINSP 
               
               
                   
                 VRDLNGVCFD SRFQNPEWPM 
               
               
                   
               
               
                 1261 
                 DADANGAYHI ALKGQLLLNH LKESKDLKLQ NGISNQDWLA 
               
               
                   
                 YIQELRN 
               
            
           
         
       
     
     In some embodiments of the compositions of the disclosure, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a CRISPR Cas protein or RNA-binding portion thereof. In some embodiments, the CRISPR Cas protein comprises a Type VI CRISPR Cas protein. In some embodiments, the Type VI CRISPR Cas protein comprises a Cas13 protein. Exemplary Cas13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, bacteria or archaea. Exemplary Cas13 proteins of the disclosure may be isolated or derived from any species, including, but not limited to,  Leptotrichia wadei, Listeria seeligeri  serovar 1/2b (strain ATCC 35967/DSM 20751/CIP 100100/SLCC 3954).  Lachnospiraceae bacterium, Clostridium aminophilum  DSM 10710 , Carnobacterium gallinarum  DSM 4847 , Paludibacter propionicigenes  WB4.  Listeria weihenstephanensis  FSL R9-0317,  Listeria weihenstephanensis  FSL R9-0317, bacterium FSL M6-0635 ( Listeria newyorkensis ),  Leptotrichia wadei  F0279,  Rhodobacter capsulatus  SB 1003,  Rhodobacter capsulatus  R121 , Rhodobacter capsulatus  DE442 and  Corynebacterium ulcerans . Exemplary Cas13 proteins of the disclosure may be DNA nuclease inactivated. Exemplary Cas13 proteins of the disclosure include, but are not limited to, Cas13a, Cas13b, Cas13c, Cas13d, and orthologs thereof. Exemplary Cas13b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively. 
     Exemplary Cas13a proteins include, but are not limited to: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Cas13a 
                 Cas13a 
                   
                   
               
               
                 number 
                 abbreviation 
                 Organism name 
                 Accession number 
               
               
                   
               
             
            
               
                 Cas13a1 
                 LshCas13a 
                 Leptotrichia shahii 
                 WP_018451595.1 (SEQ ID NO: 338) 
               
               
                 Cas13a2 
                 LwaCas13a 
                 Leptotrichia wadei 
                 WP_021746774.1 (SEQ ID NO: 339) 
               
               
                 Cas13a3 
                 LseCas13a 
                 Listeria seeligeri 
                 WP_012985477.1 (SEQ ID NO: 340) 
               
               
                 Cas13a4 
                 LbmCas13a 
                 Lachnospiraceae bacterium 
                 WP_044921188.1 (SEQ ID NO: 341)  
               
               
                   
                   
                 MA2020 
                   
               
               
                 Cas13a5 
                 LbnCas13a 
                 Lachnospiraceae bacterium 
                 WP_022785443.1 (SEQ ID NO: 342) 
               
               
                   
                   
                 NK4A179 
                   
               
               
                 Cas13a6 
                 CamCas13a 
                 [Clostridium] aminophilum  
                 WP_031473346.1 (SEQ ID NO: 343) 
               
               
                   
                   
                 DSM 10710 
                   
               
               
                 Cas13a7 
                 CgaCas13a 
                 Carnobacterium gallinarum  
                 WP_034560163.1 (SEQ ID NO: 344) 
               
               
                   
                   
                 DSM 4847 
                   
               
               
                 Cas13a8 
                 Cga2Cas13a 
                 Carnobacterium gallinarum  
                 WP_034563842.1 (SEQ ID NO: 345) 
               
               
                   
                   
                 DSM 4847 
                   
               
               
                 Cas13a9 
                 Pprcas13a 
                 Paludibacter propionicigenes 
                 WP_013443710.1 (SEQ ID NO: 346) 
               
               
                   
                   
                 WB4 
                   
               
               
                 Cas13a10 
                 LweCas13a 
                 Listeria weihenstephanensis 
                 WP_036059185.1 (SEQ ID NO: 347) 
               
               
                   
                   
                 FSL R9-0317 
                   
               
               
                 Cas13a11 
                 LbfCas13a 
                 Listeriaceae bacterium FSL  
                 WP_036091002.1 (SEQ ID NO: 348) 
               
               
                   
                   
                 M6-0635 (Listeria newyorkensis) 
                   
               
               
                 Cas13a12 
                 Lwa2cas13a 
                 Leptotrichia wadei F0279 
                 WP_021746774.1 (SEQ ID NO: 349) 
               
               
                 Cas13a13 
                 RcsCas13a 
                 Rhodobacter capsulatus  
                 WP_013067728.1 (SEQ ID NO: 350) 
               
               
                   
                   
                 SB 1003 
                   
               
               
                 Cas13a14 
                 RcrCas13a 
                 Rhodobacter capsulatus R121 
                 WP_023911507.1 (SEQ ID NO: 351) 
               
               
                 Cas13a15 
                 RcdCas13a 
                 Rhodobacter capsulatus DE442 
                 WP_023911507.1 (SEQ ID NO: 352) 
               
               
                   
               
            
           
         
       
     
     Exemplary wild type Cas13a proteins of the disclosure may comprise or consist of the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 353) 
               
            
           
           
               
               
            
               
                 1 
                 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN YDGNKYILNI 
               
               
                   
                 NENNNKEKID NNKFIRKYIN 
               
               
                   
               
               
                 61 
                 YKKNDNILKE FTRKFHAGNI LFKLKGKEGI IRIENNDDFL 
               
               
                   
                 ETEEVVLYIE AYGKSEKLKA 
               
               
                   
               
               
                 121 
                 LGITKKKIID EAIRQGITKD DKKIEIKRQE NEEEIEIDIR 
               
               
                   
                 DEYTNKTLND CSIILRIIEN 
               
               
                   
               
               
                 181 
                 DELETKKSIY EIFKNINMSL YKIIEKIIEN ETEKVFENRY 
               
               
                   
                 YEEHLREKLL KDDKIDVILT 
               
               
                   
               
               
                 241 
                 NFMEIREKIK SNLEILGFVK FYLNVGGDKK KSKNKKMLVE 
               
               
                   
                 KILNINVDLT VEDIADFVIK 
               
               
                   
               
               
                 301 
                 ELEFWNITKR IEKVKKVNNE FLEKRRNRTY IKSYVLLDKH 
               
               
                   
                 EKFKIERENK KDKIVKFFVE 
               
               
                   
               
               
                 361 
                 NIKNNSIKEK IEKILAEFKI DELIKKLEKE LKKGNCDTEI 
               
               
                   
                 FGIFKKHYKV NFDSKKFSKK 
               
               
                   
               
               
                 421 
                 SDEEKELYKI IYRYLKGRIE KILVNEQKVR LKKMEKIEIE 
               
               
                   
                 KILNESILSE KILKRVKQYT 
               
               
                   
               
               
                 481 
                 LEHIMYLGKL RHNDIDMTTV NTDDFSRLHA KEELDLELIT 
               
               
                   
                 FFASTNMELN KIFSRENINN 
               
               
                   
               
               
                 541 
                 DENIDFFGGD REKNYVLDKK ILNSKIKIIR DLDFIDNKNN 
               
               
                   
                 ITNNFIRKFT KIGTNERNRI 
               
               
                   
               
               
                 601 
                 LHAISKERDL QGTQDDYNKV INIIQNLKIS DEEVSKALNL 
               
               
                   
                 DVVFKDKKNI ITKINDIKIS 
               
               
                   
               
               
                 661 
                 EENNNDIKYL PSFSKVLPEI LNLYRNNPKN EPFDTIETEK 
               
               
                   
                 IVLNALIYVN KELYKKLILE 
               
               
                   
               
               
                 721 
                 DDLEENESKN IFLQELKKTL GNIDEIDENI IENYYKNAQI 
               
               
                   
                 SASKGNNKAI KKYQKKVIEC 
               
               
                   
               
               
                 781 
                 YIGYLRKNYE ELFDFSDFKM NIQEIKKQIK DINDNKTYER 
               
               
                   
                 ITVKTSDKTI VINDDFEYII 
               
               
                   
               
               
                 841 
                 SIFALLNSNA VINKIRNRFF ATSVWLNTSE YQNIIDILDE 
               
               
                   
                 IMQLNTLRNE CITENWNLNL 
               
               
                   
               
               
                 901 
                 EEFIQKMKEI EKDFDDFKIQ TKKEIFNNYY EDIKNNILTE 
               
               
                   
                 FKDDINGCDV LEKKLEKIVI 
               
               
                   
               
               
                 961 
                 FDDETKFEID KKSNILQDEQ RKLSNINKKD LKKKVDQYIK 
               
               
                   
                 DKDQEIKSKI LCRIIFNSDF 
               
               
                   
               
               
                 1021 
                 LKKYKKEIDN LIEDMESENE NKFQEIYYPK ERKNELYIYK 
               
               
                   
                 KNLFLNIGNP NFDKIYGLIS 
               
               
                   
               
               
                 1081 
                 NDIKMADAKF LFNIDGKNIR KNKISEIDAI LKNLNDKLNG 
               
               
                   
                 YSKEYKEKYI KKLKENDDFF 
               
               
                   
               
               
                 1141 
                 AKNIQNKNYK SFEKDYNRVS EYKKIRDLVE FNYLNKIESY 
               
               
                   
                 LIDINWKLAI QMARFERDMH 
               
               
                   
               
               
                 1201 
                 YIVNGLRELG IIKLSGYNTG ISRAYPKRNG SDGFYTTTAY 
               
               
                   
                 YKFFDEESYK KFEKICYGFG 
               
               
                   
               
               
                 1261 
                 IDLSENSEIN KPENESIRNY ISHFYIVRNP FADYSIAEQI 
               
               
                   
                 DRVSNLLSYS TRYNNSTYAS 
               
               
                   
               
               
                 1321 
                 VFEVFKKDVN LDYDELKKKF KLIGNNDILE RLMKPKKVSV 
               
               
                   
                 LELESYNSDY IKNLIIELLT  
               
               
                   
               
               
                 1381 
                 KIENTNDTL 
               
            
           
         
       
     
     Exemplary Cas13b proteins include, but are not limited to: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 Cas13b 
                 Cas13b 
               
               
                 Species 
                 Accession 
                 Size (aa) 
               
               
                   
               
             
            
               
                 Paludibacter propionicigenes WB4 
                 WP_013446107.1 
                 1155 
               
               
                   
                 (SEQ ID NO: 354) 
                   
               
               
                 Prevotella sp. P5-60 
                 WP_044074780.1 
                 1091 
               
               
                   
                 (SEQ ID NO: 355) 
                   
               
               
                 Prevotella sp. P4-76 
                 WP_044072147.1 
                 1091 
               
               
                   
                 (SEQ ID NO: 356) 
                   
               
               
                 Prevotella sp. P5-125 
                 WP_044065294.1 
                 1091 
               
               
                   
                 (SEQ ID NO: 357) 
                   
               
               
                 Prevotella sp. P5-119 
                 WP_042518169.1 
                 1091 
               
               
                   
                 (SEQ ID NO: 358) 
                   
               
               
                 Capnocytophaga canimorsus Cc5 
                 WP_013997271.1 
                 1200 
               
               
                   
                 (SEQ ID NO: 359) 
                   
               
               
                 Phaeodactylibacter xiamenensis 
                 WP_044218239.1 
                 1132 
               
               
                   
                 (SEQ ID NO: 360) 
                   
               
               
                 Porphyromonas gingivalis W83 
                 WP_005873511.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 361) 
                   
               
               
                 Porphyromonas gingivalis F0570 
                 WP_021665475.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 362) 
                   
               
               
                 Porphyromonas gingivalis  
                 WP_012458151.1 
                 1136 
               
               
                 ATCC 33277 
                 (SEQ ID NO: 363) 
                   
               
               
