US20220220473A1

US20220220473A1 - Protein translational control

Info

Publication number: US20220220473A1
Application number: US17/604,128
Authority: US
Inventors: Eugene Yeo; Frederick Tan
Original assignee: University of California
Current assignee: University of California
Priority date: 2019-04-16
Filing date: 2020-04-16
Publication date: 2022-07-14
Also published as: US20220204978A1; WO2020214806A1; WO2020214830A1

Abstract

Provided herein are compositions and methods for regulating protein translation. The compositions include a Cas polypeptide and a capped-sgRNA that includes (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. The disclosure further provides 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.

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 10⁻⁶M, less than 10⁻⁷M, less than 10⁻⁸M, less than 10⁻⁹M, less than 10⁻¹⁰M, less than 10⁻¹¹M, less than 10⁻¹²M, less than 10⁻¹³M, less than 10⁻¹⁴M, or less than 10⁻¹⁵M. 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's solution, 6×SSPE, 0.2% SDS at 22° C., followed by washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt'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 —OCH₃. 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., m₃ ^2.2.7GP₃G), and m₂ ^7,3′-OGP₃(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 & 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's disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, hereditary nonpolyposis colorectal cancer, hereditary multiple exostoses, Von Willebrand disease, and acute intermittent porphyria. In some embodiments, the single-gene disease or disorder is an autosomal recessive disease or disorder including, but not limited to, Albinism, Medium-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis, sickle-cell disease, Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy, and Roberts syndrome. In some embodiments, the single-gene disease or disorder is X-linked disease or disorder including, but not limited to, muscular dystrophy, Duchenne muscular dystrophy, Hemophilia, Adrenoleukodystrophy (ALD), Rett syndrome, and Hemophilia A. In some embodiments, the single-gene disease or disorder is a mitochondrial disorder including, but not limited to, Leber's hereditary optic neuropathy.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an immune disease or disorder. In some embodiments, the immune disease or disorder is an immunodeficiency disease or disorder including, but not limited to, B-cell deficiency, T-cell deficiency, neutropenia, asplenia, complement deficiency, acquired immunodeficiency syndrome (AIDS) and immunodeficiency due to medical intervention (immunosuppression as an intended or adverse effect of a medical therapy). In some embodiments, the immune disease or disorder is an autoimmune disease or disorder including, but not limited to, Achalasia, Addison's disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet's disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme disease chronic, Meniere's disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (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's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, or Wegener's granulomatosis.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an inflammatory disease or disorder.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a metabolic disease or disorder.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a degenerative or a progressive disease or disorder. In some embodiments, the degenerative or a progressive disease or disorder includes, but is not limited to, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and aging.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, an infectious disease or disorder.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a pediatric or a developmental disease or disorder.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a cardiovascular disease or disorder.
In some embodiments of the compositions and methods of the disclosure, a disease or disorder of the disclosure includes, but is not limited to, a proliferative disease or disorder. In some embodiments, the proliferative disease or disorder is a cancer. In some embodiments, the cancer includes, but is not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer, Gastrointestinal Carcinoid Tumors, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Central Nervous System (Brain Cancer), Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma, Osteosarcoma, Malignant Fibrous Histiocytoma, Brain Tumors, Breast Cancer, Burkitt Lymphoma, Carcinoid Tumor, Carcinoma, Cardiac (Heart) Tumors, Embryonal Tumors, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Embryonal Tumors, Endometrial Cancer (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.

Claims

1. A complex 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.

2. The complex of claim 1, wherein the RNA molecule is a messenger RNA (mRNA).

3. The complex of claim 2, wherein the mRNA has an endogenous m7G cap.

4. The complex of claim 3, wherein the target sequence is downstream of the endogenous m7G cap of the mRNA.

5.-12. (canceled)

13. The complex of claim 1, wherein the spacer is at least 80% complementary to the target sequence.

14.-17. (canceled)

18. The complex of claim 1, wherein the spacer is connected to the m7G cap or analog thereof via a linker.

19.-22. (canceled)

23. The complex of claim 1, wherein 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.

24. The complex of claim 23, wherein the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d.

25. The complex of claim 24, wherein the direct repeat is capable of binding to a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d.

26. The complex of claim 23, wherein the nuclease-deficient Cas polypeptide is a nuclease-deficient Cas9 (dCas9) polypeptide.

27. (canceled)

28. A nucleic acid comprising a sequence encoding the capped-sgRNA in the complex of claim 1.

29. (canceled)

30. A nucleic acid comprising a sequence encoding a capped-sgRNA, wherein the capped-sgRNA comprises:

(i) an m7G cap or analog thereof;

(iii) a direct repeat capable of binding to a Cas polypeptide.

31. The nucleic acid of claim 30, wherein the RNA molecule is an mRNA.

32.-54. (canceled)

55. The nucleic acid of claim 30, further comprising a sequence encoding the Cas polypeptide.

56. The nucleic acid of claim 55, wherein the Cas polypeptide is a nuclease-deficient Cas polypeptide.

57.-60. (canceled)

61. The nucleic acid of claim 55, wherein the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from the same promoter.

62. The nucleic acid of claim 55, wherein the sequence encoding the capped-sgRNA and the sequence encoding the Cas polypeptide are expressed from different promoters.

63. A vector comprising the nucleic acid of claim 30.

64. (canceled)

65. A cell comprising the nucleic acid of claim 30.

66. A method 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;

(iii) a direct repeat capable of binding to the Cas polypeptide.

67.-70. (canceled)