Engineered acr proteins for modulating crispr activity

Abstract

Provided are compositions and methods that include an engineered anti-CRISPR (Acr) protein that can achieve a balance in which Cas proteins retain activity to perform on-target nucleic acid targeting functions (e.g., DNA cleavage for gene editing applications), but are inhibited by a subject Acr protein to a degree that decreases off-target activity – thus resulting in an increased ratio of on-target to off-target nucleic acid targeting events. In some cases, a subject composition (e.g., a subject system) includes a CRISPR nuclease or a nucleic acid encoding it. Methods of using a subject Acr polypeptide together with a CRISPR nuclease to achieve target nucleic acid modification are provided.

Description

ENGINEERED ACR PROTEINS FOR MODULATING CRISPR ACTIVITY CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,494, filed June 15, 2022, which application is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE OFSEQUENCELISTINGPROVIDED AS ASEQUENCELISTINGXML FILE

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “ACRG- 006WO_SEQ_LIST.xml” created on June 5, 2023 and having a size of 557,231 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. I. INTRODUCTION

[0003] CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas systems are found in diverse bacterial and archaeal species, serving as an immune defense mechanism against phage infection. The simplicity, programmability, and versatility of CRISPR-Cas systems (e.g., Cas9 and Cas12 systems) have facilitated the genetic modification of many organisms and offer immense therapeutic potential for the treatment of human disease. However, in practice CRISPR-Cas mediated genome editing can be associated with off-targeting, e.g., introduction of unintended mutations, insertions, or deletions, and DNA restructuring at unintended “off-target” sites. Off-target editing caused by CRISPR-Cas systems has been reported in various cell and animal models, including in human cells, and such events might accumulate in vivo with prolonged nuclease activity. Further, human genetic variations and uncertain Cas protein expression lifetimes in vivo add to the unpredictability of off-target events, which should be addressed for safe clinical translation of CRISPR-Cas systems. Unintended editing events can potentially lead to genomic instability, disrupt the functionality of genes, and cause serious adverse events including cell death and cancer.

[0004] There is a need for compositions and methods that modify Cas activity, e.g., decrease off-target events (e.g., relative to on-target events), thus leading to enhancednucleic acid targeting specificity. The present disclosure provides such compositions and methods. II. SUMMARY

[0005] Anti-CRISPR (Acr) proteins were discovered in phages for their ability to bind to and inhibit certain Cas proteins, thwarting the CRISPR system’s attempt to cleave invading phage DNA. Thus, Acr proteins are apparently used by phage to evade CRISPR-Cas systems.

[0006] The inventors have generated engineered Acr proteins (ErAcrs) that are not naturally occurring (see, e.g., SEQ ID NOs.: 126-132 and 264-267) and can be used to achieve a balance in which Cas proteins retain sufficient activity to perform a desired on-target nucleic acid functions (e.g., loading, complex assembling, binding and cleavage), but are inhibited to a degree that decreases off-target activity. Thus, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (an ErAcr), or a nucleic acid encoding the Acr polypeptide, where the Acr polypeptide includes an amino acid sequence having 75% or more 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) with the sequence set forth in any one of SEQ ID Nos.: 126-132. Likewise, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (an ErAcr), or a nucleic acid encoding the Acr polypeptide, where the Acr polypeptide includes an amino acid sequence having 75% or more 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) with the sequence set forth in any one of SEQ ID Nos.: 264- 267. In some cases, a subject Acr polypeptide has 90% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.: 126-132. In some cases, a subject Acr polypeptide comprises the sequence set forth in any one of SEQ ID Nos.: 126-132. In some cases, a subject Acr polypeptide has 90% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.: 264-267. In some cases, a subject Acr polypeptide comprises the sequence set forth in any one of SEQ ID Nos.: 264-267.

[0007] In general, an Acr polypeptide is capable of decreasing activity of a CRISPR nuclease. For example, in some embodiments, a subject Acr polypeptide decreases OFF target CRISPR nuclease activity by 10% or more (e.g., 20% or more, 30% or more 50% or more, etc.) as compared with OFF target (off-target) CRISPR nuclease activity in the absence of the Acr polypeptide. In some embodiments, a subject Acr polypeptidedecreases ON target (on-target) CRISPR nuclease activity by no more than 40% (e.g., no more than 30%, no more than 20%, etc.) as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide. In some embodiments, a subject Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity as compared with the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide (e.g., by at least 1.25x, 1.5x, 2x, 3x, etc.).

[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, a subject Acr polypeptide decreases (inhibits) activity of a Cas12a nuclease (see, e.g., SEQ ID NOs.: 175 and 245-262). In some cases, a subject Acr polypeptide decreases activity of a CRISPR nuclease having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 175 and 245-262. In some cases, a subject Acr polypeptide decreases activity of a CRISPR nuclease having 90% or more sequence identity (e.g., 95% or more, or 100% sequence identity) with the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 175 and 245-262. In some cases, a subject Acr polypeptide decreases activity of a CRISPR nuclease having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the Cas12a nuclease amino acid sequence set forth in SEQ ID NO: 175. In some cases, a subject Acr polypeptide decreases activity of a CRISPR nuclease having 90% or more sequence identity (e.g., 95% or more, or 100% sequence identity) with the Cas12a nuclease amino acid sequence set forth in SEQ ID NO: 175.

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

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

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

[0012] Also provided herein are systems that include a subject Acr polypeptide (e.g., an ErAcr) or a nucleic acid encoding the Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease. In such systems, the Acr polypeptide can decrease (inhibit) activity of the CRISPR nuclease. The Acr polypeptide of such systems can be any of those discussed above (e.g., having 75% or more sequence identity with SEQ ID NOs.: 126-132, e.g., having 75% or more sequence identity with SEQ ID NOs.: 264-267), and the CRISPR nuclease can also be any of those discussed above (e.g., a Cas12a nuclease such as SEQ ID NOs.: 175 and 245-262, a NUX protein such as SEQ ID NOs: 1-86 and 176-244, and the like – see, e.g., the CRISPR nuclease of SEQ ID NO: 263). In some cases, a subject system will also include 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 a subject system (or method) can include an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the Acr amino acid sequence set forth in any one of SEQ ID NOs.: 165-169. In some embodiments, the Acr polypeptide of a subject system (or method) can include an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with 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 the system do not naturally occur together. In some cases, the Acr polypeptide of a subject system (or method) includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the Acr amino acid sequence set forth inany one of SEQ ID NOs.: 165-169, and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the NUX protein amino acid sequence set forth in any one of SEQ ID NOs.: 1-86 and 176-244 or with 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 a subject system (or method) includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the Acr amino acid sequence set forth in any one of SEQ ID NOs.: 165-169, and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) with the amino acid sequence set forth in 263.

[0014] The Acr polypeptide and the CRISPR nuclease can each, independently, be provided as a nucleic acid encoding (DNA, RNA) the protein or can be provided in protein form. When both are provided as nucleic acids, the Acr polypeptide and the CRISPR nuclease can be encoded on separate nucleic acids (e.g., provided as two separate vectors), or can be encoded on the same nucleic acid (e.g., provided on the same vector). In either case, a translation control element (e.g., an IRES sequence, a 2A peptide encoding sequence, a non-canonical start codon, or any combination thereof) can be operably linked to the Acr polypeptide coding sequence and / or to 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, a nucleic acid (encoding the Acr polypeptide, the CRISPR nuclease, or both) is a viral vector, e.g., an AAV vector. Whether the Acr polypeptide and / or the CRISPR nuclease are provided as a nucleic acid or in protein form, either can In some cases, by included in a lipid nanoparticle (LNP) formulation.

[0015] Also provided herein are methods that utilize the above compositions / systems. For example, provided are methods of modifying a target nucleic acid, where the method includes contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease (see above), where the contacting results in a modification to the nucleotide sequence (e.g., substitution, insertion, deletion) of the target nucleic acid. Insome cases, the contacting includes delivery of the CRISPR nuclease and the Acr polypeptide at a 1:1 ratio. In other cases, the contacting includes delivery of the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) in a range of from 1:1.25 to 1:10. In yet other cases, the contacting includes delivery of the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) in a range of from 1:1.25 to 1:10. The contacting can occur in vitro, ex vivo, and in vivo, and the contacting can occur in a cell, e.g., a eukaryotic cell, an animal cell, a mammalian cell, a human cell, etc. In some embodiments, the target nucleic acid encodes a gene product (e.g., a non-coding RNA, an mRNA / protein, and the like).

[0016] The on-target / off-target CRISPR activity referred to in a subject composition, system, or method can include editing of the target nucleic acid (e.g., via DNA cleavage in the presence or absence of a donor polynucleotide), e.g., genome editing. In some cases, editing efficiency of ON target CRISPR nuclease activity is greater than editing efficiency of OFF target CRISPR nuclease activity (e.g., at least 2 times, at least 4 times, at least 5 times, or at least 10 times greater). In some cases, the OFF target CRISPR nuclease activity is at an off-target site that includes no more than 5 mismatches as 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 decreased when compared with 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 case, the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased as compared with the ON target activity of the CRISPR nuclease in the absence of the Acr polypeptide (e.g., by no more than 10%, by no more than 20%, by no more than 30%, by no more than 40%, etc.).

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

[0018] FIG.1 depicts editing efficiency of AsCas12a (SEQ ID NO: 175) in the presence of Acrs - tested in HEK293T cells. Acx-105 represents a negative control; this Acr inhibits Ca9 but does not inhibit AsCas12a.

[0019] FIG.2 depicts editing efficiency of NUX (SEQ ID NO: 176) in the presence of Acrs - tested in HEK293T cells.

