Engineered Acr proteins for modulating CRISPR activity

Abstract

A composition and method are provided that include an engineered anti-CRISPR (Acr) protein, where the Cas protein retains activity to perform an on-target nucleic acid targeting function (e.g., DNA cleavage for gene editing applications), but is inhibited by the subject Acr protein to an extent that off-target activity is reduced, resulting in an increase in the ratio of on-target:off-target nucleic acid targeting events. In some cases, the subject composition (e.g., the subject system) includes a CRISPR nuclease or a nucleic acid encoding the same. A method is provided for achieving target nucleic acid modification using the subject Acr polypeptide together with a CRISPR nuclease.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,494, filed on June 15, 2022, which is hereby incorporated by reference in its entirety.

[0002] Incorporation by reference of the Sequence Listing (provided as a Sequence Listing XML file) The Sequence Listing is provided with this specification as the Sequence Listing XML “ACRG-006WO_SEQ_LIST.xml” (created on June 5, 2023, size 557,231 bytes). The contents of this Sequence Listing XML are hereby incorporated by reference in their entirety.

Background Art

[0003] I. Introduction The CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas system is found in diverse bacterial and archaeal species and functions as an immune defense mechanism against phage infection. The simplicity, programmability, and versatility of the CRISPR-Cas system (e.g., Cas9 and Cas12 systems) have facilitated gene modification in many organisms and have brought great potential for the treatment of human diseases. However, in practical terms, genome editing via CRISPR-Cas can be associated with off-targeting (e.g., introduction of unintended mutations, insertions, or deletions, and DNA rearrangement at unintended "off-target" sites). Off-target editing caused by the CRISPR-Cas system has been reported in various cells and animal models, including human cells, and such events may accumulate in vivo with the persistence of nuclease activity. Furthermore, the unpredictability of off-target events is added due to the uncertainty of human genetic variations and the in vivo Cas protein expression period. This problem should be addressed for the safe clinical translation of the CRISPR-Cas system. Unintended editing events can, in some cases, cause genomic instability, disrupt gene functionality, and lead to serious adverse events including cell death and cancer.

[0004] There is a need for compositions and methods that modify Cas activity and, for example, reduce off-target events (e.g., compared to on-target events) to result in enhanced nucleic acid targeting specificity. The present disclosure provides such compositions and methods. SUMMARY OF THE INVENTION

[0005] II. SUMMARY Anti-CRISPR (Acr) proteins have been discovered to have the ability to bind to specific Cas proteins within a phage and inhibit them, thereby interfering with the CRISPR system's attempt to cleave the invading phage's DNA. Thus, Acr proteins are thought to be used by phages to evade the CRISPR-Cas system.

[0006] The inventors generated engineered Acr proteins (ErAcr) that do not occur in nature (see, e.g., SEQ ID NOs: 126-132 and 264-267). Using this, a balance can be achieved such that the Cas protein retains sufficient activity to carry out the desired on-target nucleic acid functions (e.g., loading, complex construction, binding, and cleavage), but is inhibited to such an extent as to reduce off-target activity. Accordingly, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (ErAcr), or a nucleic acid encoding this Acr polypeptide, wherein the Acr polypeptide has at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the sequence set forth in any one of SEQ ID NOs: 126-132, and comprises an amino acid sequence. Similarly, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (ErAcr), or a nucleic acid encoding this Acr polypeptide, wherein the Acr polypeptide has at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the sequence set forth in any one of SEQ ID NOs: 264-267, and comprises an amino acid sequence. In some cases, the subject Acr polypeptide has at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr polypeptide has at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 264-267.

[0007] Generally, an Acr polypeptide can reduce the activity of a CRISPR nuclease. For example, in some embodiments, a subject Acr polypeptide reduces off-target CRISPR nuclease activity by at least 10% (e.g., at least 20%, at least 30%, at least 50%, etc.) compared to off-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some embodiments, a subject Acr polypeptide reduces on-target CRISPR nuclease activity by no more than 40% (e.g., no more than 30%, no more than 20%, etc.) compared to on-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some embodiments, a subject Acr polypeptide increases the on-target:off-target CRISPR nuclease activity ratio (e.g., by at least 1.25-fold, 1.5-fold, 2-fold, 3-fold, etc.) compared to the on-target:off-target CRISPR nuclease activity ratio in the absence of the Acr polypeptide.

[0008] In some embodiments, the CRISPR nuclease is a Cas12a protein. In some embodiments, the CRISPR nuclease is a NUX protein. For example, in some cases, the subject Acr polypeptide reduces (inhibits) the activity of a Cas12a nuclease (see, e.g., SEQ ID NOs: 175 and 245-262). In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 90% sequence identity (e.g., at least 95% or 100% sequence identity) to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Cas12a nuclease amino acid sequence set forth in SEQ ID NO: 175. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 90% sequence identity (e.g., at least 95% or 100% sequence identity) to the Cas12a nuclease amino acid sequence set forth in SEQ ID NO: 175.

[0009] In some embodiments, the subject Acr polypeptide reduces (inhibits) the activity of a NUX protein (see, e.g., SEQ ID NOs: 1-86 and 176-244). In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 90% sequence identity (e.g., at least 95%, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 90% sequence identity (e.g., at least 95%, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 176-178.

[0010] In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having at least 90% sequence identity (e.g., at least 95%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263.

[0011] The target Acr polypeptide can be fused to a fusion partner. In some such cases, the target Acr polypeptide is fused to a nuclear localization signal (NLS).

[0012] Also provided herein are systems comprising a target Acr polypeptide (e.g., ErAcr) or a nucleic acid encoding an Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding a CRISPR nuclease. In such systems, the Acr polypeptide can reduce (inhibit) the activity of the CRISPR nuclease. The Acr polypeptide of such a system can be any of those described above (e.g., having at least 75% sequence identity to SEQ ID NOs: 126-132, e.g., having at least 75% sequence identity to SEQ ID NOs: 264-267), and the CRISPR nuclease can also be any of those described above (e.g., Cas12a nucleases such as SEQ ID NOs: 175 and 245-262, NUX proteins such as SEQ ID NOs: 1-86 and 176-244 (see, e.g., the CRISPR nuclease of SEQ ID NO: 263)). In some cases, the subject system also includes a guide RNA for targeting the CRISPR nuclease to a target sequence of a target nucleic acid.

[0013] In some embodiments, the Acr polypeptide of the subject system (or method) can comprise an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some embodiments, the Acr polypeptide of the subject system (or method) can comprise an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some cases, the CRISPR nuclease and the Acr polypeptide of this system do not naturally occur together. In some cases, the Acr polypeptide of the subject system (or method) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244, or to the Cas12a amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system (or method) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity) to the amino acid sequence set forth in 263.

[0014] The Acr polypeptide and the CRISPR nuclease can each be provided independently as a nucleic acid (DNA, RNA) encoding a protein or in the form of a protein. When both are provided as nucleic acids, the Acr polypeptide and the CRISPR nuclease may be encoded on separate nucleic acids (e.g., provided as two separate vectors) or may be encoded on the same nucleic acid (e.g., provided on the same vector). In any case, a translational control element (e.g., an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or any combination thereof) can be operably linked to the Acr polypeptide coding sequence and / or the CRISPR nuclease coding sequence. In some cases, a first promoter is operably linked to the Acr polypeptide coding sequence and a second promoter is operably linked to the CRISPR nuclease coding sequence. In some such embodiments, the first promoter is stronger than the second promoter, and in other embodiments, the second promoter is stronger than the first promoter. Suitable promoters include, but are not limited to, CMV, miniCMV, EF1A, CAG, CBh, EFS, SV40, and hPGK. In some cases, the nucleic acid (encoding the Acr polypeptide, the CRISPR nuclease, or both) is a viral vector, such as an AAV vector. Whether the Acr polypeptide and / or the CRISPR nuclease is provided as a nucleic acid or in the form of a protein, in some cases, both can be included in a lipid nanoparticle (LNP) formulation.

[0015] Also provided herein are methods of using the above-described compositions / systems. For example, a method of modifying a target nucleic acid is provided, the method comprising contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease (see above), the contacting resulting in a modification (e.g., substitution, insertion, deletion) to the nucleotide sequence of the target nucleic acid. In some cases, the contacting comprises delivering the CRISPR nuclease and the Acr polypeptide in a 1:1 ratio. In other cases, the contacting comprises delivering the CRISPR nuclease and the Acr polypeptide in a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:10. In still other cases, the contacting comprises delivering the Acr polypeptide and the CRISPR nuclease in a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10. The contacting can be performed in vitro, ex vivo, and in vivo, and the contacting can be performed in cells (e.g., eukaryotic cells, animal cells, mammalian cells, human cells, etc.). In some embodiments, the target nucleic acid encodes a gene product (e.g., non-coding RNA, mRNA / protein, etc.).

[0016] The on-target / off-target CRISPR activity referred to in the subject composition, system, or method can include editing of a target nucleic acid (e.g., editing via DNA cleavage in the presence or absence of a donor polynucleotide), e.g., genome editing. In some cases, the editing efficiency of on-target CRISPR nuclease activity is greater than the editing efficiency of off-target CRISPR nuclease activity (e.g., at least 2-fold, at least 4-fold, at least 5-fold, or at least 10-fold greater). In some cases, the off-target CRISPR nuclease activity is at an off-target site that contains five or fewer mismatches compared to the on-target site. In some cases, the off-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide (e.g., by 10% or more, 20% or more, 30% or more, etc.). In some cases, the on-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide (e.g., by 10% or less, 20% or less, 30% or less, 40% or less, etc.).

[0017] Thus, in some cases, the subject method includes measuring the editing efficiency at the on-target site. In some cases, the subject method includes measuring the editing efficiency at one or more off-target sites. III. BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

[0019] IV. Definitions The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length (either ribonucleotides or deoxyribonucleotides). Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically, or biochemically modified non-natural or derivatized nucleotide bases.

[0020] "Hybridizable," "hybridizes," "complementary," or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) is capable of non-covalently binding (i.e., forming Watson-Crick base pairs and / or G / U base pairs), "annealing," or "hybridizing" in a sequence-specific and antiparallel manner to another nucleic acid (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions. Standard Watson-Crick base pairs include the pair of adenine (A) and thymidine (T), the pair of adenine (A) and uracil (U), and the pair of guanine (G) and cytosine (C) [DNA, RNA]. Further, in hybridization between two RNA molecules (e.g., dsRNA), and in hybridization between a DNA molecule and an RNA molecule (e.g., when a DNA target nucleic acid forms base pairs with a guide RNA), guanine (G) can also form base pairs with uracil (U). For example, G / U base pair formation is at least partially involved in the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pair formation with codons in mRNA. Thus, in the context of the present disclosure, guanine (G) (e.g., in a dsRNA duplex of a guide RNA molecule, a base pair between a guide RNA and a target nucleic acid, etc.) is considered complementary to both uracil (U) and adenine (A). For example, if a G / U base pair can be formed at a given nucleotide position in a dsRNA duplex of a guide RNA molecule, this position is considered complementary rather than non-complementary.

[0021] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases can occur. Conditions appropriate for hybridization between two nucleic acids depend on variables well known in the art, such as the length of the nucleic acids and the degree of complementarity. The greater the degree of complementarity between two nucleotide sequences, the higher the melting temperature (Tm) value of the hybrid of the nucleic acids having these sequences. For hybridization between nucleic acids having short stretches of complementarity (e.g., complementarity of 35 nucleotides or less, 30 nucleotides or less, 25 nucleotides or less, 22 nucleotides or less, 20 nucleotides or less, or 18 nucleotides or less), the position of the mismatch can be important (see Sambrook et al. (supra) 11.7-11.8). Typically, the length of hybridizable nucleic acids is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 17 nucleotides or more, 18 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Temperature, salt concentration of the wash solution, and other conditions can be adjusted as needed according to factors such as the length of the complementary region and the degree of complementarity.

[0022] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0023] As used herein, "binding" (e.g., with respect to a nucleic acid binding domain of a polypeptide, binding to a target nucleic acid, etc.) refers to non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid (e.g., DNA or RNA)). In the state of non-covalent interactions, the macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when it is said that molecule X interacts with molecule Y, it means that molecule X binds non-covalently to molecule Y). Not all components of a binding interaction need to be sequence-specific (e.g., contacts with phosphate residues within the DNA backbone), but some portion of the binding interaction can be sequence-specific. A binding interaction is generally characterized by a dissociation constant (K -6 <10 -7 <10 -8 <10 -9 <10 -10 <10 -11 <10 -12 <10 -13 <10 -14 <10 -15 <) that is less than 10 D <M, less than 10 D <M, less than 10

[0024]

[0024] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For the purposes of the present disclosure, a promoter sequence extends upstream (5' direction) to include the minimum number of bases or elements necessary to bind at its 3' end by the transcription start site and initiate transcription at a detectable level above background. Eukaryotic promoters often, but not always, contain a "TATA" box and a "CAT" box. A variety of promoters, including constitutive, tissue-specific, and inducible promoters, can be used to drive expression by the various vectors of the present disclosure. The expression level of a given promoter can be described as weak, moderate, or strong, and thus the promoter can be classified as a weak, moderate, or strong promoter.

[0025] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship that enables them to function in their intended manner. For example, if a promoter affects the transcription of a nucleotide sequence, the promoter is operably linked to the nucleotide sequence (it can also be said that the nucleotide sequence is operably linked to the promoter). As another example, if a translation control element affects the translation of a protein from a protein coding sequence, the translation control element is operably linked to the protein coding sequence (it can also be said that the protein coding sequence is operably linked to the translation control element).

[0026] As used herein, the term "co-delivery system" refers to the co-delivery of Acr protein and Cas nuclease. The co-delivery system comprises one or more nucleic acids (e.g., vectors) for expressing the Acr protein and the CRISPR nuclease (e.g., within a host cell). In some cases, the co-delivery system provides a single nucleic acid (e.g., a vector) for expressing the Acr protein and the CRISPR nuclease. In some cases, the co-delivery system provides multiple nucleic acids (e.g., vectors) for the expression of the Acr protein and the Cas nuclease, and the expression and / or function are coordinated as in the case of providing split Cas from two vectors. In some cases, the expression and / or function of the Acr protein and the Cas nuclease of the co-delivery system are coupled (i.e., coordinated) by translational control elements selected to regulate the translation of the Acr protein and / or the CRISPR nuclease.

[0027] As used herein, terms such as "treatment", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease or an adverse effect attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (e.g., a human), including (a) preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., arresting its development, or (c) alleviating the disease, i.e., causing regression of the disease.

[0028] The terms "subject" and "host" are used interchangeably herein and refer to an individual organism that expresses, or is intended to express, the co-delivery system and / or the Cas nuclease and / or the Acr protein described herein. Hosts include, but are not limited to, fungi (e.g., yeast), plants, algae, insects, animals, such as birds and mammals (e.g., mammals including, but not limited to, mice, monkeys, humans, mammalian livestock, mammalian sport animals, and mammalian pets). Hosts include, but are not limited to, microorganisms (e.g., bacteria and fungi (e.g., yeast)), plants, algae, insects, animals, such as birds and mammals (e.g., mammals including, but not limited to, mice, monkeys, humans, mammalian livestock, mammalian sport animals, and mammalian pets).

[0029] The terms "on-target" (or "ON target") and "off-target" (or "OFF target") are used herein to refer to the location of CRISPR complex activity (e.g., target DNA cleavage, DNA editing) within the target DNA. Both location types (on- and off-target) are based on the guide sequence of the guide RNA. A CRISPR complex-mediated event occurring at a location based on a 100% match to the guide sequence is considered "on-target", while an event occurring at a (undesired) location not based on a 100% match to the guide sequence is considered "off-target". If the nucleotide sequence of the target DNA is known (e.g., most of the genome of the target cell has been sequenced), sites that may be off-target can be predicted for a given guide sequence. Generally, off-target events are more likely to occur at sequences with a percentage identity closer to 100% between the guide RNA and the target than at sequences with a lower percentage identity between the guide RNA and the target. Thus, most off-target events tend to occur at sequences having 50% or more (e.g., 75% or more) sequence identity to the intended target sequence. For example, an off-target event may occur if there is one mismatch between the guide RNA and the target, two mismatches between the guide RNA and the target, or three mismatches between the guide RNA and the target. Thus, sequence analysis of the target DNA can provide a list of sites within the target DNA that are predicted to be potentially off-target. The number of predicted off-target sites depends on the target DNA sequence, but in some cases, the number of predicted off-target sites ranges from 10 to 200 predicted sites (e.g., 10 to 150, 10 to 100, 10 to 50, 15 to 200, 15 to 150, 15 to 100, 15 to 80, 20 to 200, 20 to 150, 20 to 100, or 20 to 80 predicted sites).

DETAILED DESCRIPTION OF THE INVENTION

[0030] V. DETAILED DESCRIPTION Before further describing the present invention, it is to be understood that the present invention is not limited to the specific embodiments described, and of course it can itself change. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only, and since the scope of the present invention is limited only by the appended claims, such terms are not intended to be limiting.

[0031] When a range of values is indicated, each value that exists between them (up to one-tenth of the unit of the lower limit, unless otherwise clearly indicated by context between the upper and lower limits of the range), and the described values or the values that exist between them, are to be understood as being included in the present invention. The upper and lower limits in these smaller ranges can be independently included in the smaller ranges, and these are also included in the present invention and are subject to any specifically excluded limitations in the described range. When the described range includes one or both of the upper and lower limits, ranges excluding one or both of the included upper and lower limits are also included in the present invention.

[0032] In this specification, a specific range may be indicated by using the term "about" before a numerical value. The term "about" is used herein to provide literal support for the exact number that follows and for numbers close to or approximating the number that follows this term. When determining whether a number is close to or approximates a specifically recited number, the unrecited close or approximate number can be a number that results in a substantial equivalent of the specifically recited number in the context in which it is presented.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs. When practicing or testing the present invention, any methods and materials similar to or equivalent to those described herein can be used, but representative exemplary methods and materials are described herein.

[0034] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or substances cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention has no right to antedate such publication by virtue of prior disclosure. Further, the publication date indicated may be different from the actual publication date, which may need to be independently confirmed.