                 Porphyromonas gingivalis F0185 
                 ERJ81987.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 364) 
                   
               
               
                 Porphyromonas gingivalis F0185 
                 WP_021677657.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 365) 
                   
               
               
                 Porphyromonas gingivalis SJD2 
                 WP_023846767.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 366) 
                   
               
               
                 Porphyromonas gingivalis F0568 
                 ERJ65637.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 367) 
                   
               
               
                 Porphyromonas gingivalis W4087 
                 ERJ87335.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 368) 
                   
               
               
                 Porphyromonas gingivalis W4087 
                 WP_021680012.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 369) 
                   
               
               
                 Porphyromonas gingivalis F0568 
                 WP_021663197.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 370) 
                   
               
               
                 Porphyromonas gingivalis 
                 WP_061156637.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 371) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039445055.1 
                 1136 
               
               
                   
                 (SEQ ID NO: 372) 
                   
               
               
                 Bacteroides pyogenes F0041 
                 ERI81700.1 
                 1116 
               
               
                   
                 (SEQ ID NO: 373) 
                   
               
               
                 Bacteroides pyogenes JCM 10003 
                 WP_034542281.1 
                 1116 
               
               
                   
                 (SEQ ID NO: 374) 
                   
               
               
                 Alistipes sp. ZOR0009 
                 WP_047447901.1 
                  954 
               
               
                   
                 (SEQ ID NO: 375) 
                   
               
               
                 Flavobacterium branchiophilum  
                 WP_014084666.1 
                 1151 
               
               
                 FL-15 
                 (SEQ ID NO: 376) 
                   
               
               
                 Prevotella sp. MA2016 
                 WP_036929175.1 
                 1323 
               
               
                   
                 (SEQ ID NO: 377) 
                   
               
               
                 Myroides odoratimimus  
                 EHO06562.1 
                 1160 
               
               
                 CCUG 10230 
                 (SEQ ID NO: 378) 
                   
               
               
                 Myroides odoratimimus  
                 EKB06014.1 
                 1158 
               
               
                 CCUG 3837 
                 (SEQ ID NO: 379) 
                   
               
               
                 Myroides odoratimimus  
                 WP_006265509.1 
                 1158 
               
               
                 CCUG 3837 
                 (SEQ ID NO: 380) 
                   
               
               
                 Myroides odoratimimus  
                 WP_006261414.1 
                 1158 
               
               
                 CCUG 12901 
                 (SEQ ID NO: 381) 
                   
               
               
                 Myroides odoratimimus  
                 EHO08761.1 
                 1158 
               
               
                 CCUG 12901 
                 (SEQ ID NO: 382) 
                   
               
               
                 Myroides odoratimimus 
                 WP_058700060.1 
                 1160 
               
               
                 (NZ_CP013690.1) 
                 (SEQ ID NO: 383) 
                   
               
               
                 Bergeyella zoohelcum  
                 EKB54193.1 
                 1225 
               
               
                 ATCC 43767 
                 (SEQ ID NO: 384) 
                   
               
               
                 Capnocytophaga cynodegmi 
                 WP_041989581.1 
                 1219 
               
               
                   
                 (SEQ ID NO: 385) 
                   
               
               
                 Bergeyella zoohelcum  
                 WP_002664492.1 
                 1225 
               
               
                 ATCC 43767 
                 (SEQ ID NO: 386) 
                   
               
               
                 Flavobacterium sp. 316 
                 WP_045968377.1 
                 1156 
               
               
                   
                 (SEQ ID NO: 387) 
                   
               
               
                 Psychroflexus torquis  
                 WP_015024765.1 
                 1146 
               
               
                 ATCC 700755 
                 (SEQ ID NO: 388) 
                   
               
               
                 Flavobacterium columnare  
                 WP_014165541.1 
                 1180 
               
               
                 ATCC 49512 
                 (SEQ ID NO: 389) 
                   
               
               
                 Flavobacterium columnare 
                 WP_060381855.1 
                 1214 
               
               
                   
                 (SEQ ID NO: 390) 
                   
               
               
                 Flavobacterium columnare 
                 WP_063744070.1 
                 1214 
               
               
                   
                 (SEQ ID NO: 391) 
                   
               
               
                 Flavobacterium columnare 
                 WP_065213424.1 
                 1215 
               
               
                   
                 (SEQ ID NO: 392) 
                   
               
               
                 Chryseobacterium sp. YR477 
                 WP_047431796.1 
                 1146 
               
               
                   
                 (SEQ ID NO: 393) 
                   
               
               
                 Riemerella anatipestifer ATCC  
                 WP_004919755.1 
                 1096 
               
               
                 11845 = DSM 15868 
                 (SEQ ID NO: 394) 
                   
               
               
                 Riemerella anatipestifer  
                 WP_015345620.1 
                  949 
               
               
                 RA-CH-2 
                 (SEQ ID NO: 395) 
                   
               
               
                 Riemerella anatipestifer 
                 WP_049354263.1 
                  949 
               
               
                   
                 (SEQ ID NO: 396) 
                   
               
               
                 Riemerella anatipestifer 
                 WP_061710138.1 
                  951 
               
               
                   
                 (SEQ ID NO: 397) 
                   
               
               
                 Riemerella anatipestifer 
                 WP_064970887.1 
                 1096 
               
               
                   
                 (SEQ ID NO: 398) 
                   
               
               
                 Prevotella saccharolytica  
                 EKY00089.1 
                 1151 
               
               
                 F0055 
                 (SEQ ID NO: 399) 
                   
               
               
                 Prevotella saccharolytica  
                 WP_051522484.1 
                 1152 
               
               
                 JCM 17484 
                 (SEQ ID NO: 400) 
                   
               
               
                 Prevotella buccae  
                 EFU31981.1 
                 1128 
               
               
                 ATCC 33574 
                 (SEQ ID NO: 401) 
                   
               
               
                 Prevotella buccae  
                 WP_004343973.1 
                 1128 
               
               
                 ATCC 33574 
                 (SEQ ID NO: 402) 
                   
               
               
                 Prevotella buccae D17 
                 WP_004343581.1 
                 1128 
               
               
                   
                 (SEQ ID NO: 403) 
                   
               
               
                 Prevotella sp. MSX73 
                 WP_007412163.1 
                 1128 
               
               
                   
                 (SEQ ID NO: 404) 
                   
               
               
                 Prevotella pallens  
                 EGQ18444.1 
                 1126 
               
               
                 ATCC 700821 
                 (SEQ ID NO: 405) 
                   
               
               
                 Prevotella pallens  
                 WP_006044833.1 
                 1126 
               
               
                 ATCC 700821 
                 (SEQ ID NO: 406) 
                   
               
               
                 Prevotella intermedia ATCC  
                 WP_036860899.1 
                 1127 
               
               
                 25611 = DSM 20706 
                 (SEQ ID NO: 407) 
                   
               
               
                 Prevotella intermedia 
                 WP_061868553.1 
                 1121 
               
               
                   
                 (SEQ ID NO: 408) 
                   
               
               
                 Prevotella intermedia 17 
                 AFJ07523.1 
                 1135 
               
               
                   
                 (SEQ ID NO: 409) 
                   
               
               
                 Prevotella intermedia 
                 WP_050955369.1 
                 1133 
               
               
                   
                 (SEQ ID NO: 410) 
                   
               
               
                 Prevotella intermedia 
                 BAU18623.1 
                 1134 
               
               
                   
                 (SEQ ID NO: 411) 
                   
               
               
                 Prevotella intermedia ZT 
                 KJJ86756.1 
                 1126 
               
               
                   
                 (SEQ ID NO: 412) 
                   
               
               
                 Prevotella aurantiaca  
                 WP_025000926.1 
                 1125 
               
               
                 JCM 15754 
                 (SEQ ID NO: 413) 
                   
               
               
                 Prevotella pleuritidis F0068 
                 WP_021584635.1 
                 1140 
               
               
                   
                 (SEQ ID NO: 414) 
                   
               
               
                 Prevotella pleuritidis  
                 WP_036931485.1 
                 1117 
               
               
                 JCM 14110 
                 (SEQ ID NO: 415) 
                   
               
               
                 Prevotella falsenii DSM  
                 WP_036884929.1 
                 1134 
               
               
                 22864 = JCM 15124 
                 (SEQ ID NO: 416) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039418912.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 417) 
                   
               
               
                 Porphyromonas sp.  
                 WP_039428968.1 
                 1176 
               
               
                 COT-052 OH4946 
                 (SEQ ID NO: 418) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039442171.1 
                 1175 
               
               
                   
                 (SEQ ID NO: 419) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039431778.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 420) 
                   
               
               
                 Porphyromonas gulae 
                 WP_046201018.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 421) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039434803.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 422) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039419792.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 423) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039426176.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 424) 
                   
               
               
                 Porphyromonas gulae 
                 WP_039437199.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 425) 
                   
               
               
                 Porphyromonas gingivalis  
                 WP_013816155.1 
                 1120 
               
               
                 TDC60 
                 (SEQ ID NO: 426) 
                   
               
               
                 Porphyromonas gingivalis  
                 WP_012458414.1 
                 1120 
               
               
                 ATCC 33277 
                 (SEQ ID NO: 427) 
                   
               
               
                 Porphyromonas gingivalis  
                 WP_058019250.1 
                 1176 
               
               
                 A7A1-28 
                 (SEQ ID NO: 428) 
                   
               
               
                 Porphyromonas gingivalis  
                 EOA10535.1 
                 1176 
               
               
                 JCVI SC001 
                 (SEQ ID NO: 429) 
                   
               
               
                 Porphyromonas gingivalis W50 
                 WP_005874195.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 430) 
                   
               
               
                 Porphyromonas gingivalis 
                 WP_052912312.1 
                 1176 
               
               
                   
                 (SEQ ID NO: 431) 
                   
               
               
                 Porphyromonas gingivalis AJW4 
                 WP_053444417.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 432) 
                   
               
               
                 Porphyromonas gingivalis 
                 WP_039417390.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 433) 
                   
               
               
                 Porphyromonas gingivalis 
                 WP_061156470.1 
                 1120 
               
               
                   
                 (SEQ ID NO: 434) 
               
               
                   
               
            
           
         
       
     
     Exemplary wild type  Bergeyella zoohelcum  ATCC 43767 Cas13b (BzCas13b) proteins of the disclosure may comprise or consist of the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 435) 
               
            
           
           
               
               
            
               
                 1 
                 menktslgnn iyynpfkpqd ksyfagyfna amentdsvfr 
               
               
                   
                 elgkrlkgke ytsenffdai 
               
               
                   
               
               
                 61 
                 fkenislvey eryvkllsdy fpmarlldkk evpikerken 
               
               
                   
                 fkknfkgiik avrdlrnfyt 
               
               
                   
               
               
                 121 
                 hkehgeveit deifgvldem lkstvltvkk kkvktdktke 
               
               
                   
                 ilkksiekql dilcqkkley 
               
               
                   
               
               
                 181 
                 lrdtarkiee krrnqrerge kelvapfkys dkrddliaai 
               
               
                   
                 yndafdvyid kkkdslkess 
               
               
                   
               
               
                 241 
                 kakyntksdp qqeegdlkip iskngvvfll slfltkqeih 
               
               
                   
                 afkskiagfk atvideatvs 
               
               
                   
               
               
                 301 
                 eatvshgkns icfmatheif shlaykklkr kvrtaeinyg 
               
               
                   
                 eaenaeqlsv yaketlmmqm 
               
               
                   
               
               
                 361 
                 ldelskvpdv vyqnlsedvq ktfiedwney lkenngdvgt 
               
               
                   
                 meeeqvihpv irkryedkfn 
               
               
                   
               
               
                 421 
                 yfairfldef aqfptlrfqv hignylhdsr pkenlisdrr 
               
               
                   
                 ikekitvfgr lselehkkal 
               
               
                   