[0020] FIG.3 depicts results from dosing experiments using the CRISPR nuclease AsCas12a with increasing amounts (left to right) of the Acr Acx-175, using a guide that matched the DNMT1 (for on-targeting editing) and one guide that was mismatched at position 9 (to measure off-target editing). Acx-105 (which does not inhibit AsCas12a) was added to keep the relative amount of Acr protein consistent across the samples.

[0021] FIG.4 depicts results from dosing experiments using the CRISPR nuclease NUX (SEQ ID NO: 177) with the Acr Acx-175 (SEQ ID NO: 165) to observe editing activity targeting DNMT1. Off target editing was measured using a guide RNA with a single base change at position 9 (MM9) and a guide RNA with a single base change at position 5 (MM5).

[0022] FIG.5 depicts results from using seven engineered Acrs (ErAcrs) (Table 4, Acx- 305, -306, -307, -308, -310, -311) that were transferred into a CMV expressing vector and tested in HEK293T cells with NUX (SEQ ID NO: 178) and DNMT1 for on-target editing and with a guide RNA with a single base change at position 8 (MM8) for off- target editing..

[0023] FIG.6A-6C depict results from using four of the engineered Acrs (Acx-306, Acx- 308, Acx-310 and Acx-311). Three different ratios of Nux:Acx (2:1 (FIG.6A), 1:1 (FIG. 6B) and 1:2 (FIG.6C)) were transfected into HEK293T cells. The target was DNMT1. On-target editing was measured with a DNMT1 guide RNA and off-target editing was measured with a guide RNA with a single base change at position 9.

[0024] FIG.7 depicts results in HEK293T cells comparing Acrs, Acx-175, Acx-306, Acx- 310, and negative control Acx-105 with the nuclease NUX (SEQ ID NO: 178) using guide RNAs with either a perfect match to the target, or a mismatch of one base at different positions in the guide RNA.

[0025] FIG.8A-8B depict results from using the Acr Acx-315 (SEQ ID NO: 132) with NUX (SEQ ID NO: 178) in HEK293T cells over a range of Nux:Acx ratios for on-target editing for DNMT1and off-target editing (with MM8 guide RNA). Figure 8A shows the results by % editing efficiency and Figure 8B shows the results as a ratio of on-target to off-target editing.

[0026] FIG.9A-9D depict schematics of non-limiting examples of arrangements of components in a subject nucleic acid. “P1” and “P2” are promoters, “Cas” is the Cas- encoding sequence, “Acr” is the Acr-encoding sequence, “2A” is a 2A peptide encoding sequence, and “X” is a ‘spacer’ sequence.

[0027] FIG.10A-10F depict schematics of non-limiting examples of arrangements of components in a subject nucleic acid. “P1” and “P2” are promoters, “Cas” is the Cas- encoding sequence, “Acr” is the Acr-encoding sequence, “IRES” is an IRES sequence, and “X” is a ‘spacer’ sequence.

[0028] FIG.11A-11B depict schematics of two non-limiting examples of arrangements of components in a subject nucleic acid. “P1” and “P2” are promoters, “Cas” is the Cas- encoding sequence, “Acr” is the Acr-encoding sequence, Asterisks denotes a non-AUG start codon.

[0029] FIG.12 depicts results from on-target vs off-target editing experiments using ErAcr3, ErAcr4, ErAcr13, and ErAcr20.

[0030] FIG.13 depicts a sequence alignment of ErAcr3, ErAcr4, ErAcr13, and ErAcr20 (SEQ ID Nos.: 264-27, respectively). IV. DEFINITIONS

[0031] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0032] By "hybridizable", "hybridizes", “complementary”, or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence- specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G / U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of thisdisclosure, a guanine (G) (e.g., of dsRNA duplex of a guide RNA molecule; of a guide RNA base pairing with a target nucleic acid, etc.) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G / U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0033] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid 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, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0034] The terms ''peptide," ''polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form 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.

[0035] "Binding" as used herein (e.g., with reference to a nucleic acid binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid such as DNA or RNA). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non- covalent manner). Not all components of a binding interaction need be sequence- specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions can generally becharacterized by a dissociation constant (KD) of less than 106M, less than 10 M, less than 10-8M, less than 10-9M, less than 10-10M, less than 10-11M, less than 10-12M, less than 10-13M, less than 10-14M, or less than 10-15M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD.

[0036] As used herein, a “promoter” or a "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 purposes of the present disclosure, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including constitutive, tissue-specific, and inducible promoters may be used to drive expression by the various vectors of the present disclosure. The level of expression of a given promoter can be described as weak, medium, or strong – and thus, promoters can be categorized as weak, medium, or strong promoters.

[0037] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a nucleotide sequence (the nucleotide sequence can also be said to be operably linked to the promoter) if the promoter affects transcription of said nucleotide sequence. As another example, a translational control element is operably linked to a protein-coding sequence (the protein-coding sequence can also be said to be operably linked to the translational control element) if the translational control element affects translation of protein from the protein-coding sequence.

[0038] A “coordinated delivery system” as used herein refers to the coordinated delivery of a an Acr protein and a Cas nuclease. A coordinated delivery system includes 1 or more nucleic acids (e.g., vectors) for expression of an Acr protein and a CRISPR nuclease, e.g., in a host cell. In some cases, a coordinated delivery system provides a single nucleic acid (e.g., vector) for expression of an Acr protein and a CRISPR nuclease. In some cases, a coordinated delivery system provides more than one nucleic acid (e.g., vector) for expression of an Acr protein and a Cas nuclease and the expression and / or function is coordinated such as with the provision of a split-Cas from 2 vectors. In some cases, of the coordinated delivery system the expression and / or function of an Acr protein and a Cas nuclease is linked (i.e., coordinated) byvirtue of translational control element selected to regulate the translation of the Acr protein and / or CRISPR nuclease.

[0039] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0040] The terms "subject," and "host,"," used interchangeably herein, refer to an individual organism that expresses or is intended to express the coordinated delivery system and / or the Cas nuclease and / or Acr proteins decribed herein. Hosts include, but are not limited to fungi (such as yeasts), plants, algae, insects, animals such as birds and mammals, (e.g., a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets). Hosts include, but are not limited to, microbes such as bacteria and fungi (such as yeasts), plants, algae, insects, animals such as birds and mammals, (e.g., a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets).

[0041] The terms “on-target” (or “ON target”) and “off-target” (or “OFF target”) are used herein to refer to the locations of CRISPR complex activity (e.g., target DNA cleavage, DNA editing) within a target DNA. Both location types (on- and off- target) are based on the guide sequence of the guide RNA. CRISPR complex mediated events that take place at a location based on a 100% match with the guide sequence are considered “on-target” while those that take place at (undesired) locations that are not based on a 100% match with the guide sequence are “off-target”. If the sequence of the target DNA is known (e.g., a large portion of the genome of a target cell has been sequenced), then likely off-target sites can be predicted for a given guide sequence. In general, off- target events are more likely to take place at sequences that are closer to a 100% match between the guide RNA and target than sequences that are at lower percentages of identity between the guide RNA and target. As such, most off-target events tend to take place at sequences with 50% or more (e.g., 75% or more) sequence identity with the intended target sequence. For example, an off-target event may occur where thereis 1 mismatch between the guide RNA and target, 2 mismatches between the guide RNA and target or 3 mismatches between the guide RNA and target. As such, a sequence analysis of the target DNA can provide a list of expected possible off-target sites within a target DNA. The number of predicted off-target sites will depend on the target DNA sequence, but In some cases, the number of predicted off-target sites will be in a range of from 10-200 predicted sites (e.g., from 10-150, 10-100, 10-50, 15-200, 15-150, 15-100, 15-80, 20-200, 20-150, 20-100, or 20-80 predicted sites). V. DETAILED DESCRIPTION

[0042] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0043] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0044] Certain ranges may be presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0046] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are 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 is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0047] It is noted that as used herein and in 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 a plurality of such proteins and reference to “the protein” includes reference to one or more such proteins and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0048] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. For example, as will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0049] As noted above, the present disclosure provides Acrs, including non-naturally occurring engineered Acr proteins (ErAcrs), that can be used to achieve a balance in which Cas proteins retain sufficient activity to perform a desired on-target nucleic acid functions (e.g., loading, complex assembling, binding and cleavage), but are inhibited to a degree that decreases off-target activity as compared to the activity of the Cas protein without the presence of the Acr (see, e.g., SEQ ID NOs.: 126-132). Thus, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide or a nucleic acid encoding the Acr polypeptide, where the Acr polypeptide includes an amino acid sequence having 75% or more 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) with the sequence set forth in any one of SEQ ID Nos.: 126-132.

[0050] Also provided herein are systems and methods of using those systems to modify target nucleic acid, where the system includes a subject Acr polypeptide (e.g., ErAcrs of SEQ ID NOs.: 126-132, and / or Acrs of SEQ ID NOs.: 165-169) or a nucleic acid encoding the Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease. In some cases, the CRISPR nuclease and the Acr polypeptide of the system do not naturally occur together. CRISPR complex

[0051] The terms “CRISPR complex” and “effector complex” as used herein refer to the protein-RNA complex that is guided to a specific sequence within a target nucleic acid (e.g., target genomic DNA) by the RNA component – often referred to as a “guide RNA”. In Class 2 CRISPR systems, the functions of the effector complex are carried out by a single protein (which can be referred to as an “effector protein”, also referred to herein as a “CRISPR nuclease” or simply a “Cas protein”) – where the natural protein is an endonuclease (e.g., see 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).