[0035] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes reference to a plurality of such proteins, and reference to "the protein" includes reference to one or more proteins and their equivalents known to those of ordinary skill in the art, and so forth. Further, it should be noted that the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as a basis for the use of exclusive terms such as "solely", "only", or the use of "negative" limitations in connection with the recitation of claim elements.

[0036] It should be understood that some features of the present invention described in the context of separate embodiments for clarity may be provided in combination in a single embodiment. Conversely, various features of the present invention described in the context of a single embodiment for brevity may be provided separately or in any suitable sub-combination. For example, as will be apparent to those skilled in the art upon reading this disclosure, the individual embodiments described and illustrated herein have separate elements and features, and these can be readily separated from or combined with the features of any of several other embodiments without departing from the scope and spirit of the present invention. All combinations in embodiments related to the present invention are clearly encompassed by the present invention, and all such combinations are disclosed herein as if each combination was individually and explicitly disclosed. In addition, all sub-combinations in various embodiments and their elements are also clearly encompassed by the present invention, and all such sub-combinations are disclosed herein as if each such combination was individually and explicitly disclosed herein.

[0037] As mentioned above, the present disclosure provides Acr (including the engineered Acr protein (ErAcr) that does not naturally exist), and using this, compared to the activity of the Cas protein without this Acr (see, for example, SEQ ID NOs: 126 - 132), the Cas protein retains sufficient activity to perform the desired on-target nucleic acid functions (e.g., loading, complex construction, binding, and cleavage), but is inhibited to such an extent as to reduce off-target activity, such that a balance can be achieved. Accordingly, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide or a nucleic acid encoding an Acr polypeptide, and this Acr polypeptide has at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the sequence set forth in any one of SEQ ID NOs: 126 - 132.

[0038] Also provided herein are systems for modifying a target nucleic acid and methods of using such systems, the systems comprising a subject Acr polypeptide (e.g., ErAcr of SEQ ID NOs: 126-132, and / or Acr of SEQ ID NOs: 165-169) or a nucleic acid encoding an Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding a CRISPR nuclease. In some cases, the CRISPR nuclease and the Acr polypeptide of the system do not naturally occur together.

[0039] CRISPR complex As used herein, the terms "CRISPR complex" and "effector complex" refer to protein-RNA complexes that are directed by an RNA component (often referred to as "guide RNA") to a specific sequence within a target nucleic acid (e.g., a target genomic DNA). In class 2 CRISPR systems, the function of the effector complex is a single protein (sometimes referred to as the "effector protein" and also referred to herein as the "CRISPR nuclease" or simply the "Cas protein"), and the native protein is an endonuclease (see, e.g., Zetsche et al, Cell. 2015 Oct 22;163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Shmakov et al., Nat Rev Microbiol. 2017 Mar;15(3):169-182; Koonin et al., Curr Opin Microbiol. 2017 Jun;37:67-78; and Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83).

[0040] Accordingly, the terms “Class 2 CRISPR / Cas protein” or “CRISPR / Cas effector protein” or “Cas effector protein”, or more simply “CRISPR nuclease” or “Cas protein” are used herein to include effector proteins from Class 2 CRISPR systems. Non-limiting examples of CRISPR nucleases include Cas12a protein and NUX protein (described elsewhere herein).

[0041] Acr protein An Acr protein is a protein that inhibits (reduces the activity of) a Cas protein and acts as a negative regulator of the CRISPR complex. In some cases, the Acr protein of interest is an inhibitor of the Cas protein of a Class 2 CRISPR complex (e.g., a Class 2 effector protein, e.g., a Cas12 protein, e.g., Cas12a (also known as Cpf1)), thereby directly regulating the effector protein of the CRISPR complex.

[0042] Effector CRISPR-Cas nucleases that complex with gRNAs are highly diverse and span six different types (types I-VI). Anti-CRISPR proteins (Acr) that inhibit CRISPR types I, II, and V systems have been discovered to date. SpCas9, a CRISPR-Cas II-A type ortholog from S. pyogenes, is the most widely used CRISPR-Cas enzyme for biotechnology applications and has also been adopted for DNA-binding applications. Acr proteins that function against type II-A systems were discovered by a bioinformatics approach investigating self-targeting of the bacterial genome. Among the individual Acr protein families discovered to date, clear inhibition mechanisms have been identified in some of them (e.g., AcrIE1, AcrIF1-3, AcrIF10, AcrIIA2, AcrIIA4, AcrIIC1-3, and AcrVA5). The known mechanisms are highly diverse and present a pool of off-switch modalities to be exploited. Acr proteins can act on three different CRISPR-Cas-mediated immunity steps, including 1) inhibition of guide RNA loading, 2) inhibition of DNA binding, and 3) prevention of DNA cleavage. The most common mechanism observed to date is that the anti-CRISPR protein occupies the DNA-binding site on the Cas protein, mimics DNA, and inhibits the DNA-binding and cleavage activities of the protein.

[0043] However, the mechanisms by which Acr blocks DNA binding can be diverse. For example, AcrIF1, AcrIF2, and AcrIF10 bind to different subunits of the Cascade effector complex of the I-F type CRISPR-Cas system, and all of these inhibit DNA binding to the complex. AcrIIC3 also inhibits DNA binding but uses a fourth, different mechanism (promotion of Cas9 dimerization). For the most potent SpCas9 inhibitor, AcrIIA4, cryo-electron structures at 3.9 Å resolution have revealed that AcrIIA4 of the Cas9-sgRNA-AcrIIA4 complex binds to the PAM interaction domain of Cas9 to inhibit target DNA binding. Interestingly, AcrIIA4 binds only to the assembled Cas9-sgRNA complex and does not bind to the Cas9 protein alone or to the pre-formed Cas9-sgRNA-DNA complex.

[0044] Throughout this disclosure, when describing a particular protein such as a Cas or Acr protein (e.g., "Cas12a", "Nux", "ErAcr", "Acr") and when presenting such terms in the claims, it should be understood that such terms are intended to encompass modified / mutated versions of such proteins that maintain their intended functions. These terms are intended to encompass embodiments in which the Cas and / or Acr proteins are fused with one or more heterologous proteins (e.g., a fluorescent protein such as GFP, one or more nuclear localization signals (NLS), and / or tags such as MBP, CBP, strep tag, GST, HA, poly(His), Myc, V5, Spot, NE, AviTag).

[0045] Thus, in some embodiments, the subject “Acr protein” includes a wild-type (native) sequence. In some cases, the subject Acr protein is mutated. As mentioned above, the inventors generated engineered Acr proteins (ErAcr) that do not occur in nature (see, e.g., SEQ ID NOs: 126-132). Using this, a balance can be achieved such that the Cas protein retains sufficient activity to perform the desired on-target nucleic acid functions (e.g., loading, complex construction, binding, and cleavage), but is inhibited to the extent of reducing off-target activity. Accordingly, the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (ErAcr) or a nucleic acid encoding an Acr polypeptide.

[0046] Examples of the engineered Acr proteins (ErAcr) provided herein include SEQ ID NOs: 126-132 (see, e.g., the working examples). Further examples of ErAcr provided herein include SEQ ID NOs: 264-267 (see, e.g., Example 7 below). Further examples of Acr proteins provided herein include the proteins described as SEQ ID NOs: 165-169.

[0047] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126 - 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126 - 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126 - 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126 - 132. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126 - 132.

[0048] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 126.

[0049] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 127.

[0050] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 128.

[0051] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 129.

[0052] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 130.

[0053] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 131.

[0054] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 132.

[0055] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267.

[0056] In some cases, the target Acr protein (i.e., polypeptide) (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the target Acr protein comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the target Acr protein comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the target Acr protein comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the target Acr protein comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267.

[0057] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 264. In some cases, the subject Acr protein comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 264. In some cases, the subject Acr protein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 264. In some cases, the subject Acr protein comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 264. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 264.

[0058] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 265.

[0059] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 266.

[0060] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 267.

[0061] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165 - 169. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165 - 169. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165 - 169. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165 - 169. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165 - 169.

[0062] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 165.

[0063] In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 165, but has an amino acid variation at at least one position from amino acid 2 to amino acid 159 (inclusive) of SEQ ID NO: 165 (see, e.g., Table 6). In some cases, the Acr polypeptide comprises one or more of the amino acid variations set forth in Table 6. In some cases, the Acr polypeptide comprises one or more of the amino acid variations set forth in Table 7.

[0064] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 166.

[0065] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 167.

[0066] In some cases, the Acr protein (i.e., polypeptide) of interest (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 85% sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 90% sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the Acr protein of interest comprises an amino acid sequence having at least 95% sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the Acr protein of interest comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 168.

[0067] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the protein of the compositions, systems, or methods disclosed herein) comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 169. In some cases, the subject Acr protein comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 169. In some cases, the subject Acr protein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 169. In some cases, the subject Acr protein comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the Acr protein amino acid sequence set forth in SEQ ID NO: 169. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 169.

[0068] CRISPR nuclease Similar to the target Acr polypeptide, in some cases, the target CRISPR nuclease contains a wild-type (natural) sequence. In some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease has an amino acid sequence with at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the CRISPR nuclease has an amino acid sequence with at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the CRISPR nuclease has an amino acid sequence with at least 90% sequence identity (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the CRISPR nuclease has an amino acid sequence with at least 95% sequence identity (e.g., at least 97%, at least 98%, at least 99%, or 100% sequence identity) to a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the CRISPR nuclease contains the amino acid sequence of a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the target CRISPR nuclease has low overall sequence homology with the natural CRISPR nuclease but has "evolved" to retain the distinguishable characteristic domain(s) of the protein. In some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease is a Cas12a protein (see, e.g., SEQ ID NOs: 175 and 245 - 262).In some embodiments (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease is a NUX protein (see, e.g., SEQ ID NOs: 1-86 and 176-244). In some cases, the CRISPR nuclease is SEQ ID NO: 263.

[0069] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 263.

[0070] Examples of CRISPR nucleases include those in Table 1.

[0071]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

[0072] As mentioned above, in some cases, the Cas effector protein (i.e., the CRISPR nuclease) is variant (modified / mutated) (i.e., contains one or more amino acid mutations such as substitution(s), insertion(s), deletion(s), etc. relative to the wild-type Cas effector protein). For example, in some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease has one or more amino acid mutations relative to the wild-type protein.

[0073] Cas12a protein Therefore, in some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262.

[0074] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 245 to 262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 245 to 262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 245 to 262. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 245 to 262. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 245 to 262.

[0075] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 175. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 175. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 175. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 175. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 175.

[0076] NUX protein The term "NUX" protein is used herein to mean a nuclease having Cas-like activity (RNA-guided CRISPR effector protein) and having an amino acid sequence of any one of SEQ ID NOs: 1 to 86 and 176 to 244, or having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity to an amino acid sequence of any one of SEQ ID NOs: 1 to 86 and 176 to 244. In some embodiments, the amino acid sequence of the Nux protein lacks identity or significant amino acid homology to a particular known Cas nuclease. In some embodiments, the amino acid sequence of the NUX protein lacks identity or significant amino acid homology to the Cas12a (Cpf1) protein. In some embodiments, the amino acid sequence of the NUX protein lacks identity or significant amino acid homology to Cas9 such as SaCas9 or SpyCas9. In some embodiments, the amino acid sequence of the NUX protein has less than 50%, less than 48%, less than 45%, less than 40%, less than 35%, or less than 34% identity to the Cas12a (Cpf1) protein, SaCas9, or SpyCas9 protein.

[0077] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244.

[0078] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 176-178.

[0079] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 176. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 176. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 176. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 176. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 176.

[0080] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 177. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 177. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 177. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 177. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 177.

[0081] In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 178. In some cases, the CRISPR nuclease comprises an amino acid sequence having at least 95% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in SEQ ID NO: 178. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 178.

[0082] guide RNA In some embodiments, the subject composition, system, or method comprises a guide RNA (or a nucleic acid encoding the guide RNA). For example, in some cases, the subject composition, system, or method (e.g., a vector or vector system) comprises an expression cassette comprising a promoter operably linked to a sequence encoding the guide RNA. In some such cases, the promoter is an RNA polymerase III promoter (e.g., U6, H1), which can be used to express non-coding RNA in eukaryotic cells.

[0083] A "guide RNA" is a nucleic acid that binds to a Cas protein (e.g., a class 2 CRISPR nuclease such as Cas12a) to form a CRISPR complex (a protein-RNA effector complex), and can target the CRISPR complex to a specific "on-target" target sequence within a target nucleic acid (e.g., genomic DNA, e.g., eukaryotic or prokaryotic genomic DNA). In some cases, a hybrid DNA / RNA may be made such that the guide RNA contains DNA bases in addition to RNA bases, but it should be understood that the term "guide RNA" is used herein to encompass such hybrid molecules.

[0084] A guide RNA confers target specificity to the CRISPR complex by including a targeting segment that includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the sequence of the target nucleic acid. Thus, a given guide RNA includes (i) a guide sequence (also referred to as a "spacer" or "targeting sequence") that hybridizes to a target sequence (also referred to as a "protospacer") of a target nucleic acid, e.g., a target DNA, and (ii) a constant region (e.g., a region adjacent to the guide sequence that binds to the Cas protein). The "constant region" may also be referred to herein as a "protein-binding segment" or a "handle". Thus, the location of an on-target event (e.g., target DNA cleavage, transcriptional regulation, DNA methylation, histone modification) is in effect determined by the guide sequence of the guide RNA. A CRISPR complex-mediated event that occurs at a position that does not match 100% with the guide sequence is referred to herein as an off-target event.

[0085] A guide RNA can be referred to according to the corresponding protein. For example, when a guide RNA binds to and guides a class 2 CRISPR / Cas effector protein, the guide RNA can be referred to as a "class 2 guide RNA". When the class 2 CRISPR / Cas effector protein is a Cpf1 (Cas12a) protein, the corresponding guide RNA can be referred to as a "Cpf1 guide RNA" or a "Cas12a guide RNA".

[0086] In some embodiments, a guide RNA comprises two separate nucleic acid molecules: an "activator" (e.g., tracrRNA) and a "targeter" (e.g., crRNA), and is herein referred to as a "dual guide RNA", a "two-molecule guide RNA", a "two-component guide RNA", or a "dgRNA". In some embodiments, the guide RNA is a single molecule. For example, in some class 2 CRISPR / Cas systems, the corresponding guide RNA is naturally a single molecule, while in other class 2 CRISPR / Cas systems, the corresponding guide RNA is naturally two separate molecules (e.g., crRNA and tracrRNA), and these two molecules (the activator (e.g., tracrRNA) and the targeter (e.g., crRNA)) can covalently bond to each other, for example, via a chemical bond or intervening nucleotides. When the guide RNA is a single molecule, the guide RNA can be referred to as a "single guide RNA", a "one-molecule guide RNA", a "one-component guide RNA", or simply an "sgRNA". "Guide RNA" (or "gRNA") is a general term encompassing dual guide and single guide forms.

[0087] The guide sequence is complementary to (hybridizes with) the target sequence of the target nucleic acid (e.g., target DNA). In some cases, the guide sequence is 15 to 28 nucleotides (nt) in length (e.g., 15 to 26, 15 to 24, 15 to 22, 15 to 20, 15 to 18, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 17 to 26, 17 to 24, 17 to 22, 17 to 21, 17 to 20, 17 to 19, 17 to 18, 18 to 26, 18 to 24, 18 to 22, 18 to 20, or 19 to 21 nt in length). In some cases, the guide sequence is 18 to 24 nucleotides (nt) in length. In some cases, the guide sequence is 17 to 18 nucleotides (nt) in length. In some cases, the guide sequence is at least 15 nt in length (e.g., at least 16, 18, 20, 22 nt in length). In some cases, the guide sequence is at least 17 nt in length. In some cases, the guide sequence is at least 18 nt in length. In some cases, the guide sequence is at least 20 nt in length. In some cases, the guide sequence is 20 nt in length.

[0088] In some cases, the constant region of the guide RNA (also called the scaffold) is 15 nucleotides (nt) in length or more (e.g., 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 nt or more, 32 or more, 33 or more, 34 or more, 35 or more, 40 or more, 45 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more nt in length). In some cases, the constant region of the guide RNA is 18 nt in length or more.

[0089] Guide RNAs with various modifications (e.g., modifications via chemical modifications, changes in spacer length, sequence modifications of the spacer or scaffold, fusions with additional DNA or RNA components, partial substitutions with DNA, etc.) to increase efficiency compared to naturally occurring guide RNAs are known in the art and are readily available to those of skill in the art. See, for example, Moon et al., Trends Biotechnol. 2019 Aug;37(8):870-881, “Improving CRISPR Genome Editing by Engineering Guide RNAs”. As used herein, the term “guide RNA” encompasses such modifications and any convenient guide RNA can be used with the methods and compositions disclosed herein (e.g., as part of the system of interest, e.g., as RNA or as encoded by a nucleic acid of interest).

[0090] Combinations of Acr / CRISPR nucleases (“systems”) Generally, the term “system” is used herein to refer to a combination of an Acr polypeptide (in the form of a protein and / or nucleic acid as described herein) and a CRISPR nuclease (in the form of a protein and / or nucleic acid as described herein) that it inhibits. As mentioned above, in some cases, the Acr polypeptide and the CRISPR nuclease do not occur together naturally. In some cases, the system of interest further comprises a guide RNA (which directs the CRISPR nuclease to the target sequence) or a nucleic acid encoding the guide RNA.

[0091] In some embodiments (e.g., in the systems and / or methods disclosed herein), the Acr polypeptide and the CRISPR nuclease do not naturally occur together (i.e., are heterologous to each other). In some cases, the Acr polypeptide and / or the CRISPR nuclease comprises a variant amino acid sequence (e.g., the engineered Acr polypeptide disclosed herein). In some cases, the Acr polypeptide and the CRISPR nuclease are naturally occurring proteins, but the two proteins do not occur together in nature (e.g., the Acr polypeptide can be from a phage that does not naturally infect bacterial species that have the CRISPR nuclease).