               
               
                 481 
                 fikntetned rehyweifpn pnydfpkeni svndkdfpia 
               
               
                   
                 gsildrekqp vagkigikvk 
               
               
                   
               
               
                 541 
                 llnqqyvsev dkavkahqlk qrkaskpsiq niieeivpin 
               
               
                   
                 esnpkeaivf ggqptaylsm 
               
               
                   
               
               
                 601 
                 ndihsilyef fdkwekkkek lekkgekelr keigkelekk 
               
               
                   
                 ivgkiqaqiq qiidkdtnak 
               
               
                   
               
               
                 661 
                 ilkpyqdgns taidkeklik dikqeqnilq klkdeqtvre 
               
               
                   
                 keyndfiayq dknreinkvr 
               
               
                   
               
               
                 721 
                 drnhkqylkd nlkrkypeap arkevlyyre kgkvavwlan 
               
               
                   
                 dikrfmptdf knewkgeqhs 
               
               
                   
               
               
                 781 
                 llqkslayye qckeelknll pekvfqhlpf klggyfqqky 
               
               
                   
                 lyqfytcyld krleyisglv 
               
               
                   
               
               
                 841 
                 qqaenfksen kvfkkvenec fkflkkqnyt hkeldarvqs 
               
               
                   
                 ilgypifler gfmdekptii 
               
               
                   
               
               
                 901 
                 kgktfkgnea lfadwfryyk eyqnfqtfyd tenyplvele 
               
               
                   
                 kkqadrkrkt kiyqqkkndv 
               
               
                   
               
               
                 961 
                 ftllmakhif ksvfkqdsid qfsledlyqs reerlgnqer 
               
               
                   
                 arqtgerntn yiwnktvdlk 
               
               
                   
               
               
                 1021 
                 lcdgkitven vklknvgdfi kyeydqrvqa flkyeeniew 
               
               
                   
                 qaflikeske eenypyvver 
               
               
                   
               
               
                 1081 
                 eieqyekvrr eellkevhli eeyilekvkd keilkkgdnq 
               
               
                   
                 nfkyyilngl lkqlknedve 
               
               
                   
               
               
                 1141 
                 sykvfnlnte pedvninqlk qeatdleqka fvltyirnkf 
               
               
                   
                 ahnqlpkkef wdycqekygk 
               
               
                   
               
               
                 1201 
                 iekektyaey faevfkkeke alik 
               
            
           
         
       
     
     An exemplary nuclease deficient Cas13b (dCas13b) nucleic acid sequence with C-terminal nuclear export sequence is: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 436)  
                   
               
               
                 ATGaacatccccgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccatgctgaacgct 
                   
               
               
                   
               
               
                 cagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcagaacgagaacaacgagaatctgtgg 
               
               
                   
               
               
                 tttcaccccgtgatgagccacctgtacaacgccaagaacggctacgacaagcagcccgagaaaaccatgttcatc 
               
               
                   
               
               
                 atcgagcggctgcagagctacttcccattcctgaagatcatggccgagaaccagagagagtacagcaacggcaag 
               
               
                   
               
               
                 tacaagcagaaccgcgtggaagtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaag 
               
               
                   
               
               
                 atgtacagggacctgaccaacgcAtacaagacctacgaggaaaagctgaacgacggctgcgagttcctgaccagc 
               
               
                   
               
               
                 acagagcaacctctgagcggcatgatcaacaactactacacagtggccctgcggaacatgaacgagagatacggc 
               
               
                   
               
               
                 tacaagacagaggacctggccttcatccaggacaagcggttcaagttcgtgaaggacgcctacggcaagaaaaag 
               
               
                   
               
               
                 tcccaagtgaataccggattcttcctgagcctgcaggactacaacggcgacacacagaagaagctgcacctgagc 
               
               
                   
               
               
                 ggagtgggaatcgccctgctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctgccc 
               
               
                   
               
               
                 atcttctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcggcatcaacagcatcaag 
               
               
                   
               
               
                 ctgcccaaggaccggatccacagcgagaagtccaacaagagcgtggccatggatatgctcaacgaagtgaagcgg 
               
               
                   
               
               
                 tgccccgacgagctgttcacaacactgtctgccgagaagcagtcccggttcagaatcatcagcgacgaccacaat 
               
               
                   
               
               
                 gaagtgctgatgaagcggagcagcgacagattcgtgcctctgctgctgcagtatatcgattacggcaagctgttc 
               
               
                   
               
               
                 gaccacatcaggttccacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatcgacggc 
               
               
                   
               
               
                 cagaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaagaggccgagacaatgcggaag 
               
               
                   
               
               
                 caagagaacggcaccttcggcaacagoggcatccggatcagagacttcgagaacatgaagcgggacgacgccaat 
               
               
                   
               
               
                 cctgccaactatccctacatcgtggacacctacacacactacatcctggaaaacaacaaggtcgagatgtttatc 
               
               
                   
               
               
                 aacgacaaagaggacagcgccccactgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagc 
               
               
                   
               
               
                 tgccggatgagcaccctggaaattccagccatggccttccacatgfttctgttcggcagcaagaaaaccgagaag 
               
               
                   
               
               
                 ctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaaagaagaagtgaccgccgagaat 
               
               
                   
               
               
                 atcgccagcttcggaatcgccgagagcgacctgcctcagaagatcctggatctgatcagcggcaatgcccacggc 
               
               
                   
               
               
                 aaggatgtggacgccttcatcagactgaccgtggacgacatgctgaccgacaccgagcggagaatcaagagattc 
               
               
                   
               
               
                 aaggacgaccggaagtccattcggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggc 
               
               
                   
               
               
                 aagctggccgacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgagaacaagatcacc 
               
               
                   
               
               
                 ggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcggcgacgattacgaggccaagcagcag 
               
               
                   
               
               
                 ttcaagctgatgttcgagaaggcccggctgatcggcaagggcacaacagagcctcatccatttctgtacaaggtg 
               
               
                   
               
               
                 ttcgcccgcagcatccccgccaatgccgtcgagttctacgagcgctacctgatcgagcggaagttctacctgacc 
               
               
                   
               
               
                 ggcctgtccaacgagatcaagaaaggcaacagagtggatgtgcccttcatcoggcgggaccagaacaagtggaaa 
               
               
                   
               
               
                 acacccgccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgcccagacagatgttcgac 
               
               
                   
               
               
                 aatgagatcaagtcccacctgaagtccctgccacagatggaaggcatcgacttcaacaatgccaacgtgacctat 
               
               
                   
               
               
                 ctgatcgccgagtacatgaagagagtgctggacgacgacttccagaccttctaccagtggaaccgcaactaccgg 
               
               
                   
               
               
                 tacatggacatgcttaagggcgagtacgacagaaagggctccctgcagcactgcttcaccagcgtggaagagaga 
               
               
                   
               
               
                 gaaggcctctggaaagagcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgcagcaac 
               
               
                   
               
               
                 cggcagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctgagcaacagccggaacgag 
               
               
                   
               
               
                 taccagaaaagcgagaaagtgatccggcgctacagagtgcaggatgccctgctgtttctgctggccaaaaagacc 
               
               
                   
               
               
                 ctgaccgaactggccgatttcgacggcgagaggttcaaactgaaagaaatcatgcccgacgccgagaagggaatc 
               
               
                   
               
               
                 ctgagcgagatcatgcccatgagcttcaccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatg 
               
               
                   
               
               
                 aagctgaagaactacggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaactcgtgggc 
               
               
                   
               
               
                 agcgacatcgtgtccaaagaggatatcatggaagagttcaacaaatacgaccagtgcaggcccgagatcagctcc 
               
               
                   
               
               
                 atcgtgttcaacctggaaaagtgggccttcgacacataccccgagctgtctgccagagtggaccgggaagagaag 
               
               
                   
               
               
                 gtggacttcaagagcatcctgaaaatcctgctgaacaacaagaacatcaacaaagagcagagcgacatcctgcgg 
               
               
                   
               
               
                 aagatccggaacgccttcgatgcAaacaattaccccgacaaaggcgtggtggaaatcaaggccctgcctgagatc 
               
               
                   
               
               
                 gccatgagcatcaagaaggcctttggggagtacgccatcatgaagggatccCTTCAACTGCCTCCACTTGAAAGA 
               
               
                   
               
               
                 CTGACACTGctcgagAGAGATTAG 
               
            
           
         
       
     
     An exemplary nuclease deficient Cas13b (dCas13b) nucleic acid sequence with stop codon (making it an independent reading frame) is as follows: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 437) 
                   
               
               
                 ATGaacatccccgctctggtggaaaaccagaagaagtactttggcacctacagcgtgatggccatgctgaacgct 
                   
               
               
                   
               
               
                 cagaccgtgctggaccacatccagaaggtggccgatattgagggcgagcagaacgagaacaacgagaatctgtgg 
               
               
                   
               
               
                 tttcaccccgtgatgagccacctgtacaacgccaagaacggctacgacaagcagcccgagaaaaccatgttcatc 
               
               
                   
               
               
                 atcgagcggctgcagagctacttcccattcctgaagatcatggccgagaaccagagagagtacagcaacggcaag 
               
               
                   
               
               
                 tacaagcagaaccgcgtggaagtgaacagcaacgacatcttcgaggtgctgaagcgcgccttcggcgtgctgaag 
               
               
                   
               
               
                 atgtacagggacctgaccaacgcAtacaagacctacgaggaaaagctgaacgacggctgcgagttcctgaccagc 
               
               
                   
               
               
                 acagagcaacctctgagcggcatgatcaacaactactacacagtggccctgcggaacatgaacgagagatacggc 
               
               
                   
               
               
                 tacaagacagaggacctggccttcatccaggacaagoggttcaagftcgtgaaggacgcctacggcaagaaaaag 
               
               
                   
               
               
                 tcccaagtgaataccggattcttcctgagcctgcaggactacaacggcgacacacagaagaagctgcacctgagc 
               
               
                   
               
               
                 ggagtgggaatcgccctgctgatctgcctgttcctggacaagcagtacatcaacatctttctgagcaggctgccc 
               
               
                   
               
               
                 atcttctccagctacaatgcccagagcgaggaacggcggatcatcatcagatccttcggcatcaacagcatcaag 
               
               
                   
               
               
                 ctgcccaaggaccggatccacagcgagaagtccaacaagagcgtggccatggatatgctcaacgaagtgaagcgg 
               
               
                   
               
               
                 tgccccgacgagctgttcacaacactgtctgccgagaagcagtcccggttcagaatcatcagcgacgaccacaat 
               
               
                   
               
               
                 gaagtgctgatgaagcggagcagcgacagattcgtgcctctgctgctgcagtatatcgattacggcaagctgttc 
               
               
                   
               
               
                 gaccacatcaggttccacgtgaacatgggcaagctgagatacctgctgaaggccgacaagacctgcatcgacggc 
               
               
                   
               
               
                 cagaccagagtcagagtgatcgagcagcccctgaacggcttcggcagactggaagaggccgagacaatgcggaag 
               
               
                   
               
               
                 caagagaacggcaccttcggcaacagcggcatccggatcagagacttcgagaacatgaagcgggacgacgccaat 
               
               
                   
               
               
                 cctgccaactatccctacatcgtggacacctacacacactacatcctggaaaacaacaaggtcgagatgtttatc 
               
               
                   
               
               
                 aacgacaaagaggacagcgccccactgctgcccgtgatcgaggatgatagatacgtggtcaagacaatccccagc 
               
               
                   
               
               
                 tgccggatgagcaccctggaaattccagccatggccttccacatgtttctgttcggcagcaagaaaaccgagaag 
               
               
                   
               
               
                 ctgatcgtggacgtgcacaaccggtacaagagactgttccaggccatgcagaaagaagaagtgaccgccgagaat 
               
               
                   