[0052] As such, the term “class 2 CRISPR / Cas protein” or “CRISPR / Cas effector protein” or “Cas effector protein” or more simply “CRISPR nuclease” or “Cas protein” is used herein to encompass the effector protein from class 2 CRISPR systems.Non-limiting examples of CRISPR nucleases include Cas12a proteins and NUX proteins (described elsewhere herein). Acr proteins

[0053] Acr proteins are proteins that inhibit (decrease activity of) Cas proteins – thereby acting as negative regulators of the CRISPR complex. In some cases, a subject Acr protein is an inhibitor of a Cas protein of a class 2 CRISPR complex (e.g., a class 2 effector protein such as a Cas12 protein, e.g., a Cas12a – also known as Cpf1) – thereby directly regulating the effector protein of a CRISPR complex.

[0054] The effector CRISPR-Cas nucleases that complex with gRNA are highly diverse and spread across 6 distinct types (Types I–VI). So far, anti-CRISPR proteins (Acrs) have been discovered that inhibit CRISPR Type I, II, and V systems. The CRISPR-Cas Type II-A orthologue from S. pyogenes, SpCas9, is the most widely utilized CRISPR- Cas enzyme for biotechnological applications and has also been deployed in DNA- binding applications. Acr proteins that function against the Type II-A system have been discovered by a bioinformatics approach that surveys bacterial genomes for self- targeting. Among the distinct families of Acr proteins discovered so far, specific inhibitory mechanisms have been determined for some of them (e.g., AcrIE1, AcrIF1-3, AcrIF10, AcrIIA2, AcrIIA4, AcrIIC1-3, and AcrVA5). The known mechanisms are highly diverse, presenting a pool of off-switch modalities to draw from. Acr proteins can act at 3 different steps of CRISPR-Cas-mediated immunity including 1) inhibiting guide RNA loading, 2) blocking DNA binding, and 3) preventing DNA cleavage. The most common mechanism observed to date is that the anti-CRISPR protein occupies the DNA-binding site on the Cas protein, thus mimicking DNA and inhibiting the DNA-binding and cleavage activity of the protein.

[0055] However, the mechanisms by which Acrs block DNA binding can be different. For example, even though AcrIF1, AcrIF2, and AcrIF10 bind to different subunits of the cascade effector complex of the type I-F CRISPR-Cas system, they all prevent DNA binding to the complex. AcrIIC3 also blocks DNA binding but uses a fourth and distinct mechanism - promoting dimerization of Cas9. For the most potent SpCas9 inhibitor, AcrIIA4, a 3.9-Å resolution cryo-electron structure revealed that the Cas9-sgRNA- AcrIIA4 complex has AcrIIA4 bound to the PAM-interacting domain of Cas9, thus preventing the target DNA binding. Interestingly, AcrIIA4 binds only to assembled Cas9- sgRNA complexes, not to Cas9 protein alone or to preformed Cas9-sgRNA-DNA complexes.

[0056] It is to be understood that when discussing particular proteins such as Cas or Acr proteins throughout this disclosure (e.g., “Cas12a”, “Nux”, “ErAcr”, “Acr”), and when presenting such terms in claims, such terms are intended to encompass modified / mutated versions of such proteins that maintain their intended function. The terms are intended to encompass embodiments in which is the Cas and / or Acr protein is fused to one or more heterologous proteins (e.g., a fluorescent protein such as GFP, one or more nuclear localization signals (NLSs), and / or a tag such as MBP, CBP, strep tag, GST, HA, poly(His), Myc, V5, Spot, NE, AviTag, and the like).

[0057] Thus, in some embodiments a subject “Acr protein” comprises the wild type (natural) sequence. In some cases, a subject Acr Protein is mutated. As noted above, the inventors have generated engineered Acr proteins (ErAcrs) that are not naturally occurring (see, e.g., SEQ ID NOs.: 126-132) and can be used to achieve a balance in which Cas proteins retain sufficient activity to perform a desired on-target nucleic acid functions (e.g., loading, complex assembling, binding and cleavage), but are inhibited to a degree that decreases off-target activity. Thus, many of the compositions and methods disclosed herein include an anti-CRISPR (Acr) polypeptide (an ErAcr), or a nucleic acid encoding the Acr polypeptide.

[0058] Examples of engineered Acr proteins (ErAcrs) provided herein include SEQ ID NOs: 126-132 (see, e.g., the working examples). Additional examples of ErAcrs provided herein include SEQ ID NOs: 264-267 (see, e.g., example 7 below). Additional examples of Acr proteins provided herein include those set forth as SEQ ID NOs.165- 169.

[0059] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr protein comprises an aminoacid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132.

[0060] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 126. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 126.

[0061] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequenceidentity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 127. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 127.

[0062] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 128. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 128.

[0063] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity)with the Acr protein amino acid sequence set forth in SEQ ID NO: 129. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 129.

[0064] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 130. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 130.

[0065] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 131. In some cases,the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 131.

[0066] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 132. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 132.

[0067] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs:126-132 and 264-267. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 126-132 and 264-267.

[0068] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 264-267.

[0069] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with 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 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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.

[0070] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 265. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 265.

[0071] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 266. In some cases,the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 266.

[0072] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 267. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 267.

[0073] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165-169. In some cases, the subject Acr proteincomprises the Acr protein amino acid sequence set forth in any one of SEQ ID NOs: 165-169.

[0074] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 165. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 165.

[0075] In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 165, but has an amino acid variation at least one position between and including amino acid 2 and amino acid 159 of SEQ ID NO:165 (see, e.g., Table 6). In some cases, an Acr polypeptide includes one or more amino acid variations set forth in Table 6. In some cases, an Acr polypeptide includes one or more amino acid variations set forth in Table 7.

[0076] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95%or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 166. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 166.

[0077] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 167. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 167.

[0078] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the subject Acr protein comprises an amino acid sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the subject Acr protein comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with theAcr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the subject Acr protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Acr protein amino acid sequence set forth in SEQ ID NO: 168. In some cases, the subject Acr protein comprises the Acr protein amino acid sequence set forth in SEQ ID NO: 168.

[0079] In some cases, a subject Acr protein (i.e., polypeptide) (e.g., of the compositions, systems, or methods discloses herein) comprises an amino acid sequence having 75% or more 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) with 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 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with 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. CRISPR Nucleases

[0080] As with a subject Acr polypeptide, in some cases, a subject CRISPR nuclease comprises a wild type (natural) sequence. In some cases, (e.g., in systems and / or methods disclosed herein), the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with a wild type CRISPR nuclease (e.g., a Cas12a or a Nux protein). In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with a wild type CRISPRnuclease (e.g., a Cas12a or a Nux protein). In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with a wild type CRISPR nuclease (e.g., a Cas12a or a Nux protein). In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) with a wild type CRISPR nuclease (e.g., a Cas12a or a Nux protein). In some cases, the CRISPR nuclease comprises the amino acid sequence of a wild type CRISPR nuclease (e.g., a Cas12a or a Nux protein). In some cases, a subject CRISPR nuclease has been ‘evolved’ such that it has low overall sequence homology to the natural CRISPR nuclease, but retains the identifiable characteristic domain(s) of that protein. In some cases, (e.g., in 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 cases, (e.g., in 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.

[0081] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in SEQ ID NO:263. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in SEQ ID NO:263. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in SEQ ID NO:263. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more 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) with 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.

[0082] Examples of CRISPR nucleases include those of Table 1.