[0092] Since any of the proteins can be provided as a nucleic acid (DNA or RNA) or in protein form, the subject systems can include any combination thereof. For example, in some embodiments, the subject systems include an Acr polypeptide or a nucleic acid (DNA or RNA) encoding the Acr polypeptide, and a CRISPR nuclease or a nucleic acid (DNA or RNA) encoding the CRISPR nuclease. In some cases, the subject systems include a CRISPR nuclease and a nucleic acid encoding the Acr polypeptide. In some cases, the subject systems include an Acr polypeptide and a nucleic acid encoding the CRISPR nuclease. In some cases, the subject systems include a nucleic acid encoding the Acr polypeptide and a nucleic acid encoding the CRISPR nuclease. In some such cases, the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the CRISPR nuclease are on the same nucleic acid (e.g., the same vector). In other cases, the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the CRISPR nuclease are on different nucleic acids (e.g., different vectors). For descriptions of nucleic acids (e.g., vectors, promoters, translational control elements, etc.), see other sections of this specification. The Acr polypeptide and the CRISPR nuclease of the system of interest can be any of those described herein in any desired combination.

[0093] Acr protein (SEQ ID NOs: 165-169) and CRISPR nuclease (Cas12a or NUX) In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 262. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 262. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 168, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 262.In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0094] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 165 - 169, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 175 and 245 - 262. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 - 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 175 and 245 - 262. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 - 168, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 175 and 245 - 262. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 175 and 245 - 262.In some cases, the systems described in this paragraph further include a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0095] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence described as SEQ ID NO: 175. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 175. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 168, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 175. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 175.In some cases, the systems described in this paragraph further comprise a guide RNA that directs a CRISPR nuclease to a target sequence, or a nucleic acid encoding the guide RNA.

[0096] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 176 to 244. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 176 to 244. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 168, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 176 to 244.In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0097] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 165 to 168, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 176 to 178.In some cases, the systems described in this paragraph further comprise a guide RNA that directs a CRISPR nuclease to a target sequence, or a nucleic acid encoding the guide RNA.

[0098] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 165 to 169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 176. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 165 to 167, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 176. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165 or 167, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 176.In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 176. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to a target sequence), or a nucleic acid encoding the guide RNA.

[0099] In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 177. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to a target sequence), or a nucleic acid encoding the guide RNA.

[0100] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 165-169, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 178. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as any one of SEQ ID NOs: 126-132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 178. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 165, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence set forth as SEQ ID NO: 178. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to a target sequence), or a nucleic acid encoding the guide RNA.

[0101] Acr (SEQ ID NOs: 126-132) and CRISPR nuclease In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 262. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 262. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0102] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 175 to 263. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0103] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 175 and 245 to 262. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 175 and 245 to 262. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0104] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 175. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to a target sequence), or a nucleic acid encoding the guide RNA.

[0105] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 176 to 244. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1 to 86 and 176 to 244. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0106] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0107] In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence described as SEQ ID NO: 263. In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 126 to 132, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 263. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0108] Acr (SEQ ID NOs: 264 to 267) and CRISPR nuclease In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the system described in this paragraph further comprises a guide RNA (which directs the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0109] In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 175 and 245-262. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0110] In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence described as any one of SEQ ID NOs: 264 to 267, and the CRISPR nuclease comprises an amino acid sequence described as SEQ ID NO: 175. In some cases, the Acr polypeptide of the system of interest comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264 to 267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 175. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0111] In some cases, the Acr polypeptide of the target system comprises an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the target system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0112] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 1-86, 176-244, and 263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to an amino acid sequence described as any one of SEQ ID NOs: 1-86, 176-244, and 263. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0113] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 264 to 267, and the CRISPR nuclease comprises an amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264 to 267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 176 to 178. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0114] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence described as SEQ ID NO: 263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as any one of SEQ ID NOs: 264-267, and the CRISPR nuclease comprises an amino acid sequence having at least 75% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity) to the amino acid sequence described as SEQ ID NO: 263. In some cases, the system described in this paragraph further comprises a guide RNA (which guides the CRISPR nuclease to the target sequence), or a nucleic acid encoding the guide RNA.

[0115] Fusion In some cases, a protein (e.g., an Acr protein and / or a CRISPR nuclease (e.g., Cas12a or NUX protein)) is fused to one or more heterologous polypeptides (also referred to herein as fusion partners) (e.g., one or more NLSs, protein tags, etc.). Suitable fusion partners include, but are not limited to, (i) intracellular localization sequences (e.g., one or more, two or more, or three or more nuclear localization signals (NLSs) for targeting to the nucleus, a sequence for not bringing the fusion protein close to the nucleus, e.g., a nuclear export sequence (NES), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to the chloroplast, an ER retention signal, etc.), (ii) protein tags, e.g., tags for facilitating tracking and / or purification (e.g., fluorescent proteins, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc., MBP, CBP, strep tag, GST, HA, FLAG, poly(His), Myc, V5, Spot, NE, AviTag, etc.), and (iii) polypeptides that increase or decrease stability (e.g., degrons (in some cases controllable), e.g., temperature-sensitive or drug-controllable degron sequences).

[0116] The target protein (e.g., Acr protein and / or CRISPR nuclease (e.g., Cas12a or NUX protein)) can have multiple (one or more, two or more, three or more, etc.) fusion partners in any combination. As an exemplary example, the target protein may have a fusion partner that results in tagging (e.g., GFP), or may have an intracellular localization sequence (e.g., one or more NLSs). In some cases, such fusion proteins may also have tags to facilitate tracking and / or purification. As another exemplary example, the target protein (e.g., Acr protein such as the engineered Acr protein of interest) can have one or more NLSs (e.g., two or more, three or more, four or more, five or more, one, two, three, four, or five NLSs). In some cases, the fusion partner is located at the C-terminus or near it (e.g., within about 50 amino acids from the C-terminus), near the N-terminus (e.g., within about 50 amino acids from the N-terminus), or at both the N-terminus and the C-terminus.

[0117] Non-limiting examples of NLSs include NLS sequences derived from the following: the NLS of SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 87), the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 88)), the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 89) or RQRRNELKRSP (SEQ ID NO: 90), the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 91), the sequence of the IBB domain from importin-alpha RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 92), the sequences of the myogenic T protein VSRKRPRP (SEQ ID NO: 93) and PPKKARED (SEQ ID NO: 94), the sequence of human p53 PQPKKKPL (SEQ ID NO: 95), the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO: 96), the sequences of influenza virus NS1 DRLRR (SEQ ID NO: 97) and PKQKKRK (SEQ ID NO: 98), the sequence of hepatitis delta antigen RKLKKKIKKL (SEQ ID NO: 99), the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 100), the sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 101), and the sequence of the steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: 102). In some cases, the NLS has the sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO: 125). Generally, the NLS (or NLSs) has sufficient strength to drive the accumulation of a detectable amount of Cas protein in the nucleus of eukaryotic cells.

[0118] In some cases, the fusion partner includes a "Protein Transduction Domain", i.e., PTD (also known as CPP (Cell-Penetrating Peptide)), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates the crossing of lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. A PTD conjugated to another molecule (which can range from small polar molecules to large macromolecules and / or nanoparticles) facilitates the molecule's crossing of the membrane (e.g., movement from the extracellular space into the intracellular space or from the cytoplasm into an organelle). In some embodiments, the PTD is covalently bound to the amino terminus of the polypeptide, and in some embodiments, the PTD is covalently bound to the carboxyl terminus of the polypeptide. In some cases, the PTD is internally inserted at a suitable insertion site. In some cases, the subject Cas protein comprises (is conjugated to, fused with) one or more PTDs (e.g., two or more, three or more, four or more PTDs).Exemplary PTDs include, but are not limited to, the minimal undecapeptide protein transduction domain (corresponding to residues 47 - 57 of HIV-1 TAT containing YGRKKRRQRRR (SEQ ID NO: 103)), polyarginine sequences containing a sufficient number of arginines to direct entry into cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10 - 50 arginines), the VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489 - 96), the Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732 - 1737), the truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248 - 1256), polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003 - 13008), RRQRRTSKLMKR (SEQ ID NO: 104), the transporteran GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 105), KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 106), and RQIKIWFQNRRMKWKK (SEQ ID NO: 107). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 108), RKKRRQRRR (SEQ ID NO: 109), and arginine homopolymers of 3 arginine residues to 50 arginine residues. Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 110), RKKRRQRR (SEQ ID NO: 111), YARAAARQARA (SEQ ID NO: 112), THRLPRRRRRR (SEQ ID NO: 113), and GGRRARRRRRR (SEQ ID NO: 114). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5 - 6):371 - 381).ACPP contains a polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable linker to a polyanion that matches (e.g., Glu9 or "E9"), which reduces the net charge to near zero and inhibits adhesion and uptake into cells. When the linker is cleaved, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness and activating the ACPP to cross the membrane.

[0119] Linker (e.g., for fusion partner) In some embodiments, the Cas protein of interest can be fused to a fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide can have any of a variety of amino acid sequences. Proteins can generally be linked by spacer peptides of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides that are 4 amino acids to 40 amino acids in length, or 4 amino acids to 25 amino acids in length. These linkers can be generated by binding the proteins using synthetic linker-encoding oligonucleotides or can also be encoded by nucleic acid sequences encoding fusion proteins. Peptide linkers having some degree of flexibility can be used. The binding peptide can have substantially any amino acid sequence, taking into account that preferred linkers generally result in flexible peptides. The use of small amino acids (e.g., glycine and alanine) is useful for creating flexible peptides. The creation of such sequences is routine to those skilled in the art. A variety of different linkers are commercially available and are considered suitable for use.

[0120] Examples of linker polypeptides include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , GSGGS n (SEQ ID NO: 115), GGSGGS n (SEQ ID NO: 116), and GGGS n(SEQ ID NO: 117) (where n is an integer of at least 1)), glycine-alanine polymers, alanine-serine polymers, etc. Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), GSSSG (SEQ ID NO: 123), and the like. One of ordinary skill in the art will recognize that a linker, which can be all or partially flexible, can include one or more moieties that impart a non-flexible structure in addition to the flexible linker, such that the design of a peptide conjugated to any desired element can be accomplished.

[0121] Translation control element The present disclosure provides compositions, systems, and methods comprising one or more nucleic acids that encode an Acr protein and a CRISPR nuclease, wherein the Acr protein is an inhibitor of a Cas protein (e.g., a Cas effector protein / CRISPR nuclease) and preferentially inhibits off-target editing over on-target editing, for example. In some cases, both coding sequences are present on the same nucleic acid (e.g., vector), and in some cases, they are present on separate nucleic acids (e.g., separate vectors).

[0122] The nucleic acid of interest can comprise a translation control element operably linked to an Acr protein or a Cas protein (e.g., a CRISPR nuclease, e.g., Cas12a, a NUX protein, or a variant thereof), or to either of these in order to achieve a desired balance (ratio of expression levels) between two proteins. For example, in some cases, the nucleic acid of interest comprises a translation control element operably linked to a sequence encoding an Acr protein (and thus regulating / modifying its translation). In some cases, the nucleic acid of interest comprises a translation control element operably linked to a sequence encoding a Cas protein (and thus regulating / modifying its translation). In some cases, both the sequence encoding an Acr protein and the sequence encoding a Cas protein are each, for example independently, operably linked to a translation control element (in which case the two control elements may be the same or different).

[0123] In some cases, both the sequence encoding an Acr protein and the sequence encoding a Cas protein are each operably linked to the same translation control element (e.g., an IRES element, a 2A peptide coding sequence), such that these sequences become part of a polycistronic transcript. Thus, in some cases, the translation control element of interest is a polycistronic linker. In other words, in some cases, the translation control element promotes (causes) the production of gene products (e.g., an Acr protein and a CRISPR nuclease) that are independent from the same transcript.

[0124] Thus, in some cases, a translation control element couples a first protein coding sequence (e.g., a sequence encoding an Acr protein) to a second protein coding sequence (e.g., a sequence encoding a CRISPR nuclease), such that the first and second proteins (e.g., an Acr protein and a CRISPR nuclease) are encoded by a polycistronic sequence. Thus, in such cases, both protein sequences are operably linked to the same promoter, and the RNA transcribed therefrom will contain both protein coding sequences in addition to the sequences encoded by the translation control element.

[0125] As described in more detail below, in some cases, multiple translation control elements (e.g., IRES elements, 2A peptides, non-AUG start codons) are used to control the expression of a given protein (e.g., an Acr protein, a CRISPR nuclease such as Cas12a, or a Nux protein). Any convenient combination of translation control elements can be used.

[0126] 2A peptide A non-limiting example of a translation control element that functions as a polycistronic linker and can promote the generation of separate protein products (e.g., two separate proteins) from the same single RNA transcript is the 2A peptide sequence.

[0127] The “2A peptide” refers to a small peptide sequence (usually 18 - 25 amino acids, although some such sequences may be arranged in tandem) that enables the expression (translation) of separate protein products from a single RNA transcript (through a self - “cleavage” event often referred to as “ribosome skipping”, although the disclosure herein is not restricted by and does not depend on the mechanism of action), where these separate proteins are encoded as part of the same open reading frame (ORF). 2A peptides are readily distinguishable by their consensus motif (DXEXNPGP, sometimes described as DVEXNPGP) and their ability to promote protein cleavage / skipping. Any convenient 2A peptide sequence can be used in the nucleic acid of interest. Examples of 2A peptides include, but are not limited to, 2A peptides from viruses such as foot - and - mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus - 1 (P2A), or Thosea asigna virus (T2A). See, for example, Szymczak - Workman, A. et al., “Design and Construction of 2A Peptide - Linked Multicistronic Vectors”. Cold Spring Harb Protoc. 2012 Feb 1;2012(2):199 - 204; Liu et al., Sci Rep. 2017;7:2193; Kim et al., PLOS One 6:e18556, 2011; and U.S. Patent Nos. 10,738,325; 9,655,956; 10,577,417 (the disclosures of which are incorporated herein by reference for their relevance to 2A peptides).

[0128] Typically, the 2A peptide coding sequence of interest is arranged to regulate the expression (translation) of a protein of interest (e.g., an Acr protein or a Cas protein), and thus is placed in-frame on the 5'-side (usually immediately 5'-side) of the protein coding sequence it regulates. Figure 9 shows non-limiting exemplary embodiments of embodiments in which the 2A peptide coding sequence is arranged in different ways. In some cases, the 2A peptide sequence is located at the 5'-side (usually immediately 5'-side) of the Cas protein coding sequence. In some cases, the 2A peptide sequence is located at the 5'-side (usually immediately 5'-side) of the Acr protein coding sequence.

[0129] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to the same promoter (encoded by a polycistronic sequence), arranged tandemly, with a 2A peptide sequence placed therebetween (see, e.g., FIGS. 9A and 9B). In some such cases, the Cas protein coding sequence is located on the 5'-side of the Acr coding sequence, such that the 2A peptide coding sequence is on the 3'-side of the Cas sequence and the 5'-side of the Acr sequence. In other such cases, the Acr protein coding sequence is located on the 5'-side of the Cas coding sequence, such that the 2A peptide coding sequence is on the 3'-side of the Acr sequence and the 5'-side of the Cas sequence.

[0130] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to a first promoter and a second promoter, respectively, such that they are transcribed as separate transcripts (see, e.g., FIGS. 9C and 9D). The first and second promoters (designated “P1” and “P2” in the figures) may be different from each other or the same (i.e., may be copies of the same promoter). In some such cases, the 2A peptide sequence regulates the Acr sequence (and is thus located 5′ thereto). In other cases, the 2A peptide sequence regulates the Cas sequence (and is thus located 5′ thereto). In some cases where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, each is regulated by a 2A peptide sequence (and is thus located 3′ to the 2A peptide sequence).

[0131] In some embodiments where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, a "spacer" protein coding sequence is used on the 5' side of the 2A peptide sequence, such that the "spacer" sequence is transcribed as part of a polycistronic sequence, along with the protein sequence to be regulated. For example, in FIG. 9C, the Cas protein coding sequence and the Acr protein coding sequence are operably linked to different promoters (P1 and P2). The spacer sequence (designated "X" in the drawing) is located on the 5' side of the 2A peptide sequence that is on the 5' side of the Acr coding sequence, such that the spacer sequence and the Acr sequence are transcribed as part of the same RNA. However, the presence of the 2A peptide sequence results in the Acr protein being produced as a separate protein. Similarly, in FIG. 9D, the Cas protein coding sequence and the Acr protein coding sequence are also operably linked to different promoters (P1 and P2) in this case. In this example, the spacer sequence (designated "X" in the drawing) is located on the 5' side of the 2A peptide sequence that is on the 5' side of the Cas coding sequence, such that the spacer sequence and the Cas sequence are transcribed as part of the same RNA. However, the presence of the 2A peptide sequence in this RNA results in the Cas protein being produced as a separate protein.

[0132] The "spacer" protein is simply intended to provide a sequence to be translated on the 5' side (N-terminal side) of the 2A peptide sequence, and thus can be any desired sequence. The spacer sequence can be of any convenient length, from very short to encoding the entire protein sequence. In some cases, the spacer is 2 amino acids or longer (e.g., 3 or more, 4 or more, 5 or more, 10 or more, or 20 or more amino acids). In some cases, the length of the spacer is 1 to 100 amino acids (e.g., 1 to 80, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 100, 2 to 80, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 100, 5 to 80, 5 to 50, 5 to 40, 5 to 30, 5 to 20, or 5 to 10 amino acids). Examples of spacer sequences include, but are not limited to, linker sequences, repeated single amino acids (e.g., AAAA), random sequences, protein fragments, and marker proteins (e.g., fluorescent proteins such as GFP, YFP, CFP, RFP, drug selectivity protein markers, enzymes such as beta-galactosidase, etc.).

[0133] Examples of 2A peptide sequences include, but are not limited to, the sequences described as SEQ ID NOs: 133 to 138.