               
               
                 atcgccagcttcggaatcgccgagagcgacctgcctcagaagatcctggatctgatcagcggcaatgcccacggc 
               
               
                   
               
               
                 aaggatgtggacgccttcatcagactgaccgtggacgacatgctgaccgacaccgagcggagaatcaagagattc 
               
               
                   
               
               
                 aaggacgaccggaagtccattcggagcgccgacaacaagatgggaaagagaggcttcaagcagatctccacaggc 
               
               
                   
               
               
                 aagctggccgacttcctggccaaggacatcgtgctgtttcagcccagcgtgaacgatggcgagaacaagatcacc 
               
               
                   
               
               
                 ggcctgaactaccggatcatgcagagcgccattgccgtgtacgatagcggcgacgattacgaggccaagcagcag 
               
               
                   
               
               
                 ttcaagctgatgttcgagaaggcccggctgatcggcaagggcacaacagagcctcatccatttctgtacaaggtg 
               
               
                   
               
               
                 ttcgcccgcagcatccccgccaatgccgtcgagttctacgagcgctacctgatcgageggaagttctacctgacc 
               
               
                   
               
               
                 ggcctgtccaacgagatcaagaaaggcaacagagtggatgtgcccttcatccggcgggaccagaacaagtggaaa 
               
               
                   
               
               
                 acacccgccatgaagaccctgggcagaatctacagcgaggatctgcccgtggaactgcccagacagatgttcgac 
               
               
                   
               
               
                 aatgagatcaagtcccacctgaagtccctgccacagatggaaggcatcgacttcaacaatgccaacgtgacctat 
               
               
                   
               
               
                 ctgatcgccgagtacatgaagagagtgctggacgacgacttccagaccttctaccagtggaaccgcaactaccgg 
               
               
                   
               
               
                 tacatggacatgcttaagggcgagtacgacagaaagggctccctgcagcactgcttcaccagcgtggaagagaga 
               
               
                   
               
               
                 gaaggcctctggaaagagcgggcctccagaacagagcggtacagaaagcaggccagcaacaagatccgcagcaac  
               
               
                   
               
               
                 cggcagatgagaaacgccagcagcgaagagatcgagacaatcctggataagcggctgagcaacagccggaacgag  
               
               
                   
               
               
                 taccagaaaagcgagaaagtgatccggcgctacagagtgcaggatgccctgctgtttctgctggccaaaaagacc  
               
               
                   
               
               
                 ctgaccgaactggccgatttcgacggcgagaggttcaaactgaaagaaatcatgcccgacgccgagaagggaatc  
               
               
                   
               
               
                 ctgagcgagatcatgcccatgagcttcaccttcgagaaaggcggcaagaagtacaccatcaccagcgagggcatg  
               
               
                   
               
               
                 aagctgaagaactacggcgacttctttgtgctggctagcgacaagaggatcggcaacctgctggaactcgtgggc  
               
               
                   
               
               
                 agcgacatcgtgtccaaagaggatatcatggaagagttcaacaaatacgaccagtgcaggcccgagatcagctcc  
               
               
                   
               
               
                 atcgtgttcaacctggaaaagtgggccttcgacacataccccgagctgtctgccagagtggaccgggaagagaag  
               
               
                   
               
               
                 gtggacttcaagagcatcctgaaaatcctgctgaacaacaagaacatcaacaaagagcagagcgacatcctgcgg  
               
               
                   
               
               
                 aagatccggaacgccttcgatgcAaacaattaccccgacaaaggcgtggtggaaatcaaggccctgcctgagatc  
               
               
                   
               
               
                 gccatgagcatcaagaaggcctttggggagtacgccatcatgaagTAG 
               
            
           
         
       
     
     Exemplary wild type Cas13d proteins of the disclosure may comprise or consist of the amino acid sequences: 
                                    Cas13d ( Ruminococcus     IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI         flavefaciens  XPD3002)   RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGP           VQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEY           ITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNN           NDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGN           ECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYIST           LNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRF           SIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYT           MMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSF           NDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPR           LPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQ           SFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPI           ADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKH           GMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRI           ADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGM           NYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVN           INARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAG           IDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEK           KAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTL           FANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYE           KSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEA           LFDRNEAAKFDKEKKSGNS (SEQ ID NO: 438)               Cas13d (contig e-   MKRQKTFAKRIGIKSTVAYGQGKYAITTFGKGSKAEIAVRSADPP       k87_11092736)   EETLPTESDATLSIHAKFAKAGRDGREFKCGDVDETRIHTSRSEY           ESLISNPAESPREDYLGLKGTLERKFFGDEYPKDNLRIQIIYSIL           DIQKILGLYVEDILHFVDGLQDEPEDLVGLGLGDEKMQKLLSKAL           PYMGFFGSTDVFKVTKKREERAAADEHNAKVFRALGAIRQKLAHF           KWKESLAIFGANANMPIRFFQGATGGRQLWNDVIAPLWKKRIERV           RKSFLSNSAKNLWVLYQVFKDDTDEKKKARARQYYHFSVLKEGKN           LGFNLTKTREYFLDKFFPIFHSSAPDVKRKVDTFRSKFYAILDFI           IYEASVSVANSGQMGKVAPWKGAIDNALVKLREAPDEEAKEKIYN           VLAASIRNDSLFLRLKSACDKFGAEQNRPVFPNELRNNRDIRNVR           SEWLEATQDVDAAAFVQLIAFLCNFLEGKEINELVTALIKKFEGI           QALIDLLRNLEGVDSIRFENEFALFNDDKGNMAGRIARQLRLLAS           VGKMKPDMTDAKRVLYKSALEILGAPPDEVSDEWLAENILLDKSN           NDYQKAKKTVNPFRNYIAKNVITSRSFYYLVRYAKPTAVRKLMSN           PKIVRYVLKRLPEKQVASYYSAIWTQSESNSNEMVKLIEMIDRLT           TEIAGFSFAVLKDKKDSIVSASRESRAVNLEVERLKKLTTLYMSI           AYIAVKSLVKVNARYFIAYSALERDLYFFNEKYGEEFRLHFIPYE           LNGKTCQFEYLAILKYYLARDEETLKRKCEICEEIKVGCEKHKKN           ANPPYEYDQEWIDKKKALNSERKACERRLHFSTHWAQYATKRDEN           MAKHPQKWYDILASHYDELLALQATGWLATQARNDAEHLNPVNEF           DVYIEDLRRYPEGTPKNKDYHIGSYFEIYHYIRQRAYLEEVLAKR           KEYRDSGSFTDEQLDKLQKILDDIRARGSYDKNLLKLEYLPFAYN           LPRYKNLTTEALFDDDSVSGKKRVAEWREREKTREAEREQRRQR           (SEQ ID NO: 439)               Cas13d (contig e-   GTGAGAAGTCTCCTTATGGGGAGATGCTAC (SEQ ID NO:       k87_11092736) Direct   300)       Repeat Sequence                   Cas13d   MKNSVTFKLIQAQENKEAARKKAKDIAEQARIAKRNGVVKKEENR       (160582958_gene49834)   INRIQIEIQTQKKSNTQNAYHLKSLAKAAGVKSVFAIGNDLLMTG           FGPGNDATIEKRVFQNRAIETLSSPEQYSAEFQNKQFKIKGNIKV           LNHSTQKMEEIQTELQDNYNRPHFDLLGCKNVLEQKYFGRTFSDN           IHVQIAYNIMDIEKLLTPYINNIIYTLNELMRDNSKDDFFGCDSH           FSVAYLYDELKAGYSDRLKTKPNLSKNIDRIWNNFCNYMNSDSGN           TEARLAYFGELFYKPKETGDAKSDYKTHLSNNQKEEWELKSDKEV           YNIFAILCDLRHFCTHGESITPSGKPFPYNLEKNLFPEAKQVLNS           LFEEKAESLGAEAFGKTAGKTDVSILLKVFEKEQASQKEQQALLK           EYYDFKVQKTYKNMGFSIKKLREAIMEIPDAAKFKDDLYSSLRHK           LYGLFDFILVKHFLDTSDSENLQNNDIFRQLRACRCEEEKDQVYR           SIAVKVWEKVKKKELNMFKQVVVIPSLSKDELKQMEMTKNTELLS           SIETISTQASLFSEMIFMMTYLLDGKEINLLCTSLIEKFENIASF           NEVLKSPQIGYETKYTEGYAFFKNADKTAKELRQVNNMARMTKPL           GGVNTKCVMYNEAAKILGAKPMSKAELESVFNLDNHDYTYSPSGK           KIPNKNFRNFIINNVITSRRFLYLIRYGNPEKIRKIAINPSIISF           VLKQIPDEQIKRYYPPCIGKRTDDVTLMRDELGKMLQSVNFEQFS           RVNNKQNAKQNPNGEKARLQACVRLYLTVPYLFIKNMVNINARYV           LAFHCLERDHALCFNSRKLNDDSYNEMANKFQMVRKAKKEQYEKE           YKCKKQETGTAHTKKIEKLNQQIAYIDKDIKNMHSYTCRNYRNLV           AHLNVVSKLQNYVSELPNDYQITSYFSFYHYCMQLGLMEKVSSKN           IPLVESLKNEANDAQSYSAKKTLEYFDLIEKNRTYCKDFLKALNA           PFSYNLPRFKNLSIEALFDKNIVYEQADLKKE (SEQ ID NO:           440)               Cas13d   GAACTACACCCCTCTGTTCTTGTAGGGGTCTAACAC (SEQ ID       (160582958_gene49834)   NO: 301)       Direct Repeat           Sequence                   Cas13d (contig   MKKQKSKKTVSKTSGLKEALSVQGTVIMTSFGKGNMANLSYKIPS       tpg|DJXD01000002.1|;   SQKPQNLNSSAGLKNVEVSGKKIKFQGRHPKIATTDNPLFKPQPG       uncultivated   MDLLCLKDKLEMHYFGKTFDDNIHIQLIYQILDIEKILAVHVNNI         Ruminococcus     VFTLDNVLHPQKEELTEDFIGAGGWRINLDYQTLRGQTNKYDRFK       assembly UBA7013,   NYIKRKELLYFGEAFYHENERRYEEDIFAILTLLSALRQFCFHSD       from sheep gut   LSSDESDHVNSFWLYQLEDQLSDEFKETLSILWEEVTERIDSEFL       metagenome)   KTNTVNLHILCHVFPKESKETIVRAYYEFLIKKSFKNMGFSIKKL           REIMLEQSDLKSFKEDKYNSVRAKLYKLFDFIITYYYDHHAFEKE           ALVSSLRSSLTEENKEEIYIKTARTLASALGADFKKAAADVNAKN           IRDYQKKANDYRISFEDIKIGNTGIGYFSELIYMLTLLLDGKEIN           DLLTTLINKFDNIISFIDILKKLNLEFKFKPEYADFFNMTNCRYT           LEELRVINSIARMQKPSADARKIMYRDALRILGMDNRPDEEIDRE           LERTMPVGADGKFIKGKQGFRNFIASNVIESSRFHYLVRYNNPHK           TRTLVKNPNVVKFVLEGIPETQIKRYFDVCKGQEIPPTSDKSAQI           DVLARIISSVDYKIFEDVPQSAKINKDDPSRNFSDALKKQRYQAI           VSLYLTVMYLITKNLVYVNSRYVIAFHCLERDAFLHGVTLPKMNK           KIVYSQLTTHLLTDKNYTTYGHLKNQKGHRKWYVLVKNNLQNSDI           TAVSSFRNIVAHISVVRNSNEYISGIGELHSYFELYHYLVQSMIA            KNNWYDTSHQPKTAEYLNNLKKHHTYCKDFVKAYCIPFGYVVPRY           KNLTINELFDRNNPNPEPKEEV (SEQ ID NO: 441)               Cas13d (contig   CAACTACAACCCCGTAAAAATACGGGGTTCTGAAAC (SEQ ID       tpg|DJXD01000002.1|;   NO: 442)       uncultivated             Ruminococcus             assembly, UBA7013,           from sheep gut           metagenome)                   Cas13d   SEQ ID NO: 443       (Gut_metagenome_contig6049000251)                   Cas13d   SEQ ID NO: 444       (Gut_metagenome_contig546000275)                   Cas13d   SEQ ID NO: 445       (Gut_metagenome_contig4114000374)                   Cas13d   SEQ ID NO: 446       (Gut_metagenome_contig721000619)                   Cas13d   SEQ ID NO: 447       (Gut_metagenome_contig2002000411)                   Cas13d   SEQ ID NO: 448       (Gut_metagenome_contig13552000311)                   Cas13d   SEQ ID NO: 449       (Gut_metagenome_contig10037000527)                   Cas13d (293. Cas13d   SEQ ID NO: 450       from           Gut_metagenome_contig238000329)                   Cas13d   SEQ ID NO: 451       (Gut_metagenome_contig2643000492)                   Cas13d   SEQ ID NO: 452       (Gut_metagenome_contig874000057)                   Cas13d   SEQ ID NO: 453       (Gut_metagenome_contig4781000489)                   Cas13d   SEQ ID NO: 454       (Gut_metagenome_contig12144000352)                   Cas13d   SEQ ID NO: 455       (Gut_metagenome_contig5590000448)                   Cas13d   SEQ ID NO: 456       (Gut_metagenome_contig525000349)                   Cas13d   SEQ ID NO: 457       (Gut_metagenome_contig7229000302)                   Cas13d   SEQ ID NO: 458       (Gut_metagenome_contig3227000343)                   Cas13d   SEQ ID NO: 459       (Gut_metagenome_contig7030000469)                   Cas13d   SEQ ID NO: 460       (Gut_metagenome_P17E0k2120140920,           _c87000043)                   Cas13d (Metagenomic   SEQ ID NO: 461       hit (no protein           accession): contig           emb|OBVH01003037.1,           human gut           metagenome sequence           (also found in WGS           contigs           emb|OBXZ01000094.1|           and           emb|OBJF01000033.1|))                   Cas13d (Metagenomic   SEQ ID NO: 462       hit (no protein           accession): contig           OGZC01000639.1           (human gut           metagenome           assembly))                   Cas13d (Metagenomic   SEQ ID NO: 463       hit (no protein           accession): contig           emb|OHBM01000764.1           (human gut            metagenome           assembly))                   Cas13d (Metagenomic   SEQ ID NO: 464       hit (no protein           accession): contig           emb|OHCP01000044.1           (human gut           metagenome           assembly))                   Cas13d (Metagenomic   SEQ ID NO: 465       hit (no protein           accession): contig           emb|OGDF01008514.1|           (human gut           metagenome           assembly))                   Cas13d (Metagenomic   SEQ ID NO: 466       hit (no protein           accession): contig           emb|OGPN01002610.1           (human gut           metagenome           assembly))                   Cas13d (Metagenomic   SEQ ID NO: 467        hit (no protein           accession): from contig           emb|OBLI01020244           and           emb|OBLI01038679           (from pig gut           metagenome))                   Cas13d (Metagenomic   SEQ ID NO: 468       hit (no protein           accession): contig            OIZX01000427.1)                   Cas13d (Metagenomic   SEQ ID NO: 469       hit (no protein           accession): contig           OCTW011587266.1)                   Cas13d (Metagenomic   SEQ ID NO: 470       hit (no protein           accession): contig           emb|OGNF01009141.1)                    Cas13d (Metagenomic   SEQ ID NO: 471       hit (no protein           accession): contig           emb|OIEN01002196.1|)                    Cas13d   SEQ ID NO: 472       (Ga0129306_1000735)                   Cas13d   SEQ ID NO: 473       (Ga0129317_1008067)                   Cas13d   SEQ ID NO: 474       (Ga0224415_10048792)                   Cas13d   SEQ ID NO: 475       (250twins_35838_GL0110300)                   Cas13d   SEQ ID NO: 476       (250twins_36050_GL0158985)               Additional exemplary Cas13d sequences and direct repeat sequences for Cas13d are listed as SEQ ID Nos: 1-277 in the corresponding sequence listing.            
IV. Nucleic Acids Encoding the Capped-sgRNA and/or the Cas Polypeptide
 