[0083] Table 1. Examples of CRISPR nuclease proteins Name Sequence SEQ ID NO: (AsCas12a) MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQE 175 QGFIEEDKARNDHYKELKPIIDRIYKTYADQCL QLVQLDWENLSAAIDSYRKEKTEETRNALIEE QATYRNAIHDYFIGRTDNLTDAINKRHAEIYKG LFKAELFNGKVLKQLGTVTTTEHENALLRSFD KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQD NFPKFKENCHIFTRLITAVPSLREHFENVKKAI GIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLG GISREAGTEKIKGLNEVLNLAIQKNDETAHIIAS LPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQ SFCKYKTLLRNENVLETAEALFNELNSIDLTHIF ISHKKLETISSALCDHWDTLRNALYERRISELT GKITKSAKEKVQRSLKHEDINLQEIISAAGKELS EAFKQKTSEILSHAHAALDQPLPTTLKKQEEK EILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSV EKFKLNFQMPTLASGWDVNKEKNNGAILFVK NGLYYLGIMPKQKGRYKALSFEPTEKTSEGFD KMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHT TPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQT AYAKKTGDQKGYREALCKWIDFTRDFLSKYTK TTSIDLSSLRPSSQYKDLGEYYAELNPLLYHIS FQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHH GKPNLHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAHRLGEKMLNKKLKDQKTPI PDTLYQELYDYVNHRLSHDLSDEARALLPNVI TKEVSHEIIKDRRFTSDKFFFHVPITLNYQAAN SPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYI TVIDSTGKILEQRSLNTIQQFDYQKKLDNREKE RVAARQAWSVVGTIKDLKQGYLSQVIHEIVDL MIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQ FEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLT DQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTG FVDPFVWKTIKNHESRKHFLEGFDFLHYDVKT GDFILHFKMNRNLSFQRGLPGFMPAWDIVFEK NETQFDAKGTPFIAGKRIVPVIENHRFTGRYR DLYPANELIALLEEKGIVFRDGSNILPKLLENDD SHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN NUX MNFQPFFQKFVHLYPISKTLRFELIPQGATQKF 176 ISEKQVLLQDEIRARKYPEMKQAIDGYHKDFIQ RALSNIDSQVFEQALNTFEDLFLRSQAERATD AYKKDFETAQTKLRELIVHSFEKGEFKQEYKS LFDKNLITNLLKPWVEQQNQIGDSNYTYHEDF NKFTTYFLGFHENRKNIYSKDPHKTALAYRLIH ENLPKFLENNKILLKIQNDHPSLWEQLQTLNQT MPQLFDGWDFSQLMQVSFFSNTLTQTGIDQY NTIIGGISEGENRQKIQGINELINLYNQKQDKKN RVAKLKQLYKQILSDRSTLSFLPEKFVDDTELY HAINMFYLEHLHHQSMINGHSYTLLERVQLLIN ELANYDLSKVYLAPNQLSTVSHQMFGDFGYIGName Sequence SEQ ID NO: RALNYYYMQVIQPDYEQLLASAKTTKKIEATEK LKTIFLDTPQSLVVIQAAIDEYIQLQPSTKPHTQ LTDFIISLLKQYETVADDQSIKVINVFSDIEGKY SCIKGLVNTKSESKREVLQDEKLATDIKAFMD AVNNVIKLLKPFSLNEKLVASVEKDARFYSDFE EIYQSLLIFVPLYNKVRNYITQKPYSTEKFKLNF NKPTLLSGWDANKEADNLSILLRKNGNYYLAI MDTAKGANKAFEPKTLNQLKVDDTTDCYEKM VYKLLSGPSKMFPKAFKAKNNEGNYYPTPELL TSYNNNEHLKNDKNFTLASLHAYIDWCKEYIN RNPSWHQFNFKFSPTQSFQDISQFYSEVSSQ SYKVHFQTIPSDYIDQLVAEGKLYLFQIYNKDF SPNAKGKENLHTLYFKALFSDENLKQPVFKLS GEAEMFYRPASLQLANTTIHKAGEPMAAKNPL TPNATRTLAYDIIKDRRFTTDKYLLHVPISLNFH AQESMSIKKHNDLVRQMIKHNHQDLHVIGIDR GEKHLLYVSVIDLKGNIVYQESLNSIKSEAQNF ETPYHQLLQHREEGRAQARTAWGKIENIKELK DGYLSQVVHRIQQLILKYNAIVMLEDLNFGFKR GRFKIEKQIYQKFEKALIHKLNYVVDKSTQADE LGGVRKAYQLTAPFESFEKLGKQSGVLFYVPA WNTSKIDPVTGFVDLLKPKYENLDKAQAFFNA FDSIHYNAQKNYFEFKVNLKQFAGLKAQAAQA EWTICSYGDERHVYQKKNAQQGETVIVNVTE ELKVLFAKNNIEVAQSVELKETICTQTQVDFFK RLMWLLQVLLALRYSSSKDKLDYILSPVANAQ GEFFDSRHASVQLPQDSDANGAYHIALKGLW VIEQLKAADNLDKVKLAISNDDWLHFAQQKPY LA NUX MNFQPFFQKFVHLYPISKTLRFELIPQGATQKF 177 ISEKQVLLQDEIRARKYPEMKQAIDGYHKDFIQ RALSNIDSQVFEQALNTFEDLFLRSQAERATD AYKKDFETAQTKLRELIVHSFEKGEFKQEYKS LFDKNLITNLLKPWVEQQNQIGDSNYTYHEDF NKFTTYFLGFHRNRKNIYSKDPHKTALAYRLIH ENLPKFLENNKILLKIQNDHPSLWEQLQTLNQT MPQLFDGWDFSQLMQVSFFSNTLTQTGIDQY NTIIGGISEGENRQKIQGINELINLYNQKQDKKN RVAKLKQLYKQILSDRSTLSFLPEKFVDDTELY HAINMFYLEHLHHQSMINGHSYTLLERVQLLIN ELANYDLSKVYLAPNQLSTVSHQMFGDFGYIG RALNYYYMQVIQPDYEQLLASAKTTKKIEATEK LKTIFLDTPQSLVVIQAAIDEYIQLQPSTKPHTQ LTDFIISLLKQYETVADDQSIKVINVFSDIEGKY SCIKGLVNTKSESKREVLQDEKLATDIKAFMD AVNNVIKLLKPFSLNEKLVASVEKDARFYSDFE EIYQSLLIFVPLYNKVRNYITQKPYSTEKFKLNF NKPTLLSGWDANREADNLSILLRKNGNYYLAI MDTAKGANKAFEPKTLNQLKVDDTTDCYEKM VYKLLSGPSKMFPKAFKAKNNEGNYYPTPELL TSYNNNEHLKNDKNFTLASLHAYIDWCKEYIN RNPSWHQFNFKFSPTQSFQDISQFYSEVSSQ SYKVHFQTIPSDYIDQLVAEGKLYLFQIYNKDF SPNAKGKENLHTLYFKALFSDENLKQPVFKLS GEAEMFYRPASLQLANTTIHKAGEPMAAKNPLName Sequence SEQ ID NO: TPNATRTLAYDIIKDRRFTTDKYLLHVPISLNFH AQESMSIKKHNDLVRQMIKHNHQDLHVIGIDR GEKHLLYVSVIDLKGNIVYQESLNSIKSEAQNF ETPYHQLLQHREEGRAQARTAWGKIENIKELK DGYLSQVVHRIQQLILKYNAIVMLEDLNFGFKR GRFKIEKQIYQKFEKALIHKLNYVVDKSTQADE LGGVRKAYQLTAPFESFEKLGKQSGVLFYVPA WNTSKIDPVTGFVDLLKPKYENLDKAQAFFNA FDSIHYNAQKNYFEFKVNLKQFAGLKAQAAQA EWTICSYGDERHVYQKKNAQQGETVIVNVTE ELKVLFAKNNIEVAQSVELKETICTQTQVDFFK RLMWLLQVLLALRYSSSKDKLDYILSPVANAQ GEFFDSRHASVQLPQDSDANGAYHIALKGLW VIEQLKAADNLDKVKLAISNDDWLHFAQQKPY LAGRSSDDEATADSQHAAPPKKKRKVGGSG GSGGSGGSGGSGGSGGSGGSLEGSYPYDV PDYAYPYDVPDYAYPYDVPDYA NUX MNFQPFFQKFVHLYPISKTLRFELIPQGATQKF 178 ISEKQVLLQDEIRARKYPEMKQAIDGYHKDFIQ RALSNIDSQVFEQALNTFEDLFLRSQAERATD AYKKDFETAQTKLRELIVHSFEKGEFKQEYKS LFDKNLITNLLKPWVEQQNQIGDSNYTYHEDF NKFTTYFLGFHRNRKNIYSKDPHKTALAYRLIH ENLPKFLENNKILLKIQNDHPSLWEQLQTLNQT MPQLFDGWDFSQLMQVSFFSNTLTQTGIDQY NTIIGGISEGENRQKIQGINELIALYNQKQDKKN RVAKLKQLYKQILSDRSTLSFLPEKFVDDTELY HAINMFYLEHLHHQSMINGHSYTLLERVQLLIN ELANYDLSKVYLAPNQLSTVSHQMFGDFGYIG RALNYYYMQVIQPDYEQLLASAKTTKKIEATEK LKTIFLDTPQSLVVIQAAIDEYIQLQPSTKPHTQ LTDFIISLLKQYETVADDQSIKVINVFSDIEGKY SCIKGLVNTKSESKREVLQDEKLATDIKAFMD AVNNVIKLLKPFSLNEKLVASVEKDARFYSDFE EIYQSLLIFVPLYNKVRNYITQKPYSTEKFKLNF NKPTLLSGWDANREADNLSILLRKNGNYYLAI MDTAKGANKAFEPKTLNQLKVDDTTDCYEKM VYKLLSGPSKMFPKAFKAKNNEGNYYPTPELL TSYNNNEHLKNDKNFTLASLHAYIDWCKEYIN RNPSWHQFNFKFSPTQSFQDISQFYSEVSSQ SYKVHFQTIPSDYIDQLVAEGKLYLFQIYNKDF SPNAKGKENLHTLYFKALFSDENLKQPVFKLS GEAEMFYRPASLQLANTTIHKAGEPMAAKNPL TPNATRTLAYDIIKDRRFTTDKYLLHVPISLNFH AQESMSIKKHNDLVRQMIKHNHQDLHVIGIDR GEKHLLYVSVIDLKGNIVYQESLNSIKSEAQNF ETPYHQLLQHREEGRAQARTAWGKIENIKELK DGYLSQVVHRIQQLILKYNAIVMLEDLNFGFKR GRFKIEKQIYQKFEKALIHKLNYVVDKSTQADE LGGVRKAYQLTAPFESFEKLGKQSGVLFYVPA WNTSKIDPVTGFVDLLKPKYENLDKAQAFFNA FDSIHYNAQKNYFEFKVNLKQFAGLKAQAAQA EWTICSYGDERHVYQKKNAQQGETVIVNVTE ELKVLFAKNNIEVAQSVELKETICTQTQVDFFK RLMWLLQVLLALRYSSSKDKLDYILSPVANAQName Sequence SEQ ID NO: GEFFDSRHASVQLPQDSDANGAYHIALKGLW VIEQLKAADNLDKVKLAISNDDWLHFAQQKPY LA Additional examples of NUX proteins 179- NUX 244 Mb3Cas12a 245 HkCas12a 246 CeCas12a 247 BfCas12a 248 BsCas12a 249 EeCas12a 250 ArCas12a 251 LpCas12a 252 PrCas12a 253 PxCas12a 254 Francisella 255 novicida U112 Cpf1 (FnCpf1) Prevotella 256 disiens Cpf1 (PdCpf1) Acidaminococc 257 us sp. BV3L6 Cpf1 (AsCpf1) Lachnospirace 258 ae bacterium ND2006 Cpf1 (LbCpf1) Lachnospirace 259 ae bacterium MA2020 CPF1(Lb2Cpf1) Candidatus 260 Methanoplasm a termitum CPF1 (CmtCpf1) Moraxella 261 bovoculi 237 Cpf1 (MbCpf1) Porphyromona 262 s crevioricanis Cpf1 (PcCpf1) CRISPR 263 nuclease

[0084] As noted above, in some cases, the Cas effector protein (i.e., CRISPR nuclease) is a variant (is modified / mutated) (i.e., includes one or more amino acid mutations such as substitution(s), insertion(s), deletion(s) relative to a wildtype Cas effector protein). For example, In some cases, (e.g., in systems and / or methodsdisclosed herein) the CRISPR nuclease has one or more amino acid mutations relative to a wild type protein. Cas12a protein

[0085] As such, in some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with 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 80% or more 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) with 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 90% or more 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) with 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 95% or more 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) with 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.