[0134] 2A peptide sequences can be used in tandem, and multiple different 2A peptide sequences can be arranged successively in any desired combination (see, as non-limiting examples, the above "E2A - F2A" and "T2A - E2A - F2A"). Thus, in some cases, the 2A peptide sequence is selected from the group consisting of P2A, F2A, E2A, T2A, and any combination thereof. In some embodiments, the translation control element encodes two or more 2A peptides in tandem (e.g., 3 or more, 4 or more, or 5 or more). In some embodiments, the translation control element encodes 2, 3, 4, or 5 2A peptides in tandem. In some embodiments, the translation control element encodes one 2A peptide. In some cases, the 2A peptide sequence comprises an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the amino acid sequence set forth in any one of SEQ ID NOs: 133 to 138.=

[0135] IRES In some embodiments herein, the translational control element is an internal ribosome entry site (IRES) sequence. The term "internal ribosome entry site (IRES)" means a nucleotide sequence that enables the initiation of protein translation within a messenger RNA (mRNA) sequence (i.e., downstream of the first start codon). For example, when an IRES segment is located between two open reading frames of a bicistronic eukaryotic mRNA molecule, it can drive the translation of the downstream protein coding region independent of the 5' cap structure at the 5' end of the mRNA molecule (i.e., upstream of the upstream protein coding region). In such a setup, both proteins are produced intracellularly. The protein located in the first cistron is synthesized by a cap-dependent translation initiation mechanism, and the initiation of translation of the second protein is directed by the IRES segment located in the intercistronic spacer region between the two protein coding regions. IRESs have been isolated from viral and cellular genomes. Artificially engineered IRESs are also known in the art. One of ordinary skill in the art will recognize that the sequences described herein as IRES sequences function as part of an RNA molecule and have corresponding sequences within the coding DNA molecule (e.g., the RNA sequence 5'-uuacuggc-3' corresponds to the DNA sequence 5'-ttactggc-3' and vice versa). The term "IRES sequence" or simply "IRES" is used herein to refer to any of the sequences.

[0136] Any convenient IRES can be used in the compositions, systems, and methods of interest. Examples of IRES sequences include, but are not limited to, the sequences set forth in FIGS. 13A-13D (SEQ ID NOs: 139-159). One of ordinary skill in the art will recognize that when the system of interest is used to express Cas protein and Acr protein in non-animal cells (e.g., plant (s) cells), a convenient IRES sequence suitable for the desired cell type (e.g., an IRES from Triticum mosaic virus (TriMV)) should be selected. See, for example, Urwin et al., Plant J. 2000 Dec;24(5):583-9 and U.S. Patent Nos. 8,772,465; 9,879,271 (each of which is incorporated by reference for teachings regarding the use of IRES sequences in plants).

[0137] In some cases, the IRES sequence comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to the nucleotide sequence set forth in any one of SEQ ID NOs: 139-159.

[0138] In some cases, the IRES sequence is selected from the group consisting of the following IRES sequences: EMCV, BIP, CAT-1, c-myc, HCV, VCIP, Apaf-1, mEMCV-1, mEMCV-2, HRV, NRF, FGF-1, KMI1, KMI2, (GAAA)16, (PPT19)4, EMCV variant 5 (SEQ ID NO: 140), EMCV variant 10 (SEQ ID NO: 141), EMCV variant 15 (SEQ ID NO: 142), and EMCV variant 21 (SEQ ID NO: 143) [see also, for example, International Patent Publication No. WO2022072673].

[0139] Typically, the IRES sequence of interest is placed within the nucleic acid of interest to regulate the expression (translation) of the protein of interest (e.g., Acr protein or Cas protein), and thus is placed 5' (usually immediately 5') of the protein-coding sequence regulated by this sequence. Figure 10 shows non-limiting exemplary embodiments of embodiments in which the IRES sequences are arranged in different ways. In some cases, the IRES sequence is located 5' (usually immediately 5') of the Cas protein-coding sequence. In some cases, the IRES sequence is located 5' (usually immediately 5') of the Acr protein-coding sequence.

[0140] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to the same promoter (encoded by a polycistronic sequence), arranged tandemly, and an IRES sequence is placed therebetween (see, e.g., the first and second examples in Figure 10). In some such cases, the Cas protein-coding sequence is located 5' of the Acr coding sequence, so that the IRES sequence is 3' of the Cas sequence and 5' of the Acr sequence. In some such cases, the Acr protein-coding sequence is located 5' of the Cas coding sequence, so that the IRES sequence is 3' of the Acr sequence and 5' of the Cas sequence.

[0141] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to a first promoter and a second promoter, respectively, such that they are transcribed as separate transcripts (see, e.g., C-F of FIG. 10). The first and second promoters (designated "P1" and "P2" in the drawings) may be different from each other or the same (i.e., may be copies of the same promoter). In some such cases, the IRES sequence regulates the Acr sequence (and is thus located 5' to it). In other cases, the IRES sequence regulates the Cas sequence (and is thus located 5' to it). In some cases where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, each is regulated by an IRES sequence (and is thus located 3' to the IRES sequence).

[0142] In some embodiments where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, a "spacer" protein coding sequence is used 5' to the IRES sequence, such that the "spacer" sequence is transcribed as part of a polycistronic sequence along with the protein sequence to be regulated (see, e.g., E and F of FIG. 10). For example, in FIG. 10E, the Cas protein coding sequence and the Acr protein coding sequence are operably linked to different promoters (P1 and P2). The spacer sequence (designated "X" in the drawings) is located 5' to the IRES sequence that is 5' to the Acr coding sequence, such that the spacer sequence and the Acr sequence are transcribed as part of the same RNA, but the presence of the IRES sequence results in the Acr protein being produced as a separate protein. Similarly, in FIG. 10F, the Cas protein coding sequence and the Acr protein coding sequence are also operably linked to different promoters (P1 and P2) in this case. In this example, the spacer sequence (designated "X" in the drawings) is located 5' to the IRES sequence that is 5' to the Cas coding sequence, such that the spacer sequence and the Cas sequence are transcribed as part of the same RNA, but the presence of the IRES sequence results in the Cas protein being produced as a separate protein.

[0143] Since the "spacer" protein is simply intended to provide a sequence to be translated that is on the 5' side of the IRES sequence, it can be any desired sequence. The spacer sequence can be of any convenient length, from very short to one that encodes the entire protein sequence. In some cases, the spacer is 2 amino acids or longer (e.g., 3 or more, 4 or more, 5 or more, 10 or more, or 20 or more amino acids). In some cases, the length of the spacer is 1 to 100 amino acids (e.g., 1 to 80, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 100, 2 to 80, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 100, 5 to 80, 5 to 50, 5 to 40, 5 to 30, 5 to 20, or 5 to 10 amino acids). Examples of spacer sequences include, but are not limited to, linker sequences, repeated single amino acids (e.g., AAAA), random sequences, protein fragments, and marker proteins (e.g., fluorescent proteins such as GFP, YFP, CFP, RFP, drug selectivity protein markers, enzymes such as beta-galactosidase, etc.).

[0144] In some cases where the Acr coding sequence and the Cas coding sequence are operably linked to separate promoters, the spacer sequence is not used (see, for example, FIGS. 10C and D).

[0145] Start codon In some embodiments of the present specification, the translation control element is a non-AUG start codon (also referred to as a non-AUG initiation codon). The term "non-AUG start codon" or "non-AUG initiation codon" is intended to include any non-AUG polynucleotide (typically a triplet) that functions as a translation initiation site with reduced efficiency compared to the AUG start codon. Examples of the frequency of use of naturally occurring alternative start codons are described, for example, in Kozak (1991) J. Cell Biol. 115(4):887-903; Mehdi et al. (1990) Gene 91:173-178; Kozak (1989) Mol. Cell. Biol. 9(11):5073-5080. Generally, the translation efficiency of non-AUG start codons is lower than that of AUG start codons.

[0146] In some cases, a non-AUG start codon is used as the start codon for the sequence encoding the Acr protein. In some cases, a non-AUG start codon is used as the start codon for the sequence encoding a Cas protein (e.g., a CRISPR nuclease). In some cases, the non-AUG start codon (used with an Acr sequence or a Cas sequence) is any one of CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, the non-AUG start codon (used with an Acr sequence or a Cas sequence) is any one of CUG, GUG, ACG, AUA, or UUG. For example, in some cases, the non-AUG start codon used with an Acr sequence is any one of CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, the non-AUG start codon used with an Acr sequence is any one of CUG, GUG, ACG, AUA, or UUG. As another example, in some cases, the non-AUG start codon used with a Cas sequence is any one of CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, the non-AUG start codon used with a Cas sequence is any one of CUG, GUG, ACG, AUA, or UUG. In some cases, the non-AUG start codon used with an Acr sequence is CUG. In some cases, the non-AUG start codon used with an Acr sequence is GUG. In some cases, the non-AUG start codon used with an Acr sequence is ACG. In some cases, the non-AUG start codon used with a Cas sequence is CUG. In some cases, the non-AUG start codon used with a Cas sequence is GUG. In some cases, the non-AUG start codon used with a Cas sequence is ACG.

[0147] The translation efficiency of non-AUG start codons can also be affected by their sequence context. For example, in eukaryotic cells, it has been reported that the optimal Kozak consensus sequence has a positive effect on the initiation of translation at non-AUG start codons (Mehdi et al. (1990) Gene 91:173-178; Kozak (1989) Mol. Cell. Biol. 9(11):5073-5080). The complete Kozak DNA consensus sequence is [Chemical formula] where the start codon ATG (AUG in the case of RNA) is immediately before the last "G", the A of the ATG start codon is called the +1 position, and the "R" at position -3 is a purine (A or G). The two most highly conserved positions are the purine at -3 (usually A) and the G at +4 (Kozak (1991) J Cell Biol 115(4):887-903). In some cases, the non-AUG start codon of interest (e.g., any of those described above) is associated with a disrupted Kozak sequence (i.e., a Kozak sequence that does not conform to the consensus).

[0148] For the above examples, see, for example, Kearse and Wilusz, Genes Dev. 2017 Sep 1;31(17):1717-1731; US Patent Application Publication Nos. US20060172382 and US20060141577; and US Patents Nos. 5,648,267; 5,733,779; 8,828,976; 10,030,252; 10,317,329. These disclosures are incorporated herein by reference because they are relevant to Kozak sequences and non-AUG start codons (and the assays related thereto). One of ordinary skill in the art will recognize that the sequences described herein as DNA have corresponding sequences as RNA molecules (e.g., the DNA sequence ATG corresponds to the RNA sequence AUG, and vice versa).

[0149] Typically, a subject non-AUG start codon is placed within a subject nucleic acid so as to regulate the expression (translation initiation) of a subject protein (e.g., an Acr protein or a Cas protein), and thus is placed in-frame 5’ (usually immediately 5’) to the protein coding sequence that this sequence regulates. In some cases (e.g., when the non-AUG start codon is used as the start codon in an Acr coding sequence), the sequence encoding the Acr protein does not contain its native AUG start codon. In some such cases, the sequence encoding the Acr protein does not contain an AUG codon. In some cases (e.g., when the non-AUG start codon is used as the start codon in a Cas coding sequence), the sequence encoding the CRISPR nuclease does not contain its native AUG start codon. In some cases, the sequence encoding the CRISPR nuclease does not contain an AUG codon. In some cases (e.g., when the non-AUG start codon is used as the start codon in a Cas coding sequence or an Acr coding sequence), the sequence encoding a subject protein (i.e., a CRISPR nuclease or an Acr protein) is wholly or partially codon-optimized to avoid having an out-of-frame AUG that could direct the translation machinery into an incorrect reading frame (i.e., a reading frame that does not encode the subject protein) contained within the nucleic acid encoding the Cas or Acr protein. In some such cases, the first 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 codons from the start of the subject protein are optimized to not contain an out-of-frame AUG.

[0150] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to a first promoter and a second promoter, respectively, such that they are transcribed as separate transcripts (see, e.g., FIG. 11). The first and second promoters (designated “P1” and “P2” in the figures) may be different from each other or the same (i.e., copies of the same promoter).

[0151] In some embodiments, multiple translation control elements can be used to control the expression of proteins (e.g., Acr proteins, CRISPR nucleases). For example, in some cases, multiple (e.g., two, two or more, three) translation control elements are used to control the expression of Acr proteins. In some cases, multiple (e.g., two, two or more, three) translation control elements are used to control the expression of CRISPR nucleases. Therefore, in some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRESs, non-AUG start codons) are used to control the expression of Acr proteins and / or CRISPR nucleases. In some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRESs, non-AUG start codons) are used to control the expression of Acr proteins. In some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRESs, non-AUG start codons) are used to control the expression of CRISPR nucleases.

[0152] Promoter The ACR and nuclease compositions, methods, and systems provided herein include expression cassettes (e.g., on one or more vectors), which include a promoter for driving the expression of a gene encoding a Cas protein (e.g., class 2 effector protein / CRISPR nuclease) and / or an Acr protein, and / or a guide RNA.

[0153] In some cases, the Acr coding sequence and the CRISPR nuclease sequence are operably linked to the same promoter and are thus transcribed as part of the same RNA. In other cases, the Acr coding sequence and the CRISPR nuclease sequence are operably linked to different promoters. For example, in some cases, the Acr coding sequence is operably linked to a first promoter and the CRISPR nuclease sequence is operably linked to a second promoter. In some such cases, the first promoter and the second promoter are the same, such that the two protein-coding sequences are transcribed as separate RNAs but are controlled by the same promoter sequence (i.e., there are two copies of the same promoter, one controlling the expression of one protein and the other controlling the expression of the other protein). In other such cases, the first and second promoters are different promoters. Type of promoter

[0154] Various types of promoters can be used to control the expression of, for example, the Acr protein and / or the CRISPR nuclease (e.g., Cas12a or NUX protein). The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively active / “ON”), the promoter can be an inducible promoter (i.e., a promoter whose active / “ON” or inactive / “OFF” state is controlled by an external stimulus (e.g., the presence of a specific temperature, compound, or protein)), the promoter can be a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and / or the promoter can be a temporally restricted promoter (e.g., the promoter is in the “ON” or “OFF” state during a specific stage of embryonic development or a specific stage of a biological process).

[0155] Suitable promoters can be of viral origin and are thus sometimes referred to as viral promoters, or they can be from any convenient organism. Suitable promoters can be of viral origin and are thus sometimes referred to as viral promoters, or they can be from any organism, including prokaryotic or eukaryotic organisms. Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31(17)), the human H1 promoter (H1), and the like. Pol III promoters (e.g., U6, enhanced U6, and H1) are commonly used for the expression of non-coding RNAs (e.g., guide RNAs).

[0156] Examples of inducible promoters include, but are not limited to, the heat shock promoter, the tetracycline-regulated promoter, the steroid-regulated promoter, the metal-regulated promoter, the estrogen receptor-regulated promoter, and the like. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline, estrogen receptor, estrogen receptor fusions, estrogen analogs, IPTG, and the like.

[0157] Inducible promoters suitable for use include any inducible promoter described herein or known to those skilled in the art. Examples of inducible promoters include, but are not limited to, chemical / biochemical regulation and physical regulation promoters, such as alcohol regulation promoters, tetracycline regulation promoters (e.g., anhydrotetracycline (aTc)-responsive promoters, and other tetracycline-responsive promoter systems (including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA))), steroid regulation promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, gaxidson receptor, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal regulation promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogen regulation promoters (e.g., those induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat inducible promoters (e.g., heat shock promoters), and light regulation promoters (e.g., light-responsive promoters from plant cells).

[0158] Examples of suitable promoters include, but are not limited to, the following. Mammalian (Pol II) promoter (for nuclease and Acr) ● Retroviral Rous sarcoma virus (RSV) ● LTR promoter (optionally with RSV enhancer) ● Cytomegalovirus (CMV) promoter (optionally with CMV enhancer) ● SV40 promoter ● Dihydrofolate reductase promoter ● Beta-actin promoter ● Phosphoglycerate kinase (PGK) promoter ● EF1.alpha (EF1a) promoter ● MMLV LTR promoter ● HIV LTR promoter, MCMV LTR promoter ● MND ● Ubc ● CAG ● HSV TK promoter ● fos promoter ● E2F promoter ● Polyomavirus ● Adenovirus, fowlpox virus ● Bovine papillomavirus ● Rous sarcoma virus Eukaryotic tissue-specific ● Bowman et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12115 - 12119 describes a brain-specific transferrin promoter ● The synapsin I promoter is neuron-specific (Schoch et al., 1996 J. Biol. Chem. 271, 3317 - 3323) ● The nectin promoter is specific to post-mitotic neurons (Uetsuki et al., 1996 J. Biol. Chem. 271, 918 - 924) ● The neurofilament light promoter is neuron-specific (Charron et al., 1995 J. Biol. Chem. 270, 30604 - 30610) ● The acetylcholine receptor promoter is neuron-specific (Wood et al., 1995 J. Biol. Chem. 270, 30933 - 30940) ● The potassium channel promoter is specific to high-frequency firing neurons (Gan et al., 1996 J. Biol. Chem 271, 5859 - 5865) ● The chromogranin A promoter is specific to neuroendocrine cells (Wu et al., 1995 A.J.Clin.Invest.96, 568 - 578) ● The von Willebrand factor promoter is specific to brain endothelium (Aird et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4567-4571) ● The flt-1 promoter is specific to endothelium (Morishita et al., 1995 J. Biol. Chem. 270, 27948-27953) ● The preproendothelin-1 promoter is specific to endothelium, epithelium, and muscle (Harats et al., 1995 J. Clin. Invest. 95, 1335-1344) ● The GLUT4 promoter is specific to skeletal muscle (Olson and Pessin, 1995 J. Biol. Chem. 270, 23491-23495) ● The slow / fast troponin promoter is specific to slow / fast muscle fibers (Corin et al., 1995 Proc. Natl. Acad. Sci. USA 92, 6185-6189) ● The actin promoter is specific to smooth muscle (Shimizu et al, 1995 J. Biol. Chem. 270, 7631-7643) ● The myosin heavy chain promoter is specific to smooth muscle (Kallmeier et al., 1995 J. Biol. Chem. 270, 30949-30957) ● The E-cadherin promoter is specific to epithelium (Hennig et al., 1996 J. Biol. Chem. 271, 595-602) ● The cytokeratin promoter is specific to keratinocytes (Alexander et al., 1995 B. Hum. Mol. Genet. 4, 993-999) ● The transglutaminase 3 promoter is specific to keratinocytes (J. Lee et al., 1996 J. Biol. Chem. 271, 4561-4568) ● The bullous pemphigoid antigen promoter is specific to basal keratinocytes (Tamai et al., 1995 J. Biol. Chem. 270, 7609-7614) ● The keratin 6 promoter is specific to proliferating epidermis (Ramirez et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4783-4787) ● The collagen 1 promoter is specific to hepatic stellate cells and skin / tendon fibroblasts (Houglum et al., 1995 J. Clin. Invest. 96, 2269-2276) ● The type X collagen promoter is specific to hypertrophic chondrocytes (Long & Linsenmayer, 1995 Hum. Gene Ther. 6, 419-428) ● The factor VII promoter is specific to the liver (Greenberg et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12347-1235) ● The fatty acid synthase promoter is specific to the liver and adipose tissue (Soncini et al., 1995 J. Biol. Chem. 270, 30339-3034) ● The carbamoyl phosphate synthetase I promoter is specific to portal hepatocytes and the small intestine (Christoffels et al., 1995 J. Biol. Chem. 270, 24932-24940) ● The Na-K-Cl transporter promoter is specific to the kidney (loop of Henle) (Igarashi et al., 1996 J. Biol. Chem. 271, 9666-9674) ● The scavenger receptor A promoter is specific to macrophages and foam cells (Horvai et al., 1995 Proc. Natl. Acad. Sci. USA 92, 5391-5395) ● The glycoprotein IIb promoter is specific to megakaryocytes and platelets (Block & Poncz, 1995 Stem Cells 13, 135-145) ● The yc chain promoter is specific to hematopoietic cells (Markiewicz et al., 1996 J. Biol. Chem. 271, 14849-14855) ● The CD11b promoter is specific for mature myeloid cells (Dziennis et al., 1995 Blood 85, 319 - 329)

[0159] Cell type The host cell (also referred to as the "target cell") can be ex vivo (e.g., fresh isolate (early passage)), in vivo, or in vitro under culture (e.g., immortalized cell line). In some cases, the target nucleic acid is chromosomal (e.g., the genome of the host cell), and in some cases, the target nucleic acid is from a pathogen (e.g., the genome of a pathogen within the host cell). The cell can be from an established cell line or a primary cell. At this time, "primary cell", "primary cell line", and "primary culture" are used interchangeably herein and refer to cells and cell cultures derived from a subject, grown in vitro for a limited number of passages, i.e., for splitting of the culture. For example, a primary culture is a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not a sufficient number of times to pass through the crisis stage. Typically, a primary cell line is maintained for less than 10 passages under culture.