     Some embodiments disclosed herein provide compositions comprising a nucleic acid sequence encoding the capped-sgRNAs described herein, and vectors (e.g., expression vector(s)) comprising the nucleic acid sequences. In some embodiments, nucleic acid sequences encoding the capped-sgRNAs are operably linked to one or more promoters. Suitable promoters include, without limitation, RNA polymerase II promoters such as, without limitation, CMV, PGK, and EF1α promoters. In one embodiment, the RNA polymerase II promoter is an RNA Pol II transcribed non-coding RNA. The sgRNA is transcribed by the RNAase polymerase II, acquires an m7G cap and becomes polyadenylated. Additional promoters suitable for driving expression of the capped-sgRNA are also contemplated, such as, without limitation, bacteriophage promoters (e.g., RNA polymerase T3, T7, and SP6), ubiquitous promoters, tissue-specific promoters, inducible promoters, and constitutive promoters. For example, liver-specific promoters as described in PCT/US06/00668 are contemplated herein. For example, promoters for genes encoding genes encoding c-jun; jun-b; c-fos; c-myc; serum amyloid A; apolipoprotein B editing catalytic subunit; liver regeneration factors, such as LRF-I; signal transducers; activators of transcription, such as STAT-3; serum alkaline phosphates (SAP); insulin-like growth factor-binding proteins, such as IGFBP-I; cyclin D1; active protein-1; CCAAT enhancer core binding protein; ornithine decarboxylase; phosphatase of regenerating liver-1; early growth response gene-1; hepatocyte growth factors; hemopexin; insulin-like growth factors (IGF), such as IGF-I and IGF-2; hepatocyte nuclear family 1; hepatocyte nuclear family 4; hepatocyte Arg-Ser-rich domain-containing proteins; glucose 6-phosphatase; acute phase proteins, such as serum amyloid A and serum amyloid P (SAA/SAP); steroid hydroxylases; leukotriene hydroxylases; fatty acid hydroxylases; desmolase; peptidyl isomerases; and sterol demethylases. 
     In one embodiment, vectors comprising the nucleic acids that encode the capped-sgRNA further include a sequence that encodes a Cas polypeptide (e.g., any of the Cas polypeptides described herein, such as, without limitation, a truncated nuclease-deficient Cas protein). The capped-sgRNA and the Cas transcript can be transcribed from the same promoter, or from different promoters. RNA polymerase II promoters (e.g. CMV, PGK, and EF1α promoters), for example, are suitable for driving expression of both the sgRNA and the Cas gene. In some embodiments, the Cas transcript is expressed from one promoter, such as a PGK promoter, and the capped-sgRNA is expressed from a different promoter, such as an EF1α promoter. 
     Some embodiments disclosed herein provide nucleic acids encoding the Cas polypeptides and vectors comprising the nucleic acids that encode the Cas polypeptides. Nucleic acids encoding the Cas polypeptides can be operably linked to one or more promoters. Suitable promoters include RNA polymerase II promoters (e.g., CMV, PGK, and EF1α promoters), bacteriophage promoters (e.g., RNA polymerase T3, T7, and SP6), ubiquitous promoters, tissue-specific promoters, inducible promoters, and constitutive promoters. The Cas polypeptide can be associated with or include or be in operable linkage with a tag or detectable agent, such as a fluorescent agent, a fluorescent protein, an enzyme. 
     In some embodiments, a sequence encoding a capped-guide RNA of the disclosure includes a sequence encoding a promoter to drive expression of the guide RNA. In some embodiments, a vector that includes a sequence encoding a capped-guide RNA of the disclosure includes a sequence encoding a promoter to drive expression of the guide RNA. In some embodiments, a promoter driving expression of the guide RNA includes a sequence encoding a constitutive promoter, or an inducible promoter. In some embodiments, a promoter to drive expression of the guide RNA includes a sequence encoding a hybrid or a recombinant promoter. In some embodiments, a promoter to drive expression of the guide RNA is a promoter capable of expressing the guide RNA in a mammalian cell or a human cell. In some embodiments, a promoter to drive expression of the guide RNA is a promoter capable of expressing the guide RNA and restricting the guide RNA to the nucleus of the cell. In some embodiments, a promoter to drive expression of the guide is a human RNA polymerase promoter or a sequence isolated or derived from a human RNA polymerase promoter. In some embodiments, a promoter to drive expression of the guide RNA is a U6 promoter or a sequence isolated or derived from a U6 promoter. In some embodiments, a promoter to drive expression of the guide RNA is a human tRNA promoter or a sequence isolated or derived from a human tRNA promoter. In some embodiments, a promoter to drive expression of the guide RNA is a human valine tRNA promoter or a sequence isolated or derived from a human valine tRNA promoter. 
     In some embodiments of the compositions of the disclosure, a promoter to drive expression of the capped-guide RNA further includes a regulatory element. In some embodiments, a vector that includes promoter to drive expression of the guide RNA further includes a regulatory element. In some embodiments, a regulatory element enhances expression of the guide RNA. Exemplary regulatory elements include, but are not limited to, an enhancer element, an intron, an exon, or a combination thereof. 
     In some embodiments, the nucleic acid sequences encoding the Cas polypeptides are linked to one or more localization signals. Localization signals are amino acid sequences on a protein that tags the protein for transportation to a particular location in a cell. An exemplary localization signal is a nuclear localization signal (NLS), which is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. In one embodiment, one or more localization signals is/are operably linked to the sequence encoding a Cas polypeptide. In some embodiments, the localization signal is a nuclear localization signal (NLS). An exemplary NLS is SV40 large T antigen NLS (PKKKRRV (SEQ ID NO: 477)) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO: 478)). Other NLSs are known in the art; see, e.g., Konermann et al., Cell 173:665-676, 2018; Cokol et al., EMBO Rep. 1(5):411-415 (2000); Freitas and Cunha, Curr Genomics 10(8): 550-557 (2009), incorporated herein by reference in their entirety. Without limitation, additional NLSs are those that have K(K/R)X(K/R) as a putative consensus sequence (e.g., PAAKRVKLD (SEQ ID NO: 479)). Other additional NLSs include KRSWSMAF (SEQ ID NO: 480) and KRKYF (SEQ ID NO: 481). In some embodiments, vectors comprising the nucleic acids that encode the Cas polypeptide further encode a capped-sgRNA (e.g., any of the capped-sgRNAs described herein). In some embodiments, the localization signal is a nuclear export signal (NES). Incorporating an NES is particularly suited for altering molecular machinery in the cytoplasm. In one embodiment, an NES is the HIV-REV NES or the PKI NES. 
     In other embodiments, the nucleic acids encoding the capped-sgRNA and/or the Cas polypeptide can be further operably linked to a sequence that encodes one or more reporter genes, or effector genes such as, without limitation, endonucleases that have nuclease activity. In one embodiment, a nucleic acid sequence encodes a capped-sgRNA disclosed herein and a fusion protein that includes a dCas polypeptide and an endonuclease. Any suitable reporter genes are contemplated, including but not limited to, fluorescent reporters. In addition, any suitable endonucleases are contemplated for fusing with a Cas polypeptide, in particular a dCas polypeptide. 
     V. Vectors 
     Vectors contemplated for the present disclosure can include those that are suitable for expression in a selected host, whether prokaryotic or eukaryotic, for example, phage, plasmid, and viral vectors. Viral vectors may be either replication competent or replication defective retroviral vectors. Viral propagation generally will occur only in complementing host cells comprising replication defective vectors, for example, when using replication defective retroviral vectors in methods provided herein viral replication will not occur. Vectors may comprise kozak sequences (Lodish et al., Molecular Cell Biology, 4th ed., 1999) and may also contain the ATG start codon. Promoters that function in a eukaryotic host are from, without limitation, SV40, LTR, CMV, EF-1 a, white cloud mountain minnow β-actin. 
     In some embodiments of the compositions of the disclosure, a vector of the disclosure includes one or more of a sequence encoding at least one capped-guide RNA of the disclosure, one or more promoters to drive expression of the one or more guide RNAs and a sequence encoding a regulatory element. In some embodiments of the compositions of the disclosure, the vector further includes a sequence encoding a Cas polypeptide, a dCas polypeptide or a dCas-fusion protein. 
     Copy number and positional effects are considered in designing transiently and stably expressed vectors. Copy number can be increased by, for example, dihydrofolate reductase amplification. Positional effects can be optimized by, for example, Chinese hamster elongation factor-1 vector pDEF38 (CHEF1), ubiquitous chromatin opening elements (UCOE), scaffold/matrix-attached region of human (S/MAR), and artificial chromosome expression (ACE) vectors, as well as by using site-specific integration methods known in the art. The expression constructs containing the vector can further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs can include a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. 
     Considering the above-mentioned factors, exemplary vectors suitable for expressing Cas polypeptides and/or sgRNAs in bacteria include PiggyBac transposon vectors, pTT vectors (e.g., from Biotechnology Research Institute (Montreal, Canada)), pQE70, pQE60, and pQE-9 (e.g. those available from Qiagen (Mississauga, Ontario, Canada)); vectors derived from pcDNA3, available from Invitrogen (Carlsbad, Calif.); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH6a, pNH18A, pNH46A, available from Stratagene (La Jolla, Calif.); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia (Peapack, N.J.). Among suitable eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene (La Jolla, Calif.); and pSVK3, pBPV, pMSG and pSVL, available from Pharmacia (Peapack, N.J.). 
     A pTT vector backbone can be used for expressing the Cas polypeptide and/or sgRNA (Durocher et al., Nucl. Acids Res. 30:E9 (2002)). Briefly, the backbone of a pTT vector may be prepared by obtaining pIRESpuro/EGFP (pEGFP) and pSEAP basic vector(s), for example from Clontech (Palo Alto, Calif.), and pcDNA3.1, pCDNA3.1/Myc-(His)6 and pCEP4 vectors can be obtained from, for example, Invitrogen (Carlsbad, Calif.). As used herein, the pTT5 backbone vector can generate a pTT5-Gateway vector and be used to transiently express proteins in mammalian cells. The pTT5 vector can be derivatized to pTT5-A, pTT5-B, pTT5-D, pTT5-E, pTT5-H, and pTT5-I, for example. As used herein, the pTT2 vector can generate constructs for stable expression in mammalian cell lines. 
     A pTT vector can be prepared by deleting the hygromycin (Bsml and Sail excision followed by fill-in and ligation) and EBNA1 (Clal and Nsil excision followed by fill-in and ligation) expression cassettes. The ColEI origin (Fspl-Sall fragment, including the 3′ end of the β-lactamase open reading frame (ORF) can be replaced with a Fspl-Sall fragment from pcDNA3.1 containing the pMBI origin (and the same 3′ end of β-lactamase ORF). A Myc-(His)6 C-terminal fusion tag can be added to SEAP (Hindlll-Hpal fragment from pSEAP-basic) following in-frame ligation in pcDNA3.1/Myc-His digested with Hindlll and EcoPvV. Plasmids can subsequently be amplified in  E. coli  (DH5a) grown in LB medium and purified using MAXI prep columns (Qiagen, Mississauga, Ontario, Canada). To quantify, plasmids can be subsequently diluted in, for example, 50 mM Tris-HCl pH 7.4 and absorbencies can be measured at 260 nm and 280 nm. Plasmid preparations with A260/A280 ratios between about 1.75 and about 2.00 are suitable for producing the Fc-fusion constructs. 
     The expression vector pTT5 allows for extrachromosomal replication of the cDNA driven by a cytomegalovirus (CMV) promoter. The plasmid vector pCDNA-pDEST40 is a Gateway-adapted vector which can utilize a CMV promoter for high-level expression. SuperGlo GFP variant (sgGFP) can be obtained from Q-Biogene (Carlsbad, Calif.). Preparing a pCEP5 vector can be accomplished by removing the CMV promoter and polyadenylation signal of pCEP4 by sequential digestion and self-ligation using Sail and Xbal enzymes resulting in plasmid pCEP4A. A Gblll fragment from pAdCMV5 (Massie et al., J. Virol. 72:2289-2296 (1998)), encoding the CMV5-poly(A) expression cassette ligated in Bglll-linearized pCEP4A, resulting in the pCEP5 vector. 
     Additional vectors include optimized for use in CHO-S or CHO-S-derived cells, such as pDEF38 (CHEF 1) and similar vectors (Running Deer et al., Biotechnol. Prog. 20:880-889 (2004)). The CHEF vectors contain DNA elements that lead to high and sustained expression in CHO cells and derivatives thereof. They may include, but are not limited to, elements that prevent the transcriptional silencing of transgenes. 
     Vectors may include a selectable marker for propagation in a host. A selectable marker can allow the selection of transformed cells based on their ability to thrive in the presence or absence of a chemical or other agent that inhibits an essential cell function. Selectable markers confer a phenotype on a cell expressing the marker, so that the cell can be identified under appropriate conditions. Suitable markers include genes coding for proteins which confer drug resistance or sensitivity thereto, impart color to, or change the antigenic characteristics of those cells transfected with a molecule encoding the selectable marker, when the cells are grown in an appropriate selective medium. 
     Suitable selectable markers include dihydro folate reductase or G41 8 for neomycin resistance in eukaryotic cell culture; and tetracycline, kanamycin, or ampicillin resistance genes for culturing in  E. coli  and other bacteria. Suitable selectable markers also include cytotoxic markers and drug resistance markers, whereby cells are selected by their ability to grow on media containing one or more of the cytotoxins or drugs; auxotrophic markers, by which cells are selected for their ability to grow on defined media with or without particular nutrients or supplements, such as thymidine and hypoxanthine; metabolic markers for which cells are selected, for example, for ability to grow on defined media containing a defined substance, for example, an appropriate sugar as the sole carbon source; and markers which confer the ability of cells to form colored colonies on chromogenic substrates or cause cells to fluoresce. 
     Retroviral vectors are contemplated herein. One such vector, the ROSA geo retroviral vector, which maps to mouse chromosome six, was constructed with the reporter gene in reverse orientation with respect to retroviral transcription, downstream of a splice acceptor sequence (U.S. Pat. No. 6,461,864; Zambrowicz et al., Proc. Natl. Acad. Sci. 94:3789-3794 (1997)). Infecting embryonic stem (ES) cells with ROSA geo retroviral vector resulted in the ROSA geo26 (ROSA26) mouse strain by random retroviral gene trapping in the ES cells. 
     Adeno-associated viral vectors (AAV vectors) are contemplated herein. The term“adeno-associated virus” or “AAV” as used herein can refer to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 or 12, sequentially numbered, are disclosed in the prior art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g., AAV2, AAV5, and AAV8, or variant serotypes, e.g. AAV-DJ. The AAV structural particle is composed of 60 protein molecules made up of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure. 
     AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro and Immunol. 158:97-129 (1992)). AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. AAV vectors have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther. 2009 August; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April; 20(4): 699-708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. For example, AAV2, AAV5, AAV2/5, AAV2/8 and AAV2/7 vectors have been used to introduce DNA into photoreceptor cells (see, e.g., Pang et al., Vision Research 2008, 48(3):377-385; Khani et al., Invest Ophthalmol Vis Sci. 2007 September; 48(9):3954-61; Allocca et al., J. Virol. 2007 81(20):11372-11380). In some embodiments, the AAV vector can include (or include a sequence encoding) an AAV capsid polypeptide described in PCT/US2014/060163; for example, a virus particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 of PCT/US2014/060163, and a Cas sequence and capped-guide RNA sequence as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, e.g., Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44. In some embodiments, the AAV incorporates inverted terminal repeats (ITRs) derived from the AAV2 serotype. Exemplary left and right ITRs are presented in Table 6 of WO 2018/026976 and are listed below: 
     