[0086] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in any one of SEQ ID NOs: 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in any one of SEQ ID NOs: 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in any one of SEQ ID NOs: 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the amino acid sequence set forth in any one of SEQ ID NOs: 245-262. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 245-262.

[0087] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in SEQ ID NO:175. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in SEQ ID NO:175. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in SEQ ID NO:175. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more 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) with 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. NUX protein

[0088] The term “NUX” protein is used herein to mean a nuclease having Cas-like activity (an RNA-guided CRISPR effector protein) and having an amino acid sequence of any one of SEQ ID NOs: 1-86 and 176-244, or at least 70% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% percent identity, or 99% identity to an amino acid sequence of any one of SEQ ID NOs: 1-86 and 176-244. In some embodiments, the amino acid sequence of the Nux protein lacks identity or significant amino acid homology with certain known Cas nucleases. In some embodiments, the amino acid sequence of the NUX protein lacks identity or significant amino acid homology with Cas12a (Cpf1) protein. In some embodiments, the amino acid sequence of the NUX protein lacks identity or significant amino acid homology with a 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% with Cas12a (Cpf1) protein, SaCas9 or SpyCas9 proteins.

[0089] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with 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 80% or more 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) with 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 90% or more 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) with 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 95% or more 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) with 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.

[0090] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with 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 80% or more 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) with 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 90% or more 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) with 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 95% or more 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) with 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.

[0091] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in SEQ ID NO:176. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in SEQ ID NO:176. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in SEQ ID NO:176. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more 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) with 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.

[0092] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in SEQ ID NO:177. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in SEQ ID NO:177. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in SEQ ID NO:177. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more 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) with 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.

[0093] In some cases, the CRISPR nuclease comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in SEQ ID NO:178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 80% or more 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) with the amino acid sequence set forth in SEQ ID NO:178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 90% or more 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) with the amino acid sequence set forth in SEQ ID NO:178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 95% or more 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) with 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. Guide RNA

[0094] In some embodiments, a subject composition, system, or method includes a guide RNA (or a nucleic acid encoding the guide RNA). For example, In some cases, a subject composition, system, or method (e.g., a vector or vector system) includes an expression cassette that includes a promoter operably linked to a sequence encoding a 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 RNAs in eukaryotic cells.

[0095] A “guide RNA” is nucleic acid that binds to a Cas protein (e.g., a Class 2 CRISPR nuclease such as Cas12a), thus forming 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). It is to be understood that in some cases, a hybrid DNA / RNA can be made such that a guide RNA includes DNA bases in addition to RNA bases - but the term “guide RNA” is still used herein to encompass such hybrid molecules.

[0096] A guide RNA provides target specificity to the CRISPR complex by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid. Thus, a subject 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., targetDNA; and (ii) a constant region (e.g., a region that is adjacent to the guide sequence and binds to the Cas protein). A “constant region” can 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, transcription modulation, DNA methylation, histone modification) is in effect determined by the guide sequence of the guide RNA. CRISPR complex mediated events that take place at a location that is not a 100% match with the guide sequence is referred to herein as an off-target event.

[0097] A guide RNA can be referred to by the protein to which it corresponds. For example, when the 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 “Cas12a guide RNA.”

[0098] In some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” (e.g., a tracrRNA) and a “targeter” (e.g., a crRNA) and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two- molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule. For example, for some class 2 CRISPR / Cas systems the corresponding guide RNA is naturally a single molecule, while for other class 2 CRISPR / Cas systems the corresponding guide RNA is naturally two separate molecules (e.g., a crRNA and a tracrRNA) – and the two molecules (an activator, e.g., tracrRNA, and a targeter, e.g., a crRNA) can be covalently linked to one another, e.g., via chemical linkage or intervening nucleotides. When the guide RNA is one molecule, the guide RNA can be referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.” “Guide RNA” (or “gRNA”) is a generic term that encompasses dual guide and single guide formats.

[0099] The guide sequence has complementarity with (hybridizes to) a target sequence of the target nucleic acid (e.g., target DNA). In some cases, the guide sequence is 15- 28 nucleotides (nt) in length (e.g., 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16- 24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-21, 17-20, 17-19, 17-18, 18-26, 18-24, 18-22, 18-20, or 19-21 nt in length). In some cases, the guide sequence is 18-24 nucleotides (nt) in length. In some cases, the guide sequence is 17-18 nucleotides (nt) in length. In some cases, the guide sequence is at least 15 nt long (e.g., at least 16, 18, 20, or 22 nt long). In some cases, the guide sequence is at least 17 nt long. In some cases, the guide sequence is at least 18 nt long. In some cases, the guide sequence is at least 20 nt long. In some cases, the guide sequence is 20 nt long.

[0100] In some cases, the constant region (also referred to as a scaffold) of a guide RNA is 15 or more nucleotides (nt) in length (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 or more nt, 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 a guide RNA is 18 or more nt in length.

[0101] Guide RNAs with various modifications to increase efficiency relative to naturally existing guide RNAs (e.g., via chemical modifications, alterations in the spacer length, sequence modifications in the spacer or scaffold, fusion with additional DNA or RNA components, partial replacement with DNA, and the like) are known in the art and are readily available to one of ordinary skill in art. See, e.g., Moon et al., Trends Biotechnol. 2019 Aug;37(8):870-881, “Improving CRISPR Genome Editing by Engineering Guide RNAs”. The term “guide RNA” as used herein encompasses such modifications and any convenient guide RNA can be used with the methods and compositions disclosed herein (e.g., as part of a subject system – for example as RNA or as encoded by a subject nucleic acid). Acr / CRISPR nuclease combinations – “Systems”

[0102] In general, the term “system” is used herein when referring to the combination of an Acr polypeptide (described herein) (in protein and / or nucleic acid form) and a CRISPR nuclease (described herein) (in protein and / or nucleic acid form) that it inhibits. As noted above, in some cases, the Acr polypeptide and the CRISPR nuclease do not naturally occur together. In some cases, a subject system further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0103] In some embodiments (e.g., in systems and / or methods disclosed herein), the Acr polypeptide and the CRISPR nuclease do not naturally occur together (i.e., they are heterologous to one another). In some cases, the Acr polypeptide and / or the CRISPR nuclease includes a variant amino acid sequence (e.g., an 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 exist together in nature (e.g., the Acr polypeptide may be from a phage that does not naturally infect the bacterial species which harbors the CRISPR nuclease).

[0104] Because either protein can be provided as a nucleic acid (DNA or RNA) or in protein form, subject systems can include any combination thereof. For example, in some embodiments, a subject system includes 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, a subject system includes the CRISPR nuclease and a nucleic acid encoding the Acr polypeptide. In some cases, a subject system includes the Acr polypeptide and a nucleic acid encoding the CRISPR nuclease. In some cases, a subject system includes a nucleic acid encoding the Acr polypeptide and a nucleic acid encoding the CRISPR nuclease. In some such cases, both the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding 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 CRISPR nuclease are on different nucleic acids (e.g., different vectors). See elsewhere herein for a discussion of nucleic acids (e.g., vectors, promoters, translational control elements, and the like). The Acr polypeptide and the CRISPR nuclease of a subject system can be any of those described herein, in any desired combination. Acr protein (SEQ ID NOs: 165-169) plus CRISPR nuclease (Cas12a or NUX)

[0105] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-168; and the CRISPRnuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0106] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-168; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more,99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0107] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes the amino acid sequence set forth as SEQ ID NO: 175. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 175. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-168; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 175. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 175. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0108] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-168; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0109] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 176- 178. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100%sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 176-178. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-168; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 176-178. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 176-178. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0110] In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 176. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-167; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% ormore, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 176.In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO.: 165 or 167; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 176. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 176. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0111] In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 177. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0112] In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 165-169; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more,98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 178. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 178. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 165; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 178. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA. Acrs (SEQ ID NOs: 126-132) plus CRISPR nuclease

[0113] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-262. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0114] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 175-263. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-263. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0115] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0116] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth as SEQ ID NO: 175. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% ormore, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 175. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0117] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0118] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 176- 178. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 176-178. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0119] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes the amino acid sequence set forth as SEQ ID NO: 263. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% ormore, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 126-132; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 263. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA. Acrs (SEQ ID NOs: 264-267) plus CRISPR nuclease

[0120] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 175-263. In some cases, the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 175-263. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0121] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 175 and 245-262. In some cases,a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0122] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth as SEQ ID NO: 175. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 175. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0123] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86 and 176-244. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0124] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 1-86, 176-244, and 263. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ IDNOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 1-86, 176-244, and 263. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0125] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth set forth as any one of SEQ ID NOs.: 176- 178. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 176-178. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.