[0160] Suitable host cells (which can contain target nucleic acids such as genomic DNA) include, but are not limited to, single-celled eukaryotic cells, plant cells, algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, etc.), fungal cells (e.g., yeast cells), animal cells, invertebrate-derived cells (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), insect cells (e.g., mosquitoes, bees, agricultural pests, etc.), arachnid cells (e.g., spiders, mites, etc.), vertebrate cells (e.g., fish, amphibians, reptiles, birds, mammals), mammalian cells (e.g., rodent cells, human cells, non-human mammalian cells), rodent cells (e.g., mouse, rat), lagomorph cells (e.g., rabbit), ungulate cells (e.g., cow, horse, camel, llama, vicuña, sheep, goat, etc.), marine mammalian cells (e.g., whale, seal, walrus, dolphin, sea lion, etc.), and the like. Any type of cell (e.g., stem cells, e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), adult stem cells, somatic cells, e.g., fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, in vitro or in vivo embryonic cells of embryos at any stage, e.g., zebrafish embryos at the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, etc.) may be of interest.

[0161] Suitable host cells (which can contain a target nucleic acid such as genomic DNA) include, but are not limited to, bacterial cells, archaeal cells, single-celled eukaryotic cells, plant cells, algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, etc.), fungal cells (e.g., yeast cells), animal cells, invertebrate-derived cells (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), insect cells (e.g., silkworms, bees, agricultural pests, etc.), arachnid cells (e.g., spiders, mites, etc.), vertebrate cells (e.g., fish, amphibians, reptiles, birds, mammals), mammalian cells (e.g., rodent cells, human cells, non-human mammalian cells), rodent cells (e.g., mice, rats), rabbit cells (e.g., rabbits), ungulate cells (e.g., cows, horses, camels, llamas, vicuñas, sheep, goats, etc.), marine mammalian cells (e.g., whales, seals, walruses, dolphins, sea lions, etc.), and the like. Any type of cell (e.g., stem cells, e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), adult stem cells, somatic cells, e.g., fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, in vitro or in vivo embryonic cells of an embryo at any stage, e.g., zebrafish embryos at the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, etc.) may be of interest.

[0162] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells), germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), somatic cells, e.g., fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, and the like.

[0163] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, hematopoietic stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, osteocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow-derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, pluripotent progenitor cells, unipotent progenitor cells, monocytes, cardiomyoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, heterologous cells, allogeneic cells, and postnatal stem cells.

[0164] In some cases, the cells are immune cells, neurons, epithelial cells, endothelial cells, or stem cells. In some cases, the immune cells are T cells, B cells, monocytes, natural killer cells, dendritic cells, or macrophages. In some cases, the immune cells are cytotoxic T cells. In some cases, the immune cells are helper T cells. In some cases, the immune cells are regulatory T cells (Tregs).

[0165] In some cases, the cells are stem cells. Adult stem cells are included in the stem cells. Adult stem cells are also referred to as somatic stem cells.

[0166] Adult stem cells reside in differentiated tissues but retain the properties of self-renewal and the ability to give rise to multiple cell types (usually cell types typical of the tissue in which the stem cells are present). Numerous examples of somatic stem cells are known to those skilled in the art and include muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, and the like.

[0167] Stem cells of interest include mammalian stem cells, where the term "mammal" refers to any animal classified as a mammal, including humans, non-human primates, domesticated animals and livestock, zoo animals, laboratory animals, sport animals, or pet animals such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse, rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0168] In some embodiments, the stem cells are hematopoietic stem cells (HSCs). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized by CD34 + and CD3 - . HSCs can repopulate erythrocytes, neutrophils-macrophages, megakaryocytes, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate into the same lineages seen in vivo. Thus, HSCs can be induced to differentiate into one or more of erythrocytes, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0169] In other embodiments, the stem cells are neural stem cells (NSCs). Neural stem cells (NSCs) can differentiate into neurons and glia (including oligodendrocytes and astrocytes). Neural stem cells are pluripotent stem cells capable of multiple divisions and, under specific conditions, can generate daughter cells that are neural stem cells, or neural progenitor cells (e.g., cells that are committed to becoming one or more types of neurons and glial cells each) that can become neuroblasts or glioblasts. Methods for obtaining NSCs are known in the art.

[0170] In other embodiments, the stem cells are mesenchymal stem cells (MSCs). MSCs originally derive from embryonic mesoderm and are isolated from adult bone marrow, and can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendon. Methods for isolating MSCs are known in the art, and any known method can be used to obtain MSCs. See, for example, U.S. Patent No. 5,736,396, which describes the isolation of human MSCs.

[0171] Using the promoters and translational control elements as described above, differential delivery of the Acr protein and / or CRISPR nuclease of interest at different levels to each other can be achieved. For example, in some embodiments, the goal is to deliver the CRISPR nuclease and the Acr polypeptide at a ratio of 1:1. In some embodiments, the goal is to deliver the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:10 (e.g., 1:2 to 1:10, 1:4 to 1:10, 1:5 to 1:10, 1:1.25 to 1:5, or 1:2 to 1:5). For example, the system of interest can be configured (e.g., for use in a method) to deliver the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) of, for example, 1:1.25, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0172] In some embodiments, the goal is to deliver the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10 (e.g., 1:2 to 1:10, 1:4 to 1:10, 1:5 to 1:10, 1:1.25 to 1:5, or 1:2 to 1:5). For example, the system of interest can be configured (e.g., for use in a method) to deliver the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) of, for example, 1:1.25, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0173] "Protospacer Adjacent Motif" (PAM) Wild-type CRISPR nucleases typically have nuclease activity that cleaves a target nucleic acid (e.g., double-stranded DNA (dsDNA)) at a target site defined by (i) a complementary region between the guide sequence of the guide RNA and the target nucleic acid, and (ii) a short motif called the "protospacer adjacent motif" (PAM) within the target nucleic acid.

[0174] For further information regarding CRISPR-based programmable gene editing tools (e.g., CRISPR / Cas RNA-guided proteins, CRISPR / Cas guide RNAs, and PAMs), see, for example, the following review articles and the articles cited therein: Zetsche et al, Cell. 2015 Oct 22;163(3):759-71; Makarova et al, Nat Rev Microbiol. 2015 Nov;13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Shmakov et al., Nat Rev Microbiol. 2017 Mar;15(3):169-182; Koonin et al., Curr Opin Microbiol. 2017 Jun;37:67-78; and Makarova et al., Nat Rev Microbiol. 2020 Feb;18(2):67-83 (all of which are hereby incorporated by reference in their entirety).

[0175] Vector A "vector" or "expression vector" is a replicon (e.g., plasmid, phage, virus, or cosmid) to which another DNA segment (i.e., "insert") is attached so that the attached segment can be replicated and / or expressed in a cell. An "expression cassette" contains a DNA sequence (coding or non-coding) operably linked to a promoter. In some cases, the vector of interest is a viral vector (e.g., AAV, lentivirus, adenovirus). In some cases, the vector of interest contains an origin of replication (e.g., can be a plasmid).

[0176] In some cases, both the Acr protein and its target Cas protein (the protein inhibited by Acr) are present within a single vector, thereby ensuring that all cells receiving the Cas protein (e.g., an endonuclease such as Cas9, Cas12a, etc.) also express the Acr "off-switch". Whether both proteins (Acr and Cas) are present on the same nucleic acid or not, the translation of one or both proteins can be regulated by translational control elements to achieve an appropriate balance (expression level ratio) of the two proteins.

[0177] The vector can be provided directly to the target host cell (target cell). In other words, the cell is contacted with a vector (e.g., a recombinant expression vector) containing the nucleic acid of interest so that the vector is taken up by the cell. Methods for contacting a cell with a nucleic acid vector that is a plasmid are well known in the art, such as electroporation, calcium chloride transfection, microinjection, and lipofection. For viral vector introduction, the cell can be contacted with viral particles containing the viral expression vector of interest (e.g., adeno-associated virus (AAV)).

[0178] In some embodiments, the subject vector is a viral construct, such as, for example, a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, and the like.

[0179] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (e.g., see Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655), adeno-associated virus (e.g., see Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997; Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava (WO 93 / 09239); Samulski et al., J.Vir (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., see Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., viral vectors based on J Virol 73:7812 - 7816, 1999), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses (e.g., Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus)), etc.

[0180] In some embodiments, the vector of interest is an AAV vector. Adeno - associated virus (AAV) means the virus itself or its derivatives. This term encompasses all subtypes as well as both natural and recombinant forms unless otherwise required, e.g., AAV type 1 (AAV - 1), AAV type 2 (AAV - 2), AAV type 3 (AAV - 3), AAV type 4 (AAV - 4), AAV type 5 (AAV - 5), AAV type 6 (AAV - 6), AAV type 7 (AAV - 7), AAV type 8 (AAV - 8), AAV type 9 (AAV - 9), AAV type 10 (AAV - 10), AAV type 11 (AAV - 11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non - primate AAV, ovine AAV, hybrid AAV (i.e., AAV containing the capsid protein of one AAV subtype and the genomic material of another subtype), AAV containing mutant AAV capsid proteins, or chimeric AAV capsids (i.e., capsid proteins having regions or domains or individual amino acids derived from two or more different serotypes of AAV, e.g., AAV - DJ, AAV - LK3, AAV - LK19). "Primate AAV" refers to AAV that infects primates, "non - primate AAV" refers to AAV that infects non - primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0181] In some embodiments, the vector of interest is an integrating vector, e.g., it integrates into the genome of the target cell.

[0182] The term "recombinant AAV vector" or "rAAV vector" refers to an AAV virus or AAV viral chromosome that contains a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), and typically refers to a nucleic acid sequence of interest that is incorporated into cells according to a subject method. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). In some cases, the recombinant viral vector also contains viral genes important for packaging of the recombinant viral vector material. "Packaging" refers to a series of intracellular events that result in the construction and encapsidation of viral particles (e.g., AAV viral particles). Examples of nucleic acid sequences important for AAV packaging (i.e., "packaging genes") include the AAV "rep" gene and "cap" gene, which encode the replication protein and capsidation protein of adeno-associated virus, respectively. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0183] The term "viral particle" refers to a single unit of a virus that includes a virus-based polynucleotide, such as a viral genome (as in a wild-type virus), or, for example, a targeting vector of interest (as in a recombinant virus), encapsulated in a capsid. The term "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of wild-type AAV) and an encapsulated polynucleotide AAV vector. When this particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to mammalian cells), this particle is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, the production of rAAV particles necessarily includes the production of rAAV vectors, and for this reason, the vector is contained within the rAAV particle.

[0184] rAAV virions can be constructed using methods well known in the art. See, for example, Koerber et al. (2009) Mol. Ther. 17:2088; Koerber et al. (2008) Mol Ther. 16:1703-1709; U.S. Patent Nos. 7,439,065, 6,951,758, and 6,491,907. For example, heterologous sequence(s) can be inserted directly into an AAV genome from which the major AAV open reading frame (「ORF」) has been excised. Other portions of the AAV genome can also be deleted as long as portions of the ITRs sufficient to obtain replication and packaging functions remain. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B.J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Curr. Topics Microbiol. Immunol. 158:97-129; Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med., 179:1867-1875.

[0185] To generate rAAV virions, the AAV expression vector can be introduced into a suitable host cell using known techniques (e.g., by transfection). Many transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct microinjection into cultured cells (Capecchi, M.R. (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechnigues 6:742-751), liposome-mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).

[0186] Suitable cells for the production of rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, which can be used or are being used as recipients of heterologous DNA molecules. Cells from the stable human cell line 293 (e.g., readily available from the American Type Culture Collection (Accession No. ATCC CRL1573)) can be used. For example, the human cell line 293 is a human embryonic kidney cell line transformed with adenovirus type 5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59) and expresses the adenovirus E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is easily transfectable and provides a convenient platform for the production of rAAV virions. Methods for producing AAV virions in insect cells are known in the art and can be used for the production of the rAAV virions of interest. See, for example, U.S. Patent Publication No. 2009 / 0203071; U.S. Patent No. 7,271,002; and Chen (2008) Mol. Ther. 16:924.

[0187] The AAV virus produced may or may not be replication-competent. A "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and capable of replicating in infected cells (e.g., in the presence of a helper virus or helper virus functions). In the case of AAV, replication ability generally requires the presence of functional AAV packaging genes. Generally, the rAAV vectors described herein are non-replicable in mammalian cells (particularly in human cells) because they lack one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of generating replication-competent AAV by recombination between the AAV packaging genes and the incoming rAAV vector.

[0188] Retroviruses (e.g., lentiviruses) are suitable for use in the methods of the present disclosure. Commonly used retroviral vectors are "incomplete," i.e., they cannot produce the viral proteins required for productive infection. Instead, replication of the vector requires growth in a packaging cell line. To produce virus particles containing a nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged by a packaging cell line into a viral capsid. Different packaging cell lines provide different envelope proteins (homotropic, amphotropic, or heterotropic) incorporated into the capsid, and this envelope protein determines the specificity of the virus particle for cells (homotropic for mouse and rat, amphotropic for most mammalian cell types including human, dog, and mouse, and heterotropic for most mammalian cell types except mouse cells). Appropriate packaging cell lines can be used to ensure that the packaged virus particles target cells. Methods for introducing a subject vector expression vector into a packaging cell line and methods for collecting virus particles produced by the packaging cell line are well known in the art. The nucleic acid can also be introduced directly by microinjection (e.g., injection of RNA).

[0189] Detailed descriptions of the delivery methods and formulations are presented elsewhere in this specification.

[0190] As mentioned elsewhere in this specification, instead, the protein may be provided to the cell as RNA (e.g., RNA containing translational control elements as described elsewhere in this specification). Methods for introducing RNA into cells are known in the art and include, for example, direct injection, transfection, or any other method used for the introduction of DNA.

[0191] In some cases, one or more proteins (e.g., a CRISPR nuclease and / or an Acr polypeptide) can be introduced into a cell as a protein (relative to a nucleic acid). For example, one protein coding sequence (e.g., an Acr protein coding sequence) can be introduced as a nucleic acid (RNA or DNA) (this protein coding sequence is operably linked to a translation control element), and the other protein (e.g., a Cas effector) is introduced as a polypeptide. Such a polypeptide can optionally be fused with a polypeptide domain that increases the solubility of the product. The domain can be linked to the polypeptide via a defined protease cleavage site (e.g., a TEV sequence cleaved by TEV protease). The linker may also contain one or more flexible sequences, e.g., 1 to 10 glycine residues. Examples of linkers are described elsewhere in this specification in another context, but such linkers can be used in any convenient context, including this one.

[0192] In some embodiments, cleavage of the fusion protein is carried out in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5 - 2 M urea, in the presence of a polypeptide and / or polynucleotide that increases solubility, etc. Examples of domains of interest include an endosomal degradation domain (e.g., an influenza HA domain), and other polypeptides that aid production (e.g., an IF2 domain, a GST domain, a GRPE domain, etc.). The polypeptide can be formulated to improve stability. For example, the peptide can be PEGylated, in which case the polyethyleneoxy groups improve the lifespan in the bloodstream.