       
         
           
               
            
               
                 AAV2 Left ITR 
               
               
                 (SEQ ID NO: 482) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA 
               
               
                   
               
               
                 AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA 
               
               
                   
               
               
                 GCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 
               
               
                   
               
               
                 AAV2 Right ITR 
               
               
                 (SEQ ID NO: 483) 
               
               
                 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGC 
               
               
                   
               
               
                 TCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCC 
               
               
                   
               
               
                 CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
            
           
         
       
     
     It should be noted, however, that numerous modified versions of the AAV2 ITRs are used in the field, and the ITR sequences shown below are exemplary and are not intended to be limiting. Modifications of these sequences are known in the art, or will be evident to skilled artisans, and are thus included in the scope of this disclosure. Expression of the Cas polypeptide and/or sgRNA in the AAV vector can be driven by a promoter described herein or known in the art. In some embodiments, AAV vectors capable of delivering ˜4.5 kb are used for packaging of the nucleic acids encoding capped-sgRNAs or Cas polypeptides. In some embodiments, AAVs capable of packaging larger transgenes such as about 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5.0 kb, 5.1 kb, 5.2 kb, 5.3 kb, 5.4 kb, 5.5 kb, 5.6 kb, 5.7 kb, 5.8 kb, 5.9 kb, 6.0 kb, 6.1 kb, 6.2 kb, 6.3 kb, 6.4 kb, 6.5 kb, 6.6 kb, 6.7 kb, 6.8 kb, 6.9 kb, 7.0 kb, 7.5 kb, 8.0 kb, 9.0 kb, 10.0 kb, 11.0 kb, 12.0 kb, 13.0 kb, 14.0 kb, 15.0 kb, or larger are used. 
     A DNA insert comprising nucleic acids (optionally contained in a vector or vectors) encoding Cas9 polypeptides or sgRNAs can be operatively linked to an appropriate promoter, such as the phage lambda PL promoter; the  E. coli  lac, trp, phoA, and tac promoters; the SV40 early and late promoters; and promoters of retroviral LTRs. Suitable vectors and promoters also include the pCMV vector with an enhancer, pcDNA3.1; the pCMV vector with an enhancer and an intron, pCIneo; the pCMV vector with an enhancer, an intron, and a tripartate leader, pTT2, and CHEF1. The promoter sequences include at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence may be a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. In alternative embodiments, eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. 
     The expression constructs can further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs can include a translation initiating codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated. 
     In some embodiments of the compositions and methods of the disclosure, the vector is or comprises an “RNA targeting system” comprising (a) nucleic acid sequence encoding an Cas polypeptide or dCas polypeptide or dCas polypeptide fusion protein; and (b) a capped-single guide RNA (capped-sgRNA) sequence comprising: an RNA sequence (or spacer sequence) that hybridizes to or binds to a target RNA sequence and an RNA sequence (direct repeat or scaffold sequence) capable of binding to or associating with the Cas polypeptide and wherein the RNA targeting system recognizes the target RNA and enhances translation of the target RNA. In some embodiments, the nucleic acid sequence or vector is a single vector. 
     VI. Cells 
     Some embodiments disclosed herein provide cells comprising the nucleic acid or nucleic acids (e.g., vector or vectors) that encode Cas9 polypeptides or capped-sgRNAs. In some embodiments, the cells transfected may be a prokaryotic cell, a eukaryotic cell, a yeast cell, an insect cell, an animal cell, a mammalian cell, a human cell, etc. The proteins expressed in mammalian cells have been glycosylated properly. Examples of useful mammalian host cell lines are HEK293, CHO, sp2/0, NSO, COS, BHK, and PerC6. 
     In some embodiments, the target mRNA is in a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in a subject or patient. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition comprises a vector comprising composition comprising the capped-guide RNA of the disclosure and/or a Cas polypeptide or dCas polypeptide. In some embodiments, the vector is a viral vector for transducing a cell. In some embodiments, the viral vector is an AAV vector. 
     Transfection of animal cells typically involves opening transient pores or “holes” in the cell membrane, to allow the uptake of material. There are various methods of introducing foreign DNA into a eukaryotic cell. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes, and nanoparticles. 
     VII. Methods of Modulating Protein Translation 
     Some embodiments disclosed herein provide compositions for and methods of enhancing protein translation in a cell, comprising introducing capped-sgRNAs (e.g., any of the capped-sgRNAs described herein) and Cas polypeptides (e.g., any of the Cas polypeptides described herein) into the cell. The methods of enhancing protein translation can include introducing or administering a nucleic acid or nucleic acids (e.g., vector or vectors) encoding the capped-sgRNA and the Cas polypeptides into a cell. In some embodiments, provided herein are methods of regulating translation of an mRNA in a cell that include contacting the cell with a nucleic acid comprising (a) a sequence encoding a Cas polypeptide; and (b) a sequence encoding a capped-sgRNA comprising (i) an m7G cap; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. 
     Methods of measuring levels of protein translation are known in the art. Exemplary methods include, without limitation, western blot, mass spectrometry, antibody staining, and mean fluorescence intensity flow cytometry. In instances where a reporter construct is linked to the target mRNA, protein translation can also be measured based on the levels of the reporter molecule. 
     In some embodiments, enhancing translation or increasing or upregulating gene expression refers to an increase in the amount of peptide translated from the target mRNA as compared to a control. In some embodiments, the control includes a level of peptide translated from the target mRNA in the absence of the capped-sgRNA compositions and methods. In some embodiments, the control includes the level of the peptide translated from the target mRNA prior to addition of the compositions disclosed herein. In some embodiments, translation is increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 1000 fold, or about 10,000 fold relative to the control. The amount of peptide translated can be determined by any method known in the art. 
     Gene Therapy/Therapeutic Targets 
     In certain embodiments, methods of modulating protein translation are useful for treating patients afflicted with a disease or disorder. In one embodiment, methods of using the capped-sgRNA compositions disclosed herein are haploinsufficiencies. Exemplary haploinsufficiency diseases or disorders include, without limitation, Autosomal dominant Retinitis Pigmentosa (RP11) caused by mutations in PRPF31, Autosomal dominant Retinitis Pigmentosa (RP31) caused by mutations in TOPORS, Frontotemporal dementia caused by mutations in GRN, DeVivo Syndrome (Glut1 deficiency) caused by mutations in SLC2A1, Dravet syndrome caused by mutations in SCN1A, 1q21.1 Deletion Syndrome, 5q-Syndrome in Myelodysplastic Syndrome (MDS), 22q11.2 Deletion Syndrome, CHARGE Syndrome, Cleidocrainial Dysostosis, Ehlers-Danlos Syndrome, Frontotemporal Dementia caused by mutations in Progranulin, Haploinsufficiency of A20, Holoprosencephaly (caused by haploinsufficiency in the Sonic Hedgehog gene), Holt-Oram Syndrome, Marfan Syndrome, Dyskeratosis Congenita, and Phelan-McDermid Syndrome. 
     In another embodiment, methods of using the capped-sgRNA compositions disclosed herein for treating haploinsufficiency diseases or disorders, such as, without limitation, those listed in the preceding paragraph, involving mutations which lead to introduction of a premature termination codon (PTC) resulting in degradation from mutant allele or loss of function of the protein (or less protein to be produced) are contemplated herein. 
     In another embodiment, methods of translation enhancement using the capped-sgRNA compositions disclosed herein are useful for treating cancer. In one embodiment, the methods can be used for upregulating protein expression of tumor suppressor genes (TSG) in tissue predisposed to cancer due to hereditary (or acquired) mutations of TSG. In another embodiment, the methods can be used for upregulating protein expression from genes that would prevent cancer from metastasizing (ie angiogenesis genes). In another embodiment, the methods can be used for upregulating protein expression from genes that would result in the cancer being more susceptible to follow-up treatments. In another embodiment, the methods can be used for translational enhancement to prevent cancer evasion of the immune system. 
     As used herein, the “administration” of the compositions disclosed herein (e.g., a fusion RNA, viral particle, vector, polynucleotide, cell, population of cells, or pharmaceutical composition or formulation) to a subject includes any route of introducing or delivering to a subject the agent to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, intraocularly, ophthalmically, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another. 
     In some aspects, the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a capped-sgRNA composition(s) of the disclosure. Also provided herein are methods for treating a disease or condition in a subject in need thereof, the methods comprising, consisting of, or consisting essentially of administering a nucleic acid sequence comprising/encoding (a) a capped-sgRNA disclosed herein; and (b) a dCas polypeptide, a vector comprising the nucleic acid sequence, or a viral particle comprising the vector to the subject, thereby enhancing translation of a target mRNA in the subject or patient. In some embodiments, the target mRNA is involved in the etiology of a disease or condition in the subject. 
     In some embodiments of the methods described herein, the subject or patient is an animal. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a bovine, equine, porcine, canine, feline, simian, murine, or human. In some embodiments, the subject is a human. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a genetic disease or disorder. In some embodiments, the genetic disease or disorder is a single-gene disease or disorder. In some embodiments, the single-gene disease or disorder is an autosomal dominant disease or disorder, an autosomal recessive disease or disorder, an X-chromosome linked (X-linked) disease or disorder, an X-linked dominant disease or disorder, an X-linked recessive disease or disorder, a Y-linked disease or disorder or a mitochondrial disease or disorder. In some embodiments, the genetic disease or disorder is a multiple-gene disease or disorder. In some embodiments, the genetic disease or disorder is a multiple-gene disease or disorder. In some embodiments, the single-gene disease or disorder is an autosomal dominant disease or disorder including, but not limited to, Huntington&#39;s disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, hereditary nonpolyposis colorectal cancer, hereditary multiple exostoses, Von Willebrand disease, and acute intermittent porphyria. In some embodiments, the single-gene disease or disorder is an autosomal recessive disease or disorder including, but not limited to, Albinism, Medium-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis, sickle-cell disease, Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy, and Roberts syndrome. In some embodiments, the single-gene disease or disorder is X-linked disease or disorder including, but not limited to, muscular dystrophy, Duchenne muscular dystrophy, Hemophilia, Adrenoleukodystrophy (ALD), Rett syndrome, and Hemophilia A. In some embodiments, the single-gene disease or disorder is a mitochondrial disorder including, but not limited to, Leber&#39;s hereditary optic neuropathy. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an immune disease or disorder. In some embodiments, the immune disease or disorder is an immunodeficiency disease or disorder including, but not limited to, B-cell deficiency, T-cell deficiency, neutropenia, asplenia, complement deficiency, acquired immunodeficiency syndrome (AIDS) and immunodeficiency due to medical intervention (immunosuppression as an intended or adverse effect of a medical therapy). In some embodiments, the immune disease or disorder is an autoimmune disease or disorder including, but not limited to, Achalasia, Addison&#39;s disease, Adult Still&#39;s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal &amp; neuronal neuropathy (AMAN), Balo disease, Behcet&#39;s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan&#39;s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn&#39;s disease, Dermatitis herpetiformis, Dermatomyositis, Devic&#39;s disease (neuromyelitis optica), Discoid lupus, Dressler&#39;s syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture&#39;s syndrome, Granulomatosis with Polyangiitis, Graves&#39; disease, Guillain-Barre syndrome, Hashimoto&#39;s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme disease chronic, Meniere&#39;s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren&#39;s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNJJ), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonnage-Tumer syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud&#39;s phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren&#39;s syndrome, Sperm &amp; testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac&#39;s syndrome, Sympathetic ophthalmia (SO), Takayasu&#39;s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, or Wegener&#39;s granulomatosis. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an inflammatory disease or disorder. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a metabolic disease or disorder. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a degenerative or a progressive disease or disorder. In some embodiments, the degenerative or a progressive disease or disorder includes, but is not limited to, amyotrophic lateral sclerosis (ALS), Huntington&#39;s disease, Alzheimer&#39;s disease, and aging. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an infectious disease or disorder. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a pediatric or a developmental disease or disorder. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a cardiovascular disease or disorder. 