[0126] In some cases, the Acr polypeptide of a subject system includes the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes the amino acid sequence set forth as SEQ ID NO: 263. In some cases the Acr polypeptide of a subject system includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as any one of SEQ ID NOs.: 264-267; and the CRISPR nuclease includes an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence set forth as SEQ ID NO: 263. In some cases, a system described in this paragraph further includes a guide RNA (that guides the CRISPR nuclease to a target sequence) or a nucleic acid encoding the guide RNA.Fusions

[0127] In some cases, a protein (e.g., an Acr protein and / or a CRISPR nuclease such as Cas12a or a NUX protein) is fused to one or more heterologous polypeptides (also referred to herein as fusion partners) (e.g., one or more NLSs, a protein tag, and the like). Suitable fusion partners include but are not limited to: (i) subcellular 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 to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like); (ii) protein tags, e.g., for ease of tracking and / or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like, MBP, CBP, strep tag, GST, HA, FLAG, poly(His), Myc, V5, Spot, NE, AviTag, and the like); and (iii) polypeptides that provide for increased or decreased stability (e.g., a degron, which In some cases, is controllable, e.g., a temperature sensitive or drug controllable degron sequence).

[0128] A subject protein (e.g., an Acr protein and / or a CRISPR nuclease such as Cas12a or a NUX protein) can have multiple (1 or more, 2 or more, 3 or more, etc.) fusion partners in any combination. As an illustrative example, a subject protein can have a fusion partner that provides for tagging (e.g., GFP), and can also have a subcellular localization sequence (e.g., one or more NLSs). In some cases, such a fusion protein might also have a tag for ease of tracking and / or purification. As another illustrative example, a subject protein (e.g., an Acr protein such as a subject engineered Acr protein) can have one or more NLSs (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4, or 5 NLSs). In some cases, a fusion partner is located at or near the C-terminus (e.g., within about 50 amino acids of the C-terminus), near the N- terminus (e.g., within about 50 amino acids of the N-terminus), or at both the N-terminus and C-terminus.

[0129] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the 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 with 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); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 92) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 93) and PPKKARED (SEQ ID NO: 94 of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 95) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 96) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 97) and PKQKKRK (SEQ ID NO: 98) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 99) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 100) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 101) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 102) of the steroid hormone receptors (human) glucocorticoid. In some cases, and NLS has the sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO: 125). In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of the Cas protein in a detectable amount in the nucleus of a eukaryotic cell.

[0130] In some cases, a fusion partner includes a "Protein Transduction Domain" or PTD (also known as a CPP – cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and / or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a polypeptide and in some embodiments, a PTD is covalently linked to the carboxyl terminus of a polypeptide. In some cases, the PTD is inserted internally at a suitable insertion site. In some cases, a subject Cas protein includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:103); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a 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); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (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); an arginine homopolymer of from 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). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane. Linkers (e.g., for fusion partners)

[0131] In some embodiments, a subject Cas protein can fused to a fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use.

[0132] Examples of linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 115), GGSGGSn (SEQ ID NO: 116), and GGGSn (SEQ ID NO: 117 where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers. Exemplary linkers can compriseamino 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. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. Translational Control Elements

[0133] The present disclosure provides compositions, systems, and methods that include one or more nucleic acids, where the one or more nucleic acids encode an Acr protein and a CRISPR nuclease – where the Acr protein is an inhibitor of the Cas protein (e.g., Cas effector protein / CRISPR nuclease), such as inhibiting off-target editing more preferentially than on-target editing. 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).

[0134] Subject nucleic acids can include a translational control element that is operably linked to the Acr protein or the Cas protein (e.g., CRISPR nuclease such as the Cas12a, NUX protein, or variant thereof), or both - in order to achieve a desired balance (expression level ratio) between the two proteins. For example, In some cases, a subject nucleic acid includes a translational control element that is operably linked to (and therefore regulates / modulates translation of) a sequence encoding the Acr protein. In some cases, a subject nucleic acid includes a translational control element that is operably linked to (and therefore regulates / modulates translation of) a sequence encoding the Cas protein. In some cases, the sequence encoding the Acr protein and the sequence encoding the Cas protein are both operably linked to a translational control element, e.g., independently (in which case the two control elements can be the same or different).

[0135] In some cases, the sequence encoding the Acr protein and the sequence encoding the Cas protein are both operably linked to the same translational control element (e.g., IRES element, 2A peptide encoding sequence) such that the sequences are part of a polycistronic transcript. As such, In some cases, a subject translational control element is a polycistronic linker. In other words, In some cases, the translational control element promotes (causes) the production of independent gene products (e.g., the Acr protein and the CRISPR nuclease) from the same transcript.

[0136] Thus, In some cases, a translational control element links a first protein coding sequence (e.g., a sequence encoding an Acr protein) with a second protein coding sequence (e.g., a sequenced encoding a CRISPR nuclease) such that the first and second proteins (e.g., the Acr protein and CRISPR nuclease) are encoded by a polycistronic sequence. In such cases, both protein sequences would therefore both be operably linked to the same promotor, and the RNA transcribed therefrom would include both protein-coding sequences in addition to the sequence encoded by the translational control element.

[0137] As described in more detail below, In some cases, more than one translational control element (e.g., IRES element, 2A peptide, non-AUG start codon) is used to control expression of a given protein (e.g., an Acr protein, a CRISPR nuclease such as Cas12a or a Nux protein). Any convenient combination of translational control elements can be used. 2A peptide

[0138] One non-limiting example of a translational control element that can function as a polycistronic linker and facilitate the production of separate protein products (e.g., two separate proteins) from the same single RNA transcript is a 2A peptide sequence.

[0139] By a “2A peptide” it is meant a small peptide sequence (usually 18–25 amino acids although several of such sequences can be placed in tandem) that allows for expression (translation) of discrete protein products from a single RNA transcript (e.g., through a self-“cleaving” event often referred to as “ribosome skipping” – although the disclosure herein does not rely on and is not bound by the mechanism of action), even though the separate proteins are encoding as part of the same open reading frame (ORF). 2A peptides are readily identifiable by their consensus motif (DXEXNPGP, sometimes described as DVEXNPGP) and their ability to promote protein cleavage / skipping. Any convenient 2A peptide sequence may be used in a subject nucleic acid. Examples of 2A peptides include, but are not limited to 2A peptides from a virus such as foot-and-mouth disease virus (F2A), equine Rhinitis A virus (E2A), porcine teschovirus-1 (P2A) or Thosea asigna virus (T2A). See, e.g., 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, as they relate to 2A peptides, are incorporated herein by reference.

[0140] Typically, a subject 2A peptide coding sequence will be positioned so as to regulate the expression (translation) of a subject protein (e.g., Acr protein or Cas protein) – and as such will be positioned 5’ of (usually immediately 5’ of) and in frame with the protein coding sequence which it regulates. FIG.9 provides non-limiting illustrative examples of embodiments in which a 2A peptide coding sequence is positioned in different ways. In some cases, a 2A peptide sequence is position 5’ of (and usually immediately 5’ of) a Cas protein coding sequence. In some cases, a 2A peptide sequence is position 5’ of (and usually immediately 5’ of) an Acr protein coding sequence.

[0141] In some cases, the Cas-encoding sequence and the Acr-encoding sequence are operably linked to the same promoter (encoded by a polycistronic sequence) and are positioned in tandem, with a 2A peptide sequence positioned between them (see, e.g., FIG.9A and 9B). In some such cases, the Cas protein encoding sequence is positioned 5’ of the Acr encoding sequence, and the 2A peptide encoding sequence is therefore 3’ of the Cas sequence and 5’ of the Acr sequence. In other such cases, the Acr protein encoding sequence is positioned 5’ of the Cas encoding sequence, and the 2A peptide encoding sequence is therefore 3’ of the Acr sequence and 5’ of the Cas sequence.

[0142] In some cases, the Cas-encoding sequence and the Acr-encoding 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.9C and 9D). The first and second promoters (labeled “P1” and “P2” in the figure) can be different from one another or can be the same (i.e., can be a copy of the same promoter). In some such cases, the 2A peptide sequence regulates (and is therefore positioned 5’ of) the Acr sequence. In other cases, the 2A peptide sequence regulates (and is therefore positioned 5’ of) the Cas sequence. In some cases, in which the Cas-encoding sequence and the Acr-encoding sequence are transcribed as separate sequences, each one is regulated by a 2A peptide sequence (and each is therefore positioned 3’ of the 2A peptide sequence).

[0143] In some embodiments in which the Cas-encoding sequence and the Acr- encoding sequence are transcribed as separate sequences, a “spacer” protein coding sequence is used 5’ of the 2A peptide sequence such that the ‘spacer’ sequence is transcribed as part of a polycistronic sequence with the protein sequence being regulated. For example, in FIG.9C, a Cas protein coding sequence and an Acr protein coding sequence are operably linked to different promoters (P1 and P2). A spacer sequence (labeled as “X” in the figure) is positioned 5’ of a 2A peptide sequence, whichis 5 of an Acr coding sequence and therefore the spacer sequence and the Acr sequence are transcribed as part of the same RNA. However, presence of the 2A peptide sequence results in production of the Acr protein as a separate protein. Likewise, in FIG.9D, a Cas protein coding sequence and an Acr protein coding sequence are again operably linked to different promoters (P1 and P2). In this example, a spacer sequence (labeled as “X” in the figure) is positioned 5’ of a 2A peptide sequence, which is 5’ of a Cas coding sequence – and therefore the spacer sequence and the Cas sequence are transcribed as part of the same RNA. However, presence of the 2A peptide sequence in this RNA results in the production of the Cas protein as a separate protein.