[0193] Method The present disclosure provides methods for nucleic acid targeting (e.g., modification of a target nucleic acid for cleaving DNA in genome editing applications), where an Acr polypeptide and a CRISPR nuclease are delivered to the target nucleic acid as a system of interest (e.g., in the form of a nucleic acid or a protein). In some cases, the contacting is performed in an in vitro cell-free environment. In some cases, contacting is performed in cells, e.g., ex vivo, in vivo, or in vitro (e.g., cells under culture). Thus, in some embodiments, the methods of interest include introducing an Acr protein (or a nucleic acid encoding the same) and a CRISPR nuclease (or a nucleic acid encoding the same) into a host cell, whereby the ratio of on-target:off-target nucleic acid activity (e.g., cleavage) resulting from such introduction is increased compared to the ratio of on-target:off-target nucleic acid targeting that occurs in the absence of the Acr protein. In some cases, the resulting on-target:off-target nucleic acid targeting ratio is caused by an increase in on-target activity. In some cases, the resulting on-target:off-target nucleic acid targeting ratio is caused by a decrease in off-target activity. In some cases, the resulting on-target:off-target nucleic acid targeting ratio is caused by both an increase in on-target activity and a decrease in off-target activity.

[0194] The target cell (host cell) can be any desired cell / cell type. Examples of suitable cells and promoters are described in detail elsewhere in this specification (see, for example, the "Promoter" section). For example, in some cases, the cell is a prokaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. For example, in some cases, the cell is a eukaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. In some cases, the cell is ex vivo. In some cases, the cell is in vivo. In some cases, the cell is in vitro.

[0195] In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of a pathogen (e.g., a virus), and in some cases, the pathogen is within the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of a pathogen (such as a virus, bacterium, etc.), and in some cases, the pathogen is within the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is an RNA molecule. In some cases, on-target nucleic acid targeting alters the expression of a protein within the host cell (e.g., by reducing the transcription of mRNA). In some cases, on-target nucleic acid targeting alters the expression of an RNA within the host cell (e.g., non-coding RNA, mRNA, microRNA, etc.).

[0196] In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes gene editing (e.g., correction of gene mutations in the host cell genome). In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes alteration (editing) of a gene locus from a disease-related sequence to a health-related sequence (e.g., correction from a disease-causing allele of Huntington's disease (HD), Duchenne muscular dystrophy (DMD), or alpha-1 antitrypsin deficiency (AATD) to an allele not associated with (not causative of) the disease).

[0197] In some embodiments, the subject method includes measuring the editing efficiency at an ON target site. In some embodiments, the subject method includes measuring the editing efficiency at one or more OFF target sites.

[0198] As described elsewhere in this specification, the location of an on-target (“ON-TARGET”) event (e.g., cleavage / editing of target DNA) is in effect determined by the guide sequence of the guide RNA. A CRISPR complex-mediated event that occurs at a location that does not match 100% with the guide sequence is referred to herein as an off-target (“OFF-TARGET”) event. Any convenient method can be used to measure on-target and off-target events, and the choice of method depends on the type of CRISPR complex used and the desired outcome of the complex activity (e.g., when using a nickase protein, when performing double-strand target cleavage, when using a donor polynucleotide (the target can be edited by introducing a known heterologous sequence), when not using a donor polynucleotide (which can result in a number of different indels), etc.). Examples of suitable assays include, but are not limited to, mismatch cleavage assays (e.g., surveyor assay, T7E1 mismatch assay), PCR assays, PCR / sequencing assays, direct sequencing assays (e.g., next-generation sequencing), etc. (and any combination thereof). Sequencing assays or alternative expression assays (e.g., qRT-PCR and / or microarray analysis) can be used when the activity of the CRISPR complex results in a change in the expression of the target sequence (e.g., when a promoter sequence is targeted, when a coding sequence is targeted, and when a new sequence is sensitive to nonsense-mediated decay, etc.). There are various assays for testing on-target and off-target activities, and any desired assay or combination of assays can be used. In some cases, the desired result (e.g., the desired balance between reduced ON-TARGET and OFF-TARGET CRISPR nuclease activities) can be achieved by using a specific Acr protein (see above for the description of the Acr protein of interest).In some cases, the off-target site is a predicted and / or known site, and in some cases, the off-target site can be identified retrospectively (e.g., based on a genome-wide hunt such as can be achieved using high-throughput / next-generation sequencing methods such as RNA or DNA sequencing methods).

[0199] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein reduces off-target CRISPR nuclease activity (i.e., the off-target activity of the CRISPR nuclease). For example, in some cases, the Acr polypeptide reduces off-target CRISPR nuclease activity by 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) compared to the off-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide reduces off-target CRISPR nuclease activity by 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) compared to the off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0200] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein reduces ON - target CRISPR nuclease activity (i.e., the ON - target activity of the CRISPR nuclease) compared to the ON - target CRISPR nuclease activity in the absence of the Acr polypeptide. For example, in some cases, the Acr polypeptide reduces the ON - target CRISPR nuclease activity by 50% or less (e.g., 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 2% or less, or 1% or less) compared to the ON - target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide reduces the ON - target CRISPR nuclease activity by 40% or less (e.g., 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 2% or less, or 1% or less) compared to the ON - target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0201] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein increases the ratio of ON-target:OFF-target CRISPR nuclease activity compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide. For example, in some cases, the Acr polypeptide increases the ratio of ON-target:OFF-target CRISPR nuclease activity by at least 1.25-fold (i.e., 1.25 times) (e.g., at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide increases the ratio of ON-target:OFF-target CRISPR nuclease activity by at least 1.5-fold (e.g., at least 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide increases the ratio of ON-target:OFF-target CRISPR nuclease activity by at least 2-fold (e.g., at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold) compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0202] In some embodiments, the editing efficiency of the ON-TARGET CRISPR nuclease activity is at least 4-fold (4x) (e.g., at least 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or 100-fold) greater than the editing efficiency of the OFF-TARGET CRISPR nuclease activity. In some embodiments, the editing efficiency of the ON-TARGET CRISPR nuclease activity is at least 6-fold (6x) (e.g., at least 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or 100-fold) greater than the editing efficiency of the OFF-TARGET CRISPR nuclease activity. In some embodiments, the editing efficiency of the ON-TARGET CRISPR nuclease activity is at least 10-fold (10x) (e.g., at least 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or 100-fold) greater than the editing efficiency of the OFF-TARGET CRISPR nuclease activity.

[0203] In some embodiments, the off-target CRISPR nuclease activity is at an off-target site that contains five or fewer mismatches (e.g., four or fewer, three or fewer, two or fewer, or one or fewer mismatches) compared to the on-target site (i.e., mismatches between the intended target site and the off-target site). In other words, in some cases, the off-target CRISPR nuclease activity is at an off-target site that contains five or fewer mismatches (e.g., four or fewer, three or fewer, two or fewer, or one mismatch) (i.e., mismatches between the intended target site and the off-target site) compared to the on-target site. In some embodiments, the off-target CRISPR nuclease activity is at an off-target site that contains three or fewer mismatches (e.g., two or fewer or one or fewer mismatches) compared to the on-target site (i.e., mismatches between the intended target site and the off-target site). In other words, in some cases, the off-target CRISPR nuclease activity is at an off-target site that contains three or fewer mismatches (e.g., two or fewer or one mismatch) (i.e., mismatches between the intended target site and the off-target site) compared to the on-target site.

[0204] In some cases, the desired result is an off-target rate result such that the off-target events detected per cell population (e.g., off-target cleavage events (e.g., insertions / deletions (indels)) detected per cell population) are less than 100. In some such cases, the number of cells in the cell population is in the range of 10 4 ~10 6 (e.g., in some cases, the number of cells in the cell population is about 10 5(which are cells). In some cases, the desired result is an off-target rate result where the number of off-target events detected per cell population is less than 90 (e.g., less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell). In some cases, the desired result is an off-target rate result where the number of off-target events detected per cell population is less than 50 (e.g., less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell).

[0205] In some cases, the desired result is 10 5 an off-target rate result where the number of off-target events detected per cell is less than 100. In some cases, the desired result is 10 5 an off-target rate result where the number of off-target events detected per cell is less than 90 (e.g., less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell). In some cases, the desired result is 10 5 an off-target rate result where the number of off-target events detected per cell is less than 50 (e.g., less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell).

[0206] In some cases, the desired result is that less than 50% (e.g., less than 45%, less than 40%, or less than 35%) of all measured nucleic acid targeting events (e.g., cleavage) are off-target events. In other words, in some cases, the on-target event:off-target event ratio (e.g., measured on-target event:off-target event) is greater than 1 (e.g., greater than 1.2, greater than 1.5, greater than 1.8, greater than 2, greater than 2.2, or greater than 2.5). In some cases, the events can be measured after introducing the Acr protein and the Cas protein and subculturing the host cells (e.g., for more than 10 generations in some cases). Thus, in some cases, the desired result is that less than 50% (e.g., less than 45%, less than 40%, or less than 35%) of the total nucleic acid targeting events (e.g., cleavage) measured after subculturing the host cells (e.g., for more than 10 generations) after introduction of the Acr and Cas proteins are off-target events. In other words, in some such cases, the on-target event:off-target event ratio (e.g., measured on-target event:off-target event) is greater than 1 (e.g., greater than 1.2, greater than 1.5, greater than 1.8, greater than 2, greater than 2.2, or greater than 2.5).

[0207] As mentioned above, off-target sites can, in some cases, be predicted. Generally, the rate (frequency) of off-target activity (e.g., cleavage / editing) varies by site, for example when measuring the rate of activity using a population of cells. Thus, in some cases, a desirable result is one where the measured frequency of off-target events is less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1%) compared to the off-target events measured (or predicted) in the absence of the Acr protein. As an illustrative example, the frequency of off-target events at one particular predicted or known off-target site (or any number of off-target sites (predicted / known or not predicted / unknown)) can be measured in the presence and absence of the Acr protein, and the number of off-target events when the Acr protein is present is less than 50% (e.g., less than 45%, less than 40%, or less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1%) compared to the number of off-target events when the Acr protein is absent. As a further illustrative example of the above, if a total of 100 off-target events are measured when this method is performed in the presence of the Acr protein and 200 such events are measured (or predicted) in the absence of the Acr protein, the result is that the measured frequency of off-target events in the presence of the Acr protein is 50% compared to the off-target events in the absence of the Acr protein.

[0208] Delivery As mentioned above, in some embodiments, both the Cas protein and the Acr protein are delivered to the host cell as DNA, and in some such cases, the sequences encoding the two proteins are present on the same nucleic acid (e.g., DNA vector) or on separate nucleic acids. However, in some embodiments, the protein of interest (e.g., Cas protein and / or Acr protein) is not provided as a DNA vector. For example, either protein (or both) can be introduced into the host cell as RNA encoding the protein. In such cases, the RNAs encoding the two proteins can be delivered in appropriate ratios to achieve the desired effect (i.e., an increase in the ratio of on-target:off-target CRISPR complex activity), for example, by reducing off-target activity while retaining desirable on-target activity, and one or more translational control elements may be present on the RNA.

[0209] As another example, either protein (or both) can be introduced directly into the host cell as a protein. In some such cases (e.g., when the Cas protein is a class 2 effector protein), the Cas protein can be delivered as an RNP (ribonucleoprotein complex) that is already complexed with an appropriate guide RNA. In such cases, the other protein (e.g., Acr protein) can be delivered as DNA or RNA, and its coding sequence can be operably linked to a translational control element of interest.

[0210] Thus, the Cas protein and the Acr protein can be delivered in any desired format (DNA, RNA, protein). For example, when the Cas protein is delivered as DNA, the Acr protein can be delivered as DNA, RNA, or protein; when the Cas protein is delivered as RNA, the Acr protein can be delivered as DNA, RNA, or protein; when the Cas protein is delivered as protein, the Acr protein can be delivered as DNA or RNA. Similarly, when the Acr protein is delivered as DNA, the Cas protein can be delivered as DNA, RNA, or protein; when the Acr protein is delivered as RNA, the Cas protein can be delivered as DNA, RNA, or protein; when the Acr protein is delivered as protein, the Cas protein can be delivered as DNA or RNA.

[0211] As will be readily understood by those skilled in the art, the nucleic acids (e.g., vectors) and proteins of interest can be delivered into cells using any convenient method. Methods for introducing nucleic acids and / or proteins into host cells (e.g., prokaryotic cells, eukaryotic cells, plant cells, animal cells, insect cells, mammalian cells, human cells, etc.) are known in the art and any convenient method can be used. Suitable methods include, for example, viral infection (e.g., AAV, adenovirus, lentivirus), transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et.,al Adv Drug Deliv Rev.2012 Sep 13:S0169-409X(12)00283-9), etc.

[0212] In some cases, the proteins of the present disclosure (e.g., Cas proteins, Acr proteins) are provided as nucleic acids encoding the proteins (e.g., mRNA, DNA, plasmid, expression vector, viral vector, etc.). In some cases, the target protein is provided directly as a protein (e.g., without the associated guide RNA, or together with the associated guide RNA, i.e., as a ribonucleoprotein complex). The target protein can be introduced (provided to) the cell by any convenient method, and such methods are known to those skilled in the art. As an exemplary example, the target protein can be directly injected into the cell. As another example, the target protein can be introduced into a cell (e.g., a eukaryotic cell) via nucleofection, a protein transduction domain (PTD) conjugated to the protein, etc.

[0213] In some cases, the target protein is delivered to the cell (e.g., the target host cell) with or in particles. In some cases, the target protein is delivered together with a cationic lipid and a hydrophilic polymer. For example, the cationic lipid includes 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), and / or the hydrophilic polymer includes ethylene glycol or polyethylene glycol (PEG), and / or the particle further includes cholesterol (e.g., formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; formulation 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; particles from formulation 3 = DOTAP 90, DMPC 0, PEG 5, cholesterol 5).

[0214] The target protein (RNA or DNA or protein) can be delivered using particles or a lipid envelope. For example, biodegradable core-shell structured nanoparticles in which a poly(β-amino ester) (PBAE) core is encapsulated with a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling biocompatible polymers are used. Such particles / nanoparticles can be applied to oral delivery of peptides, intravenous delivery of peptides, nasal delivery of peptides (e.g., delivery to the brain). Other embodiments, such as oral absorption and ophthalmic delivery of hydrophobic drugs are also contemplated. Molecular envelope technology (with an engineered polymer envelope that is delivered to the protected disease site) can be used.

[0215] Lipidoid compounds (e.g., those described in U.S. Patent Application No. 20110293703) are also useful for the administration of polynucleotides and can be used for the delivery of a target protein or nucleic acid (RNA or DNA). In one embodiment, an amino alcohol lipidoid compound forms microparticles, nanoparticles, liposomes, or micelles in combination with an agent to be delivered to a cell or subject. The amino alcohol lipidoid compound can form particles in combination with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. These particles can then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0216] In some cases, lipid nanoparticles (LNPs) are used to deliver a target protein or nucleic acid to target cells. Negatively charged polymers such as RNA can be loaded into LNPs at a low pH value (e.g., pH 4) where the ionizable lipid exhibits a positive charge. However, at physiological pH values, LNPs exhibit a low surface charge that is compatible with a longer circulation time. Examples of ionizable cationic lipids include, but are not limited to, 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyl-oxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyl-oxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Examples of LNP preparation are described, for example, in Rosin et al. (2011) Molecular Therapy 19:1286-2200. Cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyl-oxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyl-oxy-keto-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2''-(methoxypolyethylene glycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(omega-methoxy-(polyethylene glycol)2000) carbamoyl]-1,2-dimyristyloxyl (oxl) propyl-3-amine (PEG-C-DOMG) can be used. Nucleic acids can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at a molar ratio of 40:10:40:10). In some cases, 0.2% of SP-DiOC18 is incorporated.Lipids suitable for LNPs include, but are not limited to, DLin-KC2-DMA4, C12-200, and the co-lipids distearoyl phosphatidylcholine, cholesterol, and PEG-DMG, and these can be formulated with the protein or nucleic acid of interest using the spontaneous vesicle formation procedure. The molar ratio of the components can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoyl phosphatidylcholine / cholesterol / PEG-DMG). To ensure a narrow particle size distribution in the range of 70 - 90 nm and a low polydispersity index of 0.11 ± 0.04 (n = 56), for example, the particles can be extruded up to 3 times through an 80 nm membrane before adding RNA such as guide RNA. Particles containing the very potent amino lipid 16 may be used, in which case the molar ratio of the four lipid components 16, DSPC, cholesterol, and PEG lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance activity.

[0217] Spherical nucleic acids (SNA™) constructs and other nanoparticles, particularly gold nanoparticles, can be used to deliver a target protein or nucleic acid to a target cell. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011, 133:9254-9257; Hao et al., Small 2011, 7:3158-3162; Zhang et al., ACS Nano 2011, 5:6962-6970; Cutler et al., J. Am. Chem. Soc. 2012, 134:1376-1391; Young et al., Nano Lett. 2012, 12:3867-71; Zheng et al., Proc. Natl. Acad. Sci. USA 2012, 109:11975-80; Mirkin, Nanomedicine 2012, 7:635-638; Zhang et al., J. Am. Chem. Soc. 2012, 134:16488-1691; Weintraub, Nature 2013, 495:S14-S16; Choi et al., Proc. Natl. Acad. Sci. USA 2013, 110(19):7625-7630; Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013); and Mirkin, et al., Small, 10:186-192.

[0218] Self-assembled nanoparticles having RNA can be constructed using polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG).

[0219] Generally, a "nanoparticle" refers to any particle with a diameter less than 1000 nm. In some cases, the diameter of the nanoparticle suitable for use in delivering the target protein or nucleic acid to the target cell is 500 nm or less, for example, 25 nm - 35 nm, 35 nm - 50 nm, 50 nm - 75 nm, 75 nm - 100 nm, 100 nm - 150 nm, 150 nm - 200 nm, 200 nm - 300 nm, 300 nm - 400 nm, or 400 nm - 500 nm. In some cases, the diameter of the nanoparticle suitable for use in delivering the target protein or nucleic acid to the target cell is 25 nm - 200 nm.

[0220] Nanoparticles suitable for use in delivering the target protein or nucleic acid to the target cell can be provided in various forms, for example, as solid nanoparticles (such as metals like silver, gold, iron, titanium, etc.), non-metals, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles, as well as hybrid structures (such as core-shell nanoparticles) can be prepared. Nanoparticles made from semiconductor materials may also be classified as quantum dots when they are small enough (typically less than 10 nm) for quantization of electron energy levels to occur. Such nanoscale particles are used in biomedical applications as drug carriers or contrast agents and can be adapted for similar purposes in the present disclosure.