     In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a proliferative disease or disorder. In some embodiments, the proliferative disease or disorder is a cancer. In some embodiments, the cancer includes, but is not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer, Gastrointestinal Carcinoid Tumors, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Central Nervous System (Brain Cancer), Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma, Osteosarcoma, Malignant Fibrous Histiocytoma, Brain Tumors, Breast Cancer, Burkitt Lymphoma, Carcinoid Tumor, Carcinoma, Cardiac (Heart) Tumors, Embryonal Tumors, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Embryonal Tumors, Endometrial Cancer (UterineCancer), Ependymoma, Esophageal Cancer, Esthesioneuroblastoma (Head and Neck Cancer), Ewing Sarcoma (Bone Cancer), Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Childhood Intraocular Melanoma, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Childhood Gastrointestinal Stromal Tumors, Germ Cell Tumors, Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Heart Tumors, Hepatocellular (Liver) Cancer, Histiocytosis, Hodgkin Lymphoma, Hypopharyngeal Cancer (Head and Neck Cancer), Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), Kidney (Renal Cell) Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer (Head and Neck Cancer), Leukemia, Lip and Oral Cavity Cancer (Head and Neck Cancer), Liver Cancer, Lung Cancer (Non-Small Cell and Small Cell), Childhood Lung Cancer, Lymphoma, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma (Skin Cancer), Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary (Head and Neck Cancer), Midline Tract Carcinoma With NUT Gene Changes, Mouth Cancer (Head and Neck Cancer), Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer (Head and Neck Cancer), Nasopharyngeal Cancer (Head and Neck Cancer), Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer (Head and Neck Cancer), Pheochromocytoma, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Recurrent Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Childhood (Soft Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer), Sarcoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Childhood Vascular Tumors (Soft Tissue Sarcoma), Ewing Sarcoma (Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma (Bone Cancer), Uterine Sarcoma, Sezary Syndrome, Lymphoma, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer (Head and Neck Cancer), Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Renal Cell Cancer, Urethral Cancer, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma), Vulvar Cancer, Wilms Tumor and Other Childhood Kidney Tumors. 
     In some embodiments of the methods of the disclosure, a subject of the disclosure has been diagnosed with the disease or disorder. In some embodiments, the subject of the disclosure presents at least one sign or symptom of the disease or disorder. In some embodiments, the subject has a biomarker predictive of a risk of developing the disease or disorder. In some embodiments, the biomarker is a genetic mutation. 
     In some embodiments of the methods of the disclosure, a subject of the disclosure is female. In some embodiments of the methods of the disclosure, a subject of the disclosure is male. In some embodiments, a subject of the disclosure has two XX or XY chromosomes. In some embodiments, a subject of the disclosure has two XX or XY chromosomes and a third chromosome, either an X or a Y. 
     In some embodiments of the methods of the disclosure, a subject of the disclosure is a neonate, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of years or partial years in between of age. 
     In some embodiments of the methods of the disclosure, a subject of the disclosure is a mammal. In some embodiments, a subject of the disclosure is a non-human mammal. 
     In some embodiments of the methods of the disclosure, a subject of the disclosure is a human. 
     In some embodiments of the methods of the disclosure, a therapeutically effective amount comprises a single dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises at least one dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises one or more dose(s) of a composition of the disclosure. In some embodiments of the methods of the disclosure, a therapeutically effective amount eliminates a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount reduces a severity of a sign or symptom of the disease or disorder. 
     In some embodiments of the methods of the disclosure, a therapeutically effective amount eliminates the disease or disorder. 
     In some embodiments of the methods of the disclosure, a therapeutically effective amount prevents an onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount improves a prognosis for the subject. 
     In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject systemically. In some embodiments, the composition of the disclosure is administered to the subject by an intravenous route. In some embodiments, the composition of the disclosure is administered to the subject by an injection or an infusion. 
     In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject locally. In some embodiments, the composition of the disclosure is administered to the subject by an intraosseous, intraocular, intracerebrospinal, or intraspinal route. In some embodiments, the composition of the disclosure is administered directly to the cerebral spinal fluid of the central nervous system. In some embodiments, the composition of the disclosure is administered directly to a tissue or fluid of the eye and does not have bioavailability outside of ocular structures. In some embodiments, the composition of the disclosure is administered to the subject by an injection or an infusion. 
     Also provided herein is a pharmaceutical composition comprising any one or more of the capped-sgRNAs and Cas or dCas polypeptide, or the nucleic acid sequences encoding the polypeptide, and a carrier. In some embodiments, a composition can be one or more polynucleotides encoding a capped-guide nucleotide sequence-Cas polypeptide or fusion polypeptide. In some embodiments, a composition can be any of the nucleic acids or proteins described herein. In some embodiments, a composition can be any polynucleotide described herein. In some embodiments, the carrier is a pharmaceutically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition comprising a capped-guide nucleotide sequence-Cas polypeptide or fusion polypeptide, and a pharmaceutically acceptable carrier. In some embodiments, the composition or pharmaceutical composition further comprises one or more gRNAs, capped-sgRNAs, crRNAs, and/or tracrRNAs. 
     Briefly, pharmaceutical compositions disclosed herein may comprise a capped-guide nucleotide sequence-Cas polypeptide or fusion polypeptide or a nucleotide sequence encoding the same, optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, subpial, and/or parenteral administration. In certain embodiments, the compositions of the disclosure are formulated for intravenous administration. 
     In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject locally. In some embodiments, the composition of the disclosure is administered to the subject by an intraosseous, intraocular, intracerebrospinal, intraspinal, or subpial route. In some embodiments, the composition of the disclosure is administered directly to the cerebral spinal fluid of the central nervous system. In some embodiments, the composition of the disclosure is administered directly to a tissue or fluid of the eye and does not have bioavailability outside of ocular structures. In some embodiments, the composition of the disclosure is administered to the subject by an injection or an infusion. 
     In some embodiments, the compositions disclosed herein are formulated as pharmaceutical compositions. Briefly, pharmaceutical compositions for use as disclosed herein may include a protein(s) or a polynucleotide encoding the protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like: carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the disclosure may be formulated for oral, intravenous, topical, enteral, intraocular, and/or parenteral administration. In some embodiments, intraocular administration includes, without limitation, subretinal, intravitreal, or topical (via eye drops) administration. In certain embodiments, the compositions of the disclosure are formulated for intravenous administration. 
     EXAMPLES 
     The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. 
     Example 1: Modulation of Translation Using dCas and Capped-sgRNA 
     Exemplary constructs for generating nuclease dead Cas (dCas13b) and m7G capped-sgRNA are shown in  FIG. 1A . dCas and capped-sgRNA were transcribed from either the same promoter (i), or independent promoters (ii) by RNA polymerase II. An exemplary structure for the unprocessed capped-sgRNA is shown in  FIG. 1B . From 5′ to 3′, the capped-sgRNA contains the m7G cap, a linker of variable length, a spacer, a direct repeat, an RNase P processing site such as a tRNA-like small RNA, and a poly-A tail. An exemplary chemical composition of a capped-sgRNA is shown in  FIG. 1C . Variants of the m7G cap include either guanosine or adenine for the second nucleotide of the di-nucleotide m7G cap (at the a′ position).  FIG. 1D  shows RNase P processing to trim downstream sequences, including the poly-A tail, from the transcript. The capped-RNA complexed with dCas, binds to a site on the target messenger RNA thereby bringing the m7G cap of the sgRNA to the vicinity of a desired start codon ( FIG. 1E ). 
     A two construct system was used to test the ability of dCas and capped-sgRNA to regulate translation in trans. The first construct includes a dCas13b gene driven by a PGK promoter, followed by a NLS-Turquoise reporter which is driven by an EF1α promoter. The second construct includes a sequence encoding a capped sgRNA, a 5′UTR region of ATF4, and the ATF4 ORF linked to an NL-Citrine promoter. Both the sequence encoding a capped sgRNA and the ATF4 ORF are driven by a TRE promoter. The second construct further includes an rtTA-P2A-Puro construct linked to an NLS-Cherry reporter via an IRES sequence. The rtTA-P2A-Puro-IRES-NLS-Cherry portion is driven by the EF1α promoter. A number of different types of capped sgRNAs (sg(1), sg(2), sg(3), sg(4), sg(5), sg(6)) were generated, each included a spacer that targets the ATF4 transcript at a different site. The sequences of the sgRNAs are listed in below. The target sequences were within the “sgRNA targeting window” as shown in  FIG. 1G . Control capped-sgRNAs were also generated that do not target a sequence within the ATF4 transcript. These control capped-sgRNAs are referred to as non-targeting sgRNAs. Each capped-sgRNA was tested using dCas gradient in combination with a target ATF4 transcript gradient. The plots in  FIG. 1H  reflect log 2 fold difference in Citrine/Cherry ratio between each capped sgRNA—sg (a1), sg(a2), sg(a3), sg(a4), sg(a5) and sg(a6), and non-targeting sgRNAs. A spacer targeting a region just 3′ proximal to the start codon AUG was shown to result in the biggest increase in protein expression. The results demonstrate that optimal translational control is a function of the target sequence chosen, the expression level of the target RNA, and the expression level of dCas. 
     Uncapped-sgRNAs that correspond to each of the capped-sgRNA tested were subjected to the same gradient test. As shown in  FIG. 1I , uncapped-sgRNAs were either ineffective in enhancing translation, or only minimally enhanced translation. These results showed that the localized recruitment of the 5′ m7G cap proximal to a start codon enabled an enhancement in translation. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 sgRNA 
                 Sequence 
               