[0144] A ‘spacer’ protein can be any desired sequence – as its purpose is to simply provide a sequence to be translated that is 5’ of (N-terminal to) the 2A peptide sequence. A spacer sequence can be any convenient length, from very short to encoding an entire protein sequence. In some cases, the spacer is 2 or more amino acids long (e.g., 3 or more, 4 or more 5 or more, 10 or more, or 20 or more amino acids). In some cases, the spacer has a length of from 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 sequence include, but are not limited to: linker sequences, repeated single amino acids (e.g., AAAA), random sequences, fragments of proteins, and marker proteins (e.g., a fluorescent protein such as GFP, YFP, CFP, RFP, and the like, a drug selectable protein marker, an enzyme such as beta-galactosidase, etc.).

[0145] Examples of 2A peptide sequences include, but are not limited to those set forth as SEQ ID NOs: 133-138.

[0146] 2A peptide sequences can be used in tandem, and multiple different 2A peptide sequences can be positioned one after another, in any desired combination (see “E2A- F2A” and “T2A-E2A-F2A” above as non-limiting examples). Thus, In some cases, a 2A peptide sequence is selected from the group consisting of: P2A, F2A, E2A, T2A, and any combination thereof. In some embodiments, a translational control element encodes 2 or more 2A peptides in tandem (e.g.., 3 or more, 4 or more, or 5 or more). In some embodiments, a translational control element encodes 2, 3, 4, or 52A peptides in tandem. In some embodiments, a translational control element encodes one 2A peptide.In some cases, a 2A peptide sequence comprises an amino acid sequence having 70% or more 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) with the amino acid sequence set forth in any one of SEQ ID NOs: 133-138. = IRES

[0147] In some embodiments herein, a translational control element is an internal ribosome entry site (IRES) sequence. By an “internal ribosome entry site,” or “IRES” it is meant a nucleotide sequence that allows for the initiation of protein translation within the interior of 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 in a bicistronic eukaryotic mRNA molecule, it can drive translation of the downstream protein-coding region independently of the 5'-cap structure at the 5' end of the mRNA molecule, i.e. in front of the upstream protein coding region. In such a setup, both proteins are produced in the cell. The protein located in the first cistron is synthesized by the cap-dependent initiation mechanism, while translation initiation 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 genomes 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, which function as part of RNA molecules, will have correlative sequences in the encoding DNA molecules, e.g., RNA sequence 5’-uuacuggc-3’ would correspond to DNA sequence 5’-ttactggc-3’, and vice versa”. The term “IRES sequence” or simply “IRES” is used herein to refer to either sequence.

[0148] Any convenient IRES may be employed in the subject compositions, sytems, and methods. Examples of IRES sequences include but are not limited to those listed in FIG.13A-13D (SEQ ID NOs: 139-159). One of ordinary skill in the art will recognize that when a subject system is to be used for expressing Cas and Acr proteins in non-animal cells (e.g., plants / plant cells), they should select a convenient IRES sequence appropriate for the desired cell type (e.g., an IRES from Triticum mosaic virus (TriMV)). See, e.g., Urwin et al., Plant J.2000 Dec;24(5):583-9 as well U.S. Patent Nos. 8,772,465; 9,879,271, each of which is incorporated by reference with respect to teachings related to using IRES sequences in plant.

[0149] In some cases, an IRES sequence comprises a nucleotide sequence having 70% or more sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% ormore, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the nucleotide sequence set forth in any one of SEQ ID NOs: 139-159.

[0150] In some cases, an 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 mutant 5 (SEQ ID NO: 140), EMCV mutant 10 (SEQ ID NO: 141) EMCV mutant 15 (SEQ ID NO: 142) and EMCV mutant 21 (SEQ ID NO: 143) [also see, e.g., international patent publication WO2022072673].

[0151] Typically, a subject IRES sequence will be positioned in a subject nucleic acid so as to regulate the expression (translation) of a subject protein (e.g., Acr protein or Cas protein) – and as such will be positioned 5’ of (usually immediately 5’ of) the protein coding sequence which it regulates. FIG.10 provides non-limiting illustrative examples of embodiments in which and IRES sequence is positioned in different ways. In some cases, an IRES sequence is positioned 5’ of (and usually immediately 5’ of) a Cas protein coding sequence. In some cases, an IRES sequence is positioned 5’ of (and usually immediately 5’ of) an Acr protein coding sequence.

[0152] In some cases, the Cas-encoding sequence and the Acr-encoding sequence are operably linked to the same promoter (encoded by a polycistronic sequence) and are positioned in tandem, with an IRES sequence positioned between them (see, e.g., FIG. 10, first and second examples). In some such cases, the Cas protein encoding sequence is positioned 5’ of the Acr encoding sequence, and the IRES sequence is therefore 3’ of the Cas sequence and 5’ of the Acr sequence. In some such cases, the Acr protein encoding sequence is positioned 5’ of the Cas encoding sequence, and the IRES sequence is therefore 3’ of the Acr sequence and 5’ of the Cas sequence.

[0153] In some cases, the Cas-encoding sequence and the Acr-encoding 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.10,C-F). The first and second promoters (labeled “P1” and “P2” in the figure) can be different from one another or can be the same (i.e., can be a copy of the same promoter). In some such cases, the IRES sequence regulates (and is therefore positioned 5’ of) the Acr sequence. In other cases, the IRES sequence regulates (and is therefore positioned 5’ of) the Cas sequence. In some cases, in which the Cas-encoding sequence and the Acr-encoding sequence are transcribed as separate sequences, each one is regulated by an IRES sequence (and each is therefore positioned 3’ of an IRES sequence).

[0154] In some embodiments in which the Cas-encoding sequence and the Acr- encoding sequence are transcribed as separate sequences, a “spacer” protein coding sequence is used 5’ of the IRES sequence such that the ‘spacer’ sequence is transcribed as part of a polycistronic sequence with the protein sequence being regulated (see, e.g., the FIG.10 E and F). For example, in FIG.10E, a Cas protein coding sequence and an Acr protein coding sequence are operably linked to different promoters (P1 and P2). A spacer sequence (labeled as “X” in the figure) is positioned 5’ of an IRES sequence, which is 5’ of an Acr coding sequence – and therefore the spacer sequence and the Acr sequence are transcribed as part of the same RNA, but the presence of the IRES sequence causes the Acr protein to be produced as a separate protein. Likewise, in FIG.10F, a Cas protein coding sequence and an Acr protein coding sequence are again operably linked to different promoters (P1 and P2). In this example, a spacer sequence (labeled as “X” in the figure) is positioned 5’ of an IRES sequence, which is 5’ of a Cas coding sequence – and therefore the spacer sequence and the Cas sequence are transcribed as part of the same RNA, but the presence of the IRES sequence causes the Cas protein to be produced as a separate protein.

[0155] A ‘spacer’ protein can be any desired sequence – as its purpose is to simply provide a sequence to be translated that is 5’ of the IRES sequence. A spacer sequence can be any convenient length, from very short to encoding an entire protein sequence. In some cases, the spacer is 2 or more amino acids long (e.g., 3 or more, 4 or more 5 or more, 10 or more, or 20 or more amino acids). In some cases, the spacer has a length of from 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 sequence include, but are not limited to: linker sequences, repeated single amino acids (e.g., AAAA), random sequences, fragments of proteins, and marker proteins (e.g., a fluorescent protein such as GFP, YFP, CFP, RFP, and the like, a drug selectable protein marker, an enzyme such as beta-galactosidase, etc.).

[0156] In some cases, in which the Acr encoding sequence and the Cas encoding sequences are operably linked to separate promoters, a spacer sequence is not used (see, e.g., FIG.10C and D). Start codon

[0157] In some embodiments herein, a translational 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 meant to include any non-AUG polynucleotide (typically a triplet) that functions as a start site for translation initiation with reduced efficiency relative to that of an AUG start codon. Examples of naturally occurring alternate start codon usage 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. In general, non-AUG start codons have decreased translation efficiencies compared to that of an AUG.

[0158] In some cases, a non-AUG start codon is used as the initiation codon for the sequence encoding the Acr protein. In some cases, a non-AUG start codon is used as the initiation codon for the sequence encoding the Cas protein (e.g., CRISPR nuclease). In some cases, a non-AUG start codon (used with the Acr sequence or with the Cas sequence) is any one of: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, a non-AUG start codon (used with the Acr sequence or with the Cas sequence) is any one of: CUG, GUG, ACG, AUA, or UUG. For example, In some cases, a non-AUG start codon used with the Acr sequence is any one of: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, a non-AUG start codon used with the Acr sequence is any one of: CUG, GUG, ACG, AUA, or UUG. As another example, In some cases, a non-AUG start codon used with the Cas sequence is any one of: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC, or AUU. In some cases, a non-AUG start codon used with the Cas sequence is any one of: CUG, GUG, ACG, AUA, or UUG. In some cases, a non-AUG start codon used with the Acr sequence is CUG. In some cases, a non-AUG start codon used with the Acr sequence is GUG. In some cases, a non-AUG start codon used with the Acr sequence is ACG. In some cases, a non-AUG start codon used with the Cas sequence is CUG. In some cases, a non-AUG start codon used with the Cas sequence is GUG. In some cases, a non-AUG start codon used with the Cas sequence is ACG.

[0159] The translation efficiency of a non-AUG start codon can also be affected by its sequence context; for example, in eukaryotic cells an optimal Kozak consensus sequence has been reported to have a positive effect on translation initiation 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 GCCRCCATGG (SEQ ID NO:160), where the start codon ATG (AUG in RNA) is just prior to the final “G”, the A of the ATG start codon is designated as the +1 position, and "R" at position -3 is a purine (A or G). The two most highly conserved positions are a purine, usually an A, at - 3 and a G at +4 (Kozak (1991) J Cell Biol 115(4): 887-903). In some cases, a subjectnon-AUG start codon (e.g., any of those discussed above) is coupled with an impaired Kozak sequence (i.e., a Kozak sequence that does not conform to the consensus.