[0221] Semi-solid and soft nanoparticles are also suitable for use in delivering the target protein or nucleic acid to the target cell. A prototype nanoparticle with semi-solid properties is a liposome.

[0222] In some cases, the carrier / media can contain microparticles. Examples of microparticles include, but are not limited to, liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. In some cases, the microparticles can contain one or more of the following, in any combination: poly(lactide-co-glycolide), aliphatic polyesters (including, but not limited to, polyglycolic acid and polylactic acid), hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethane, polyacrylic acid, pseudo-poly(amino acids), polyhydroxybutyrate-related copolymers, polyanhydrides, polymethyl methacrylate, poly(ethylene oxide), lecithin, and phospholipids.

[0223] In some cases, the carrier / media may include liposomes (e.g., those capable of binding and releasing therapeutic agents (e.g., nucleic acids and / or proteins of interest)). Liposomes are microscopic spherical lipid bilayers that surround an aqueous core and are made from amphiphilic molecules such as phospholipids. For example, liposomes can trap therapeutic agents between the hydrophobic tails of phospholipid micelles. Water-soluble drugs can be trapped within the core, and lipid-soluble drugs can dissolve in the shell-like bilayer. Liposomes have special features in that they can be used to combine water-soluble and water-insoluble chemicals in a medium without using surfactants or other emulsifiers. Liposomes can form spontaneously by forcing the mixing of phospholipids in an aqueous medium. Water-soluble compounds are dissolved in a hydratable aqueous solution of phospholipids. Thus, when liposomes form, these compounds are trapped within the aqueous liposome core. The liposome wall, which is a phospholipid membrane, holds lipid-soluble materials such as oils. Liposomes provide controlled release of the incorporated compounds. Additionally, liposomes can be coated with water-soluble polymers such as polyethylene glycol to increase the pharmacokinetic half-life. Liposomes can be made from several different types of lipids, but phospholipids are most commonly used for liposome production. Liposome formation occurs spontaneously when a lipid film is mixed with an aqueous solution, but it can also be facilitated by applying force in the form of agitation by using a homogenizer, sonicator, or extrusion device. Several other additives can be added to modify the structure and properties of liposomes. For example, cholesterol or sphingomyelin can be added to the liposome mixture to stabilize the liposome structure and prevent leakage of the liposome internal cargo. Liposome formulations can consist mainly of natural phospholipids and lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside).

[0224] In some embodiments, cationic or anionic liposomes are used as part of the composition or method of interest, or liposomes having neutral lipids can also be used. Cationic liposomes can include negatively charged materials by mixing the materials and fatty acid liposome components and charge associating them. The choice of cationic or anionic liposomes depends on the desired pH of the final liposome mixture. Examples of cationic liposomes include, but are not limited to, lipofectin, lipofectamine, and lipofectace.

[0225] The protein or nucleic acid of interest can be encapsulated and delivered in PLGA microspheres as further described in U.S. Published Application Nos. 20130252281, 20130245107, and 20130244279.

[0226] In some cases, exosomes are used to deliver the protein or nucleic acid of interest to target cells. Exosomes are endogenous nanovesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs.

[0227] Poly(beta-amino alcohol) (PBAA) can be used to deliver the protein or nucleic acid of interest to target cells. U.S. Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohol) (PBAA) prepared using combinatorial polymerization.

[0228] Sugar-based particles may be used, for example, using GalNAc as described in relation to WO2014118272 (incorporated herein by reference) and Nair, J K et al, 2014, Journal of the American Chemical Society 136 (49), 16958-16961, to deliver the protein or nucleic acid of interest to target cells.

[0229] Supercharged proteins can be used to deliver a target protein or nucleic acid to a target cell. Supercharged proteins are engineered or natural proteins that have an unusually high net positive or negative charge. Both supernegative and superpositive charge proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositive charge proteins can also penetrate mammalian cells. By associating cargo with these proteins (e.g., plasmid DNA, RNA, or other proteins), the delivery of these macromolecules to mammalian cells can be facilitated both in vitro and in vivo.

[0230] Cell-penetrating peptides (CPPs) can be used to deliver a target protein or nucleic acid to a target cell. CPPs typically have an amino acid composition that has a relatively high abundance of positively charged amino acids (e.g., lysine or arginine), or has a sequence that includes an alternating pattern of polar / charged and nonpolar / hydrophobic amino acids.

[0231] Intended carriers or vehicles include materials such as gelatin, collagen, cellulose esters, dextran sulfate, sodium pentosan polysulfate, chitin, saccharides, albumin, fibrin sealant, synthetic polyvinylpyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates (including, but not limited to, 2-hydroxyethyl methacrylate), poly(orthoesters), cyanoacrylates, gelatin-resorcinol-aldehyde type bioadhesives, polyacrylic acid, and copolymers and block copolymers thereof.

[0232] Donor polynucleotide (donor template) In some cases, the subject composition or method can include a donor polynucleotide. For example, in applications where it is desirable to insert a polynucleotide sequence into a genome where the target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. A “donor sequence” or “donor polynucleotide” or “donor template” means a nucleic acid sequence that is inserted into the site targeted by the CRISPR complex (e.g., after dsDNA cleavage, after target DNA nicking, after target DNA dual nicking, etc.). In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor template is provided to the cell as double-stranded DNA. The donor template can be introduced into the cell in a linear or circular form. When introduced in linear form, the ends of the donor sequence can be protected by any convenient method (e.g., from degradation by exonucleases), such methods being known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3′ end of the linear molecule, and / or self-complementary oligonucleotides can be ligated to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. The donor template can be introduced into the cell as part of a vector molecule having additional sequences (e.g., an origin of replication, a promoter, and a gene encoding antibiotic resistance). Further, the donor template can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or delivered by a virus (e.g., an adenovirus, AAV).

[0233] Examples of non-limiting aspects of the present disclosure Aspects (including embodiments) in the subject matter of the present invention described above can be beneficial either alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are shown below (see Set A and Set B). As will be apparent to those skilled in the art, each individually numbered aspect can be used or combined with any of the individually numbered aspects before or after it. This is intended to support all such combinations of aspects and is not limited to the combinations of aspects explicitly shown below. Set A 1. An anti-CRISPR (Acr) polypeptide, or a nucleic acid encoding said Acr polypeptide, wherein said Acr polypeptide comprises an amino acid sequence having at least 75% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126 to 132, said Acr polypeptide or said nucleic acid. 2. The Acr polypeptide or nucleic acid according to 1, wherein said amino acid sequence has at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126 to 132. 3. The Acr polypeptide or nucleic acid according to 1, wherein said Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 126 to 132. 4. The Acr polypeptide or nucleic acid according to any one of 1 to 3, wherein said Acr polypeptide reduces off-target CRISPR nuclease activity by at least 10% compared to off-target CRISPR nuclease activity in the absence of said Acr polypeptide. 5. The Acr polypeptide or nucleic acid according to any one of 1 to 4, wherein said Acr polypeptide reduces on-target CRISPR nuclease activity by no more than 40% compared to on-target CRISPR nuclease activity in the absence of said Acr polypeptide. 6. The Acr polypeptide or nucleic acid according to any one of 1 to 5, wherein the Acr polypeptide increases the ratio of ON-target:OFF-target CRISPR nuclease activity by at least 1.25-fold compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide. 7. The Acr polypeptide or nucleic acid according to any one of 4 to 6, wherein the CRISPR nuclease is a Cas12a nuclease. 8. The Acr polypeptide or nucleic acid according to any one of 4 to 6, wherein the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. 9. The Acr polypeptide or nucleic acid according to any one of 4 to 6, wherein the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. 10. The Acr polypeptide or nucleic acid according to any one of 1 to 9, wherein the Acr polypeptide is fused to a nuclear localization signal (NLS). 11. (i) An Acr polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide according to any one of 1 to 10, or comprises an amino acid sequence having at least 80% sequence identity to the CRISPR nuclease amino acid sequence set forth in any one of SEQ ID NOs: 165-169, and the Acr polypeptide or the nucleic acid, (ii) A CRISPR nuclease, or a nucleic acid encoding the CRISPR nuclease and a system comprising the same. 12. The system according to 11, wherein the CRISPR nuclease is a Cas12a nuclease. 13. The system according to 11, wherein the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. 14. The system according to 11, wherein the CRISPR nuclease comprises an amino acid sequence having at least 80% sequence identity to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. 15. The system according to any one of 11-14, further comprising a guide RNA, or a nucleic acid encoding the guide RNA. 16. The system according to any one of 11-15, wherein the system comprises the nucleic acid encoding the Acr polypeptide and / or the nucleic acid encoding the CRISPR nuclease. 17. The system according to 16, comprising a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease. 18. The system according to 16, wherein the nucleic acid encoding the Acr polypeptide and the nucleic acid encoding the CRISPR nuclease are the same nucleic acid, such that the system comprises a nucleic acid encoding both the Acr polypeptide and the CRISPR nuclease. 19. The system according to any one of 16-18, wherein the translational control element is operably linked to the Acr polypeptide coding sequence or the CRISPR nuclease coding sequence. 20. The system according to 19, wherein the translational control element is selected from the group consisting of an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or a combination thereof. 21. The system according to any one of 16-18, wherein a first promoter is operably linked to the Acr polypeptide coding sequence and a second promoter is operably linked to the CRISPR nuclease coding sequence. 22. The system according to 21, wherein the first promoter is a stronger promoter than the second promoter. 23. The system according to 21, wherein the second promoter is a promoter stronger than the first promoter. 24. The system according to 21 or 22, wherein the first promoter is selected from the group consisting of CMV, miniCMV, EF1A, CAG, or CBh. 25. The system according to any one of 21 to 24, wherein the second promoter is selected from the group consisting of CMV, miniCMV, EF1A, CAG, and CBh. 26. The system according to any one of 21, 22, and 24, wherein the second promoter is selected from the group consisting of EFS, SV40, and hPGK. 27. The system according to any one of 16 to 26, wherein one or more of the nucleic acids are viral vectors. 28. The system according to 27, wherein the viral vector is an AAV vector. 29. The system according to any one of 16 to 26, wherein one or more of the nucleic acids are included in lipid nanoparticles (LNP). 30. The system according to any one of 11 to 15, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form. 31. The system according to 30, wherein the Acr polypeptide and / or the CRISPR nuclease are included in lipid nanoparticles (LNP). 32. The system according to any one of 11 to 31, wherein the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 33. The system according to 32, wherein the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is at least 1.25 times greater than the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 34. The system according to any one of 11 to 33, wherein the off-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 35. The system according to any one of 11 to 34, wherein the on-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 36. A method of modifying a target nucleic acid, comprising contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease, wherein the Acr polypeptide and the CRISPR nuclease are delivered to the target nucleic acid as the system according to any one of 11 to 35, and the contacting results in a modification to the nucleotide sequence of the target nucleic acid. 37. The method according to 36, wherein the modification results from a deletion of one or more nucleotides. 38. The method according to any one of 36 or 37, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio of 1:1. 39. The method according to any one of 36 or 37, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:10. 40. The method according to any one of 36 or 37, wherein the contacting step delivers the Acr polypeptide and the CRISPR nuclease in a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10. 41. The method according to any one of 36 to 40, wherein the contacting is performed in vivo. 42. The method according to any one of 36 to 40, wherein the contacting is performed within a eukaryotic cell. 43. The method according to 42, wherein the eukaryotic cell is an animal cell. 44. The method according to 43, wherein the eukaryotic cell is a human cell. 45. The method according to 42, wherein the eukaryotic cell is a stem cell. 46. The method according to any one of 36 to 45, wherein the target nucleic acid encodes a gene product. 47. The method according to any one of 36 to 46, wherein the editing efficiency of the ON-TARGET CRISPR nuclease activity is at least 4-fold greater than the editing efficiency of the OFF-TARGET CRISPR nuclease activity. 48. The method according to 47, wherein the OFF-TARGET CRISPR nuclease activity is at an off-target site containing 5 or fewer mismatches compared to the on-target site. 49. The method according to any one of 36 to 48, wherein the OFF-TARGET activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the OFF-TARGET activity of the CRISPR nuclease in the absence of the Acr polypeptide. 50. The method according to any one of 36 to 49, wherein the ON-TARGET activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the ON-TARGET activity of the CRISPR nuclease in the absence of the Acr polypeptide. 51. The method according to any one of 36 to 50, further comprising measuring the editing efficiency. 52. The method according to any one of 36 to 51, further comprising measuring the editing efficiency at one or more off-target sites. Set B 1. An anti-CRISPR (Acr) polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 75% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126 to 132 and 264 to 267, the Acr polypeptide or the nucleic acid. 2. The Acr polypeptide or nucleic acid according to 1, wherein the amino acid sequence has at least 90% sequence identity to the sequence described in any one of SEQ ID NOs: 126 to 132 and 264 to 267. 3. The Acr polypeptide or nucleic acid according to 1, wherein the Acr polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 126 to 132. 4. The Acr polypeptide or nucleic acid according to 1, wherein the Acr polypeptide comprises the amino acid sequence described in SEQ ID NO: 165 and has an amino acid variation at at least one position from amino acid 2 to amino acid 159 (including both ends) of SEQ ID NO: 165. 5. The Acr polypeptide or nucleic acid according to 1, wherein the Acr polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 264 to 267. 6. The Acr polypeptide or nucleic acid according to 1 or 4, wherein the Acr polypeptide comprises one or more amino acid variations described in Table 6. 7. The Acr polypeptide or nucleic acid according to 1 or 4 (1 for 4), wherein the Acr polypeptide comprises one or more amino acid variations described in Table 7. 8. The Acr polypeptide or nucleic acid according to any one of 1 to 7, wherein the Acr polypeptide reduces the off-target CRISPR nuclease activity by 10% or more compared to the off-target CRISPR nuclease activity in the absence of the Acr polypeptide. 9. The Acr polypeptide or nucleic acid according to any one of 1 to 8, wherein the Acr polypeptide reduces the on-target CRISPR nuclease activity by 40% or less compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide. 10. The Acr polypeptide or nucleic acid according to any one of 1 to 9, wherein the Acr polypeptide increases the ratio of on-target:off-target CRISPR nuclease activity by at least 1.25-fold compared to the ratio of on-target:off-target CRISPR nuclease activity in the absence of the Acr polypeptide. 11. The Acr polypeptide or nucleic acid according to any one of 8 to 10, wherein the CRISPR nuclease is a Cas12a nuclease. 12. The polypeptide or nucleic acid according to any one of 8 to 10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Cas12a nuclease amino acid sequence described in any one of SEQ ID NOs: 175 and 245 - 262. 13. The Acr polypeptide or nucleic acid according to any one of 8 to 10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the NUX protein amino acid sequence described in any one of SEQ ID NOs: 1 - 86 and 176 - 244. 14. The Acr polypeptide or nucleic acid according to any one of 1 to 13, wherein the Acr polypeptide is fused with a nuclear localization signal (NLS). 15. (i) An Acr polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide according to any one of claims 1 to 14, or comprises the amino acids described in SEQ ID NOs: 165 - 169, or comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Acr amino acid sequence described in any one of SEQ ID NOs: 165 - 169, the Acr polypeptide or the nucleic acid, and (ii) A CRISPR nuclease, or a nucleic acid encoding the CRISPR nuclease comprising a system. 16. The system according to 15, wherein the CRISPR nuclease is a Cas12a nuclease. 17. The system according to 15, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Cas12a nuclease amino acid sequence described in any one of SEQ ID NOs: 175 and 245 - 262. 18. The system according to 15, wherein the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 263, or an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the CRISPR nuclease amino acid sequence set forth in SEQ ID NO: 263. 19. The system according to 15, wherein the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 86 and 176 to 244, or an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the NUX protein amino acid sequence set forth in SEQ ID NOs: 1 to 86 and 176 to 244. 20. The system according to any one of 15 to 19, further comprising a guide RNA, or a nucleic acid encoding the guide RNA. 21. The system according to any one of 15 to 20, wherein the system comprises the nucleic acid encoding the Acr polypeptide and / or the nucleic acid encoding the CRISPR nuclease. 22. The system according to 21, comprising a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease. 23. The system according to 21, wherein the nucleic acid encoding the Acr polypeptide and the nucleic acid encoding the CRISPR nuclease are the same nucleic acid, such that the system comprises a nucleic acid encoding both the Acr polypeptide and the CRISPR nuclease. 24. The system according to any one of 21 to 23, wherein the translational control element is operably linked to the Acr polypeptide coding sequence or the CRISPR nuclease coding sequence. 25. The system according to 24, wherein the translational control element is selected from the group consisting of an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or a combination thereof. 26. The system according to any one of 21 to 23, wherein a first promoter is operably linked to the Acr polypeptide coding sequence and a second promoter is operably linked to the CRISPR nuclease coding sequence. 27. The system according to 26, wherein the first promoter is a promoter stronger than the second promoter. 28. The system according to 26, wherein the second promoter is a promoter stronger than the first promoter. 29. The system according to any one of 21 to 28, wherein one or more of the nucleic acids are viral vectors. 30. The system according to 29, wherein the viral vector is an AAV vector. 31. The system according to any one of 21 to 30, wherein one or more of the nucleic acids are contained in lipid nanoparticles (LNP). 32. The system according to any one of 15 to 20, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form. 33. The system according to 32, wherein the Acr polypeptide and / or the CRISPR nuclease are contained in lipid nanoparticles (LNP). 34. The system according to any one of 15 to 33, wherein the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 35. The system according to 34, wherein the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is at least 1.25 times greater than the ratio of the ON-target:OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 36. The system according to any one of 15 to 35, wherein the OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 37. The ON-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the ON-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. The system according to any one of 15 to 36. 38. A method for modifying a target nucleic acid, comprising contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease, wherein the Acr polypeptide and the CRISPR nuclease are delivered to the target nucleic acid as the system according to any one of 15 to 37, and the contacting results in a modification to the nucleotide sequence of the target nucleic acid. The method. 39. The method according to 38, wherein the modification results from a deletion of one or more nucleotides. 40. The method according to any one of 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio of 1:1. 41. The method according to any one of 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:10. 42. The method according to any one of 38 or 39, wherein the contacting step delivers the Acr polypeptide and the CRISPR nuclease in a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10. 43. The method according to any one of 38 to 42, wherein the contacting is performed in vivo. 44. The method according to any one of 38 to 42, wherein the contacting is performed within a eukaryotic cell. 45. The method according to 44, wherein the eukaryotic cell is an animal cell. 46. The method according to 45, wherein the eukaryotic cell is a human cell. 47. The method according to 44, wherein the eukaryotic cell is a stem cell. 48. The method according to any one of 38 to 47, wherein the target nucleic acid encodes a gene product. 49. The method according to any one of 38 to 47, wherein the editing efficiency of the ON-target CRISPR nuclease activity is at least 4 times greater than the editing efficiency of the OFF-target CRISPR nuclease activity. 50. The method according to 49, wherein the OFF-target CRISPR nuclease activity is at an off-target site containing 5 or fewer mismatches compared to the on-target site. 51. The method according to any one of 38 to 50, wherein the OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 52. The method according to any one of 38 to 51, wherein the ON-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the ON-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 53. The method according to any one of 38 to 52, further comprising measuring the editing efficiency. 54. The method according to any one of 38 to 53, further comprising measuring the editing efficiency at one or more off-target sites.