               
                   
                   
               
             
            
               
                   
                 sg(1) 
                 TTTGCTGGAATCGAGGAATGTGCTT 
               
               
                   
                   
                 (SEQ ID NO: 278) 
               
               
                   
                   
               
               
                   
                 sg(2) 
                 GTTGCGGTGCTTTGCTGGAATCGAG 
               
               
                   
                   
                 (SEQ ID NO: 279) 
               
               
                   
                   
               
               
                   
                 sg(3) 
                 TTTCGGTCATGTTGCGGTGCTTTGC 
               
               
                   
                   
                 (SEQ ID NO: 280) 
               
               
                   
                   
               
               
                   
                 sg(4) 
                 AGGAAGCTCATTTCGGTCATGTTGC 
               
               
                   
                   
                 (SEQ ID NO: 281) 
               
               
                   
                   
               
               
                   
                 sg(5) 
                 CTCGCTGCTCAGGAAGCTCATTTCG 
               
               
                   
                   
                 (SEQ ID NO: 282) 
               
               
                   
                   
               
               
                   
                 sg(6) 
                 CCACCAACACCTCGCTGCTCAGGAA 
               
               
                   
                   
                 (SEQ ID NO: 283) 
               
               
                   
                   
               
            
           
         
       
     
     Additional Embodiments 
     Embodiment 1: A capped single guide RNA (Capped-sgRNA) comprising from 5′ to 3′: 
     an m7G cap, 
     a linker, 
     a spacer complementary to a target sequence in a messenger RNA, 
     a direct repeat sequence, and 
     a Ribonuclease P (RNase P) processing site, 
     wherein the direct repeat sequence is capable of binding to a Cas protein. 
     Embodiment 2: The Capped-sgRNA of Embodiment 1, wherein the Cas protein is a nuclease dead Cas (dCas) protein.
 
Embodiment 3: The Capped-sgRNA of Embodiment 1, wherein the m7G cap comprises one or more chemical modifications relative to the structure of a naturally occurring m7G cap.
 
Embodiment 4: The Capped-sgRNA of Embodiment 1, wherein the linker comprises about 5 to about 25 nucleotides.
 
Embodiment 5: The Capped-sgRNA of Embodiment 4, wherein the linker comprises about 8 to about 20 nucleotides.
 
Embodiment 6: The Capped-sgRNA of Embodiment 1, wherein the linker is non-complementary to any messenger RNA sequence.
 
Embodiment 7: The Capped-sgRNA of Embodiment 1, wherein the linker comprises the sequence of GTCAGATCGCCTGGAATT.
 
Embodiment 8: The Capped-sgRNA of Embodiment 1, wherein the target sequence is proximal to a target start codon of the messenger RNA relative to a 5′ m7G cap of the messenger RNA.
 
Embodiment 9: The Capped-sgRNA of Embodiment 1, wherein the target sequence comprises the target start codon of the messenger RNA.
 
Embodiment 10: The Capped-sgRNA of Embodiment 8, wherein the 5′ end of the target sequence is upstream to the target start codon of the messenger RNA.
 
Embodiment 11: The Capped-sgRNA of Embodiment 8, wherein the 5′ end of the target sequence is downstream to the target start codon of the messenger RNA.
 
Embodiment 12: The Capped-sgRNA of Embodiment 1, wherein the spacer is at least 80% complementary to the target sequence in the messenger RNA.
 
Embodiment 13: The Capped-sgRNA of Embodiment 1, wherein the spacer is at least 90% complementary to the target sequence in the messenger RNA.
 
Embodiment 14: The Capped-sgRNA of Embodiment 1, further comprising a polyadenylated tail.
 
Embodiment 15: The Capped-sgRNA of Embodiment 1, having the structure:
 
     
       
         
         
             
             
         
       
     
     wherein a′ is a guanosine or adenine, b′ is the linker, and c′ is the spacer. 
     Embodiment 16: An expression vector encoding the Capped-sgRNA of Embodiment 1.
 
Embodiment 17: The expression vector of Embodiment 16, further encodes a nuclease dead Cas9.
 
Embodiment 18: A Capped-sgRNA generated by processing the Capped-sgRNA of Embodiment 1 using an RNase P.
 
Embodiment 19: An expression vector comprising a nucleic acid sequence encoding:
 
     a nuclease dead Cas (dCas), and 
     a capped single guide RNA (Capped-sgRNA) comprising
         an m7G cap,   a linker,   a spacer complementary to a target sequence in a messenger RNA, and   a direct repeat sequence, wherein the direct repeat sequence is capable of binding to the dCas protein.
 
Embodiment 20: The expression vector of Embodiment 19, wherein the Capped-sgRNA further comprises a Ribonuclease P (RNase P) processing site.
 
Embodiment 21: The expression vector of Embodiment 19, wherein the Capped-sgRNA further comprises a polyadenylated tail.
 
Embodiment 22: The expression vector of Embodiment 19, wherein the dCas and the Capped-sgRNA are under the control of the same promoter.
 
Embodiment 23; The expression vector of Embodiment 19, wherein the dCas and the Capped-sgRNA are under the control of different promoters.
 
Embodiment 24: A method of enhancing protein translation comprising:
       

     (a) providing a nuclease dead Cas (dCas) protein, and
         a capped single guide RNA (Capped-sgRNA) comprising from 5′ to 3′:
           an m7G cap,   a linker,   a spacer complementary to a target sequence in a messenger RNA, and   a direct repeat sequence,   wherein the direct repeat sequence is capable of binding to the dCas protein, and wherein the target sequence is proximal to a start codon of the messenger RNA relative to a 5′ m7G cap of the messenger RNA.   
           (b) allowing the Capped-sgRNA to bind to the target sequence and the dCas protein to bind to the direct repeat sequence of the Capped-sgRNA, thereby localizing the m7G cap of the Capped-sgRNA to the target sequence.       

     Other Embodiments 
     It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.