Example

[0234] VI. Example The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention. Nor are the following experiments all that were conducted, i.e., it is not intended to represent that no other experiments were conducted. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0235] For general methods in the biochemistry of molecules and cells, see standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are hereby incorporated by reference. Reagents, cloning vectors, cells, and kits for the methods referred to in or related to the present disclosure are available from commercial vendors (e.g., BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., etc.) and repositories (e.g., Addgene, Inc., American Type Culture Collection (ATCC), etc.).

[0236] Example 1: Construction of Vectors Nuclease Expression Vector Codon-optimized genes encoding AsCas12a, NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 177), and NUX (SEQ ID NO: 178) were synthesized and cloned into a mammalian expression vector under the CMV promoter. The vector contained an in-frame nuclear localization signal OPT NLS (GRSSDDEATADSQHAAPPKKKRKV) (SEQ ID NO: 125), followed by a linker (GGSGGSGGSGGSGGSGGSGGSGGS) (SEQ ID NO: 124), and then a 3x HA tag.

[0237] Guide vector To generate the guide vector, the direct repeat (DR) sequences of the nucleases NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 178), and AsCas12a (Table 2) were placed downstream of the U6 promoter with a starting G. DR19s are the direct repeats of NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 177), and NUX (SEQ ID NO: 178), and DR18s are the direct repeats of AsCas12a. The spacer target sequences were placed downstream of the DR sequences. The spacer sequence used for DNMT was CTGATGGTCCATGTCTGTTA (SEQ ID NO: 172), and the PAM for this was TTTC. The spacer sequence used for FANCF1 was GGCGGGGTCCAGTTCCGGGA (SEQ ID NO: 162), and the PAM for this was TTTG. Mismatch guides were generated by changing a single nucleotide at a given position to its counterpart (e.g., A→T and G→C).

Table 2

[0238] Acr vector Potent anti-CRISPR (Acr) sequences to be used with the nuclease were identified in the EBI and NCBI databases (Table 3). Codon-optimized (human) genes encoding Acr from Table 3 were synthesized and cloned into the same CMV expression vector as the nuclease.

Table 3

[0239] Example 2: Acr Activity The editing efficiency of AsCas12a (SEQ ID NO: 175) in the presence of Acr was tested in HEK293T cells. The vector constructed in Example 1 was transfected into cells using Mirus Transit X2 reagent (MirusBio; catalog number MIR6003). The test was carried out in a 96-well plate transfected with 100 ng of nuclease expression vector, 100 ng of Acr expression vector, and 50 ng of targeting guide vector according to the recommendations for Mirus Transit X2 transfection. The samples were incubated for 72 hours and harvested with Quick Extract. Genomic DNA was amplified using genomic region-specific primers. [Table 4]

[0240] The purity of the samples was confirmed on a 2% agarose gel and sequenced by Sanger sequencing. TIDE analysis was performed according to the method of Brinkman et al., 2014 and the recommendations of the TIDE website (https: / / tide.nki.nl / ). Prism software was used to plot the TIDE output data regarding the editing efficiency. Acr was compared with AcrVA1 (ACX-137) (SEQ ID NO: 67) and AcrIIA4 (ACX-105) (SEQ ID NO: 35). The results are shown in Figure 1.

[0241] Also, the activity of Acr was tested by co-transfecting the NUX (SEQ ID NO: 176) of the second nuclease. Using the expression vector, NUX (SEQ ID NO: 176) and Acr were transiently delivered into HEK293T cells according to the same protocol as above. The editing efficiency was measured as described above. The results are shown in Figure 2.

[0242] The specific Acr, Acx-175, Acx-176, Acx-177, and Acx-178 (SEQ ID NOs: 165-168 respectively) showed the ability to inhibit the editing efficiency of Cas12a. Acr showed different inhibition patterns when tested with NUX (SEQ ID NO: 176). Acx-178 showed little inhibition, and the inhibitory activity of Acx-176 was less effective compared to Acx-175, but Acx-176 was a more potent inhibitor of Cas12a than Acx-175.

[0243] Example 3: Dose-dependent nuclease inhibition characteristics of Acx-175 In a series of administration experiments, Acx-175 showed unique ErAcr characteristics of inhibiting off-target editing without substantially changing on-target activity.

[0244] The AsCas12a endonuclease was co-transfected into HEK293T cells with decreasing doses of Acx-175, and the editing activity targeting DNMT1 was observed. The analysis including transfection, incubation, and measurement of editing efficiency was performed as described in Example 2. Guide RNAs with one base change at position 9

Chemical formula

[0245] The NUX (SEQ ID NO: 177) endonuclease was co-transfected with decreasing doses of Acx-175, and the editing activity targeting DNMT1 was observed. The transfection, incubation, and analysis protocol were performed as in Example 2. Guide RNAs with one base change at position 9 (MM9) and one base change at position 5 (MM5, CTGAAGGTCCATGTCTGTTA: SEQ ID NO: 174) were used to measure off-target editing. The results are shown in Figure 4.

[0246] Example 4: Generation of engineered Acr The potent nuclease inhibitor Acx-175 (SEQ ID NO: 165) was mutagenized using a random mutagenesis kit (Genemorph II). Mutants were selected using the DEAD / ALIVE bacterial screening approach described by Huimin Zhao in 2005 (https: / / academic.oup.com / nar / article / 33 / 18 / e154 / 2401371).

[0247] Expression vectors were constructed using NUX (SEQ ID NO: 178) and the mutagenized Acx-175 library as described in Example 1. The target was DNMT1, and off-target editing was measured using a guide RNA (MM8, CTGATGGaCCATGTCTGTTA: SEQ ID NO: 161) with one base change at position 8.

[0248] As a result of the first round of screening, six Acx-175 variants (excluding Acx-315 in Table 4) were obtained. These were transferred to a CMV expression vector and tested in HEK293T cells having NUX (SEQ ID NO: 178) and DNMT1 for verification according to the method of Example 2. The results are shown in Figure 5.

Table 5-1

Table 5-2

[0249] Example 5: Dose-Dependent Nuclease Inhibition Using Engineered Acr Expression vectors were constructed using NUX (SEQ ID NO: 178) and four engineered Acrs (Acx-306, Acx-308, Acx-310, and Acx-311). Three different ratios of Nux:Acx (2:1, 1:1, and 1:2) were transfected into HEK293T cells. The target was DNMT1. Transfection, incubation, and analysis were performed as in Example 2. The results of the editing efficiency of Nux:Acx 2:1 are shown in Figure 6A. The results of the editing efficiency of Nux:Acx 1:1 are shown in Figure 6B. The results of the editing efficiency of Nux:Acx 1:2 are shown in Figure 6C.

[0250] Example 6: Measurement of off-target inhibition of Acr NUX (SEQ ID NO: 178) was co-transfected into HEK293T cells with Acx-306 or Acx-310. Editing efficiency was measured for on-target as well as off-targets at single mismatches at positions 1, 9, 19, and 20. The editing efficiency was compared with Acx-105 as a negative control and wild-type Acx-175 as a positive control. The results are shown in Figure 7. NUX (SEQ ID NO: 178) was co-transfected into HEK293T cells with Acx-315 (SEQ ID NO: 132). Editing efficiency was measured for on-target and off-targets at a single mismatch at position 8 of DNMT1. NUX (SEQ ID NO: 178) and Acx-315 were tested over a range of Nux:Acx ratios. For the results, on-target and off-target editing efficiencies are shown in Figure 8A, and on-targeting:off-targeting ratios are shown in both Figure 8B and Table 5.

Table 6

[0251] Example 7: Variant Acr activity Error-prone PCR (Genemorph II, Agilent Biosciences) was used to subject Acr Acx-175 (SEQ ID NO: 165) to random mutagenesis at codon positions 2 - 159. The resulting library of variant Acr was screened using the method described in Example 4. Sequencing of the variants yielded a set of amino acid substitutions at each position as shown in Table 6.

[0252]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

[0253] As described in Example 4, the ability of the variant to modulate on-target and off-target editing of a CRISPR nuclease (SEQ ID NO: 263) targeting DNMT1 (on-target) and DNMT1 mismatch 8 (off-target) was evaluated. The results are shown in Figure 12, and the tested variants are listed in Table 7. The results indicate that both the inhibition level and the on-target:off-target activity ratio can be modulated by changes in Acr. Figure 13 shows the amino acid sequence alignment of ErAcr shown in Figure 12.

[0254]

Table 8

[0255] Although the above invention has been described in some detail through examples and illustrations for clear understanding, it will be readily apparent to those skilled in the art that, based on the teachings of the present invention, some changes and modifications may be made to the present invention without departing from the spirit and scope of the appended claims.

[0256] Accordingly, the foregoing is merely illustrative of the principles of the present invention. It will be appreciated that those skilled in the art can devise various mechanisms that embody the principles of the present invention and fall within the spirit and scope of the present invention, even if not explicitly described or shown herein. Further, all of the examples and conditional language recited herein are principally intended to assist the reader in understanding the principles of the present invention and the concepts that the inventors contribute to the advancement of the art, and are to be construed as not being limited to such specifically recited examples and conditions. Additionally, all descriptions in this specification of the principles, aspects, and embodiments of the present invention, as well as the specific examples thereof, are intended to encompass both their structural and functional equivalents. Moreover, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future (i.e., any elements developed that perform the same function regardless of structure). Further, the disclosure herein is not intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0257] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, with respect to the limitations in the claims, the provisions of 35 U.S.C. 112, paragraph (f) or 35 U.S.C. 112, paragraph (6) are expressly defined to apply only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim, and 35 U.S.C. 112, paragraph (f) or 35 U.S.C. 112, paragraph (6) does not apply when such exact phrase is not used in the limitation in the claim.

[0258] Although the present invention has been described with reference to specific embodiments thereof, those skilled in the art should understand that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the present invention. In addition, many changes may be made to a particular situation, material, composition of matter, process, process step(s) so as to be adapted to the objectives, spirit, and scope of the present invention. All such changes are intended to be within the scope of the claims appended hereto.

Claims

**Claim 1** An anti-CRISPR (Acr) polypeptide, or a nucleic acid encoding said Acr polypeptide, wherein said Acr polypeptide comprises an amino acid sequence having at least 75% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267, said Acr polypeptide or said nucleic acid. **Claim 2** The Acr polypeptide or nucleic acid according to claim 1, wherein said amino acid sequence has at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. **Claim 3** The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 126-132. **Claim 4** The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 165 having an amino acid variation at at least one position from amino acid 2 to amino acid 159 of SEQ ID NO:

165. **Claim 5** The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 264-267. **Claim 6** The Acr polypeptide or nucleic acid according to claim 1 or claim 4, wherein said Acr polypeptide comprises one or more amino acid variations set forth in Table 6. **Claim 7** The Acr polypeptide or nucleic acid according to claim 1 or claim 4, wherein said Acr polypeptide comprises one or more amino acid variations set forth in Table 7. **Claim 8** The Acr polypeptide or nucleic acid according to any one of claims 1-7, wherein said Acr polypeptide reduces the off-target CRISPR nuclease activity by 10% or more compared to the off-target CRISPR nuclease activity in the absence of said Acr polypeptide. **Claim 9** The Acr polypeptide or nucleic acid according to any one of claims 1-8, wherein said Acr polypeptide reduces the on-target CRISPR nuclease activity by 40% or less compared to the on-target CRISPR nuclease activity in the absence of said Acr polypeptide. **Claim 10** The Acr polypeptide increases the ratio of ON-target:OFF-target CRISPR nuclease activity by at least 1.25-fold compared to the ratio of ON-target:OFF-target CRISPR nuclease activity in the absence of the Acr polypeptide, the Acr polypeptide or nucleic acid according to any one of claims 1 to 9.

11. The Acr polypeptide or nucleic acid according to any one of claims 8 to 10, wherein the CRISPR nuclease is a Cas12a nuclease.

12. The Acr polypeptide or nucleic acid according to any one of claims 8 to 10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262.

13. The Acr polypeptide or nucleic acid according to any one of claims 8 to 10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244.

14. The Acr polypeptide or nucleic acid according to any one of claims 1 to 13, wherein the Acr polypeptide is fused to a nuclear localization signal (NLS).

15. (i) An Acr polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide according to any one of claims 1 to 14, comprises the amino acids set forth in SEQ ID NOs: 165-169, or comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169, the Acr polypeptide or the nucleic acid; (ii) A CRISPR nuclease, or a nucleic acid encoding the CRISPR nuclease; A system comprising.

16. The system according to claim 15, wherein the CRISPR nuclease is a Cas12a nuclease.

17. The system according to claim 15, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262.

18. The system according to claim 15, wherein the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 263, or an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the CRISPR nuclease amino acid sequence set forth in SEQ ID NO:

263.

19. The system according to claim 15, wherein the CRISPR nuclease comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244, or an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity to the NUX protein amino acid sequence set forth in SEQ ID NOs: 1-86 and 176-244.

20. The system according to any one of claims 15-19, further comprising a guide RNA, or a nucleic acid encoding the guide RNA.

21. The system according to any one of claims 15-20, wherein the system comprises the nucleic acid encoding the Acr polypeptide and / or the nucleic acid encoding the CRISPR nuclease.

22. The system according to claim 21, comprising a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease.

23. The system according to claim 21, wherein the nucleic acid encoding the Acr polypeptide and the nucleic acid encoding the CRISPR nuclease are the same nucleic acid, such that the system comprises a nucleic acid encoding both the Acr polypeptide and the CRISPR nuclease.

24. The system according to any one of claims 21-23, wherein a translation control element is operably linked to the Acr polypeptide coding sequence or the CRISPR nuclease coding sequence.

25. The system according to claim 24, wherein the translation control element is selected from the group consisting of an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or a combination thereof.

26. The system according to any one of claims 21 to 23, wherein a first promoter is operably linked to the Acr polypeptide coding sequence, and a second promoter is operably linked to the CRISPR nuclease coding sequence. **Claim 27** The system according to claim 26, wherein the first promoter is a stronger promoter than the second promoter. **Claim 28** The system according to claim 26, wherein the second promoter is a stronger promoter than the first promoter. **Claim 29** The system according to any one of claims 21 to 28, wherein one or more of the nucleic acids are viral vectors. **Claim 30** The system according to claim 29, wherein the viral vector is an AAV vector. **Claim 31** The system according to any one of claims 21 to 30, wherein one or more of the nucleic acids are included in lipid nanoparticles (LNP). **Claim 32** The system according to any one of claims 15 to 20, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form. **Claim 33** The system according to claim 32, wherein the Acr polypeptide and / or the CRISPR nuclease are included in lipid nanoparticles (LNP). **Claim 34** The system according to any one of claims 15 to 33, wherein the ratio of the ON target:OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of the ON target:OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. **Claim 35** The system according to claim 34, wherein the ratio of the ON target:OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is at least 1.25 times greater than the ratio of the ON target:OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. **Claim 36** The system according to any one of claims 15 to 35, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. **Claim 37** The system according to any one of claims 15 to 36, wherein the ON-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the ON-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

38. A method of modifying a target nucleic acid, comprising contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease, wherein the Acr polypeptide and the CRISPR nuclease are delivered to the target nucleic acid as the system according to any one of claims 15 to 37, and the contacting results in a modification to the nucleotide sequence of the target nucleic acid.

39. The method according to claim 38, wherein the modification results from a deletion of one or more nucleotides.

40. The method according to any one of claims 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a 1:1 ratio.

41. The method according to any one of claims 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:

10.

42. The method according to any one of claims 38 or 39, wherein the contacting step delivers the Acr polypeptide and the CRISPR nuclease in a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:

10.

43. The method according to any one of claims 38 to 42, wherein the contacting is performed in vivo.

44. The method according to any one of claims 38 to 42, wherein the contacting is performed within a eukaryotic cell.

45. The method according to claim 44, wherein the eukaryotic cell is an animal cell.

46. The method according to claim 45, wherein the animal cell is a human cell.

47. The method according to claim 44, wherein the eukaryotic cell is a stem cell.

48. The method according to any one of claims 38 to 47, wherein the target nucleic acid encodes a gene product.

49. The method according to any one of claims 38 to 48, wherein the editing efficiency of the ON-target CRISPR nuclease activity is at least 4 times greater than the editing efficiency of the OFF-target CRISPR nuclease activity. Claim 50 The method according to claim 49, wherein the OFF-target CRISPR nuclease activity is in an off-target site containing five or fewer mismatches compared to the ON-target site. Claim 51 The method according to any one of claims 38 to 50, wherein the OFF-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the OFF-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. Claim 52 The method according to any one of claims 38 to 51, wherein the ON-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 40% or less compared to the ON-target activity of the CRISPR nuclease in the absence of the Acr polypeptide. Claim 53 The method according to any one of claims 38 to 52, further comprising measuring the editing efficiency. Claim 54 The method according to any one of claims 38 to 53, further comprising measuring the editing efficiency at one or more off-target sites.

Images