[0160] For examples of the above, see, e.g., Kearse and Wilusz, Genes Dev.2017 Sep 1;31(17):1717-1731; U.S. Patent Application Publication Nos. US20060172382 and US20060141577; and U.S. Patent Nos.5,648,267; 5,733,779; 8,828,976; 10,030,252; 10,317,329; the disclosures of which, as they relate to Kozak sequences and non-AUG start codons (as well as assays associated therewith), are incorporated herein by reference. One of skill in the art will recognize that the sequences described herein as DNA will have correlative sequences as RNA molecules, e.g., DNA sequence ATG would correspond to RNA sequence AUG, and vice versa.

[0161] Typically, a subject non-AUG initiation codon will be positioned in a subject nucleic acid so as to regulate the expression (translation initiation) of a subject protein (e.g., Acr protein or Cas protein) – and as such will be positioned 5’ of (usually immediately 5’ of) and in frame with the protein coding sequence which it regulates. In some cases, (e.g., when a non-AUG start codon is used as the initiation codon for the Acr encoding sequence), the sequence encoding the Acr protein does not include its native AUG start codon. In some such cases, the sequence encoding the Acr protein does not include an AUG codon. In some cases, (e.g., when a non-AUG start codon is used as the initiation codon for the Cas encoding sequence), the sequence encoding the CRISPR nuclease does not include its native AUG start codon. In some cases, the sequence encoding the CRISPR nuclease does not include an AUG codon. In some cases, (e.g., when a non-AUG start codon is used as the initiation codon for the Cas- encoding or Acr-encoding sequence), the sequence encoding the subject protein, i.e., the CRISPR nuclease or the Acr protein, is codon optimized as a whole or in part, to avoid having an out-of-frame AUG that could direct translational machinery to an incorrect reading frame contained in the nucleic acid encoding the Cas or Acr protein (i.e., a reading frame that does not encode the subject 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 so as not to include an out-of-frame AUG.

[0162] In some cases, the Cas-encoding sequence and the Acr-encoding 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 (labeled “P1” and “P2” in the figure) can be different from one another or can be the same (i.e., can be a copy of the same promoter).

[0163] In some embodiments, more than one translational control element can be used to control expression of a protein (e.g., Acr protein, CRISPR nuclease). For example, In some cases, more than one (e.g., two, two or more, three) translational control elements are used to control expression of the Acr protein. In some cases, more than one (e.g., two, two or more, three) translational control elements are used to control expression of the CRISPR nuclease. As such, In some cases, one or more (e.g., two, two or more, three) translational control elements (e.g., 2A peptide. IRES, non-AUG start codon) are used to control expression of the Acr protein and / or the CRISPR nuclease. In some cases, one or more (e.g., two, two or more, three) translational control elements (e.g., 2A peptide, IRES, non-AUG start codon) are used to control expression of the Acr protein. In some cases, one or more (e.g., two, two or more, three) translational control elements (e.g., 2A peptide. IRES, non-AUG start codon) are used to control expression of the CRISPR nuclease. Promoters

[0164] The compositions, methods and systems of ACRs and nucleases provided herein include expression cassettes, such as on one or more vectors, which expression cassettes include promoters to drive expression of the genes encoding a Cas protein (e.g., a Class 2 effector protein / CRISPR nuclease) and / or an Acr protein, and / or a guide RNA.

[0165] In some cases, an Acr coding sequence and a CRISPR nuclease sequence are operably linked to the same promoter and are therefore transcribed as part of the same RNA. In other cases, an Acr coding sequence and a CRISPR nuclease sequence are operably linked to different promoters. For example, in some cases, an Acr coding sequence is operably linked to a first promoter and a CRISPR nuclease sequence is operably linked to a second promoter. In some such cases the first and second promoters 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 expression of one protein and another controlling expression of the other). In other such cases the first and second promoters are different promoters.Promoter types

[0166] A variety of promoter types can be used, e.g., to control the expression of an Acr protein and / or CRISPR nuclease (e.g., Cas12a or NUX protein). A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / ”ON” state), it may be an inducible promoter (i.e., a promoter whose state, active / ”ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and / or it may be a temporally restricted promoter, e.g., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process.

[0167] Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any convenient organism. Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al. , Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res.2003 Sep 1;31(17)), a human H1 promoter (H1), and the like. Pol III promoters such as U6, enhanced U6, and H1, are generally used to express non-coding RNAs such as guide RNAs.

[0168] Examples of inducible promoters include, but are not limited to, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; an estrogen receptor; an estrogen receptor fusion; an estrogen analog; IPTG; etc.

[0169] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsivepromoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid- regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0170] Examples of suitable promoters include but are not limited to the following: Mammalian (Pol II) promoters (for nuclease and Acrs) • retroviral Rous sarcoma virus (RSV) • LTR promoter (optionally with the RSV enhancer), • cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) • SV40 promoter • dihydrofolate reductase promoter, • beta.-actin promoter, • phosphoglycerol kinase (PGK) promoter, • EF1.alpha. (EF1a) promoter. • MMLV LTR promoters • HIV LTR promoters, MCMV LTR promoters, • MND, • Ubc, • CAG, • HSV TK promoter, • fos promoter, • E2F promoter • polyoma virus • adenovirus, fowlpox virus • bovine papilloma virus • avian sarcoma virus Eukaryotic tissue-specific • Bowman et al., 1995 Proc. Natl. Acad. Sci. USA 92,12115-12119 describe a brain-specific transferrin promoter; • synapsin I promoter is neuron specific (Schoch et al., 1996 J. Biol. Chem.271, 3317-3323);• necdin promoter is post-mitotic neuron specific (Uetsuki et al., 1996 J. Biol. Chem.271, 918-924); • neurofilament light promoter is neuron specific (Charron et al., 1995 J. Biol. Chem.270, 30604-30610); • acetylcholine receptor promoter is neuron specific (Wood et al., 1995 J. Biol. Chem.270, 30933-30940); • potassium channel promoter is high-frequency firing neuron specific (Gan et al., 1996 J. Biol. Chem 271, 5859-5865); • chromogranin A promoter is neuroendocrine cell specific (Wu et al., 1995 A. J. Clin. Invest.96, 568-578); • Von Willebrand factor promoter is brain endothelium specific (Aird et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4567-4571); • flt-1 promoter is endothelium specific (Morishita et al., 1995 J. Biol. Chem.270, 27948-27953); • preproendothelin-1 promoter is endothelium, epithelium and muscle specific (Harats et al., 1995 J. Clin. Invest.95, 1335-1344); • GLUT4 promoter is skeletal muscle specific (Olson and Pessin, 1995 J. Biol. Chem.270, 23491-23495); • Slow / fast troponins promoter is slow / fast twitch myofibre specific (Corin et al., 1995 Proc. Natl. Acad. Sci. USA 92, 6185-6189); • Actin promoter is smooth muscle specific (Shimizu et al., 1995 J. Biol. Chem. 270, 7631-7643); • Myosin heavy chain promoter is smooth muscle specific (Kallmeier et al., 1995 J. Biol. Chem.270, 30949-30957); • E-cadherin promoter is epithelium specific (Hennig et al., 1996 J. Biol. Chem. 271, 595-602); • cytokeratins promoter is keratinocyte specific (Alexander et al., 1995 B. Hum. Mol. Genet.4, 993-999); • transglutaminase 3 promoter is keratinocyte specific (J. Lee et al., 1996 J. Biol. Chem.271, 4561-4568); • bullous pemphigoid antigen promoter is basal keratinocyte specific (Tamai et al., 1995 J. Biol. Chem.270, 7609-7614); • keratin 6 promoter is proliferating epidermis specific (Ramirez et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4783-4787); • collagen 1 promoter is hepatic stellate cell and skin / tendon fibroblast specific (Houglum et al., 1995 J. Clin. Invest.96, 2269-2276); • type X collagen promoter is hypertrophic chondrocyte specific (Long & Linsenmayer, 1995 Hum. Gene Ther.6, 419-428); • Factor VII promoter is liver specific (Greenberg et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12347-1235); • fatty acid synthase promoter is liver and adipose tissue specific (Soncini et al., 1995 J. Biol. Chem.270, 30339-3034); • carbamoyl phosphate synthetase I promoter is portal vein hepatocyte and small intestine specific (Christoffels et al., 1995 J. Biol. Chem.270, 24932-24940);• the Na--K--Cl transporter promoter is kidney (loop of Henle) specific (Igarashi et al., 1996 J. Biol. Chem.271, 9666-9674); • scavenger receptor A promoter is macrophages and foam cell specific (Horvai et al., 1995 Proc. Natl. Acad. Sci. USA 92, 5391-5395); • glycoprotein IIb promoter is megakaryocyte and platelet specific (Block & Poncz, 1995 Stem Cells 13, 135-145); • yc chain promoter is hematopoietic cell specific (Markiewicz et al., 1996 J. Biol. Chem.271, 14849-14855); • CD11b promoter is mature myeloid cell specific (Dziennis et al., 1995 Blood 85, 319-329). Cell types

[0171] Host cells (also referred to as “target cells”) can be ex vivo (e.g., fresh isolate - early passage), in vivo, or in culture in vitro (e.g., immortalized cell line). In some cases, the targeted nucleic acid is chromosomal (e.g., the host cell’s genome) and In some cases, the targeted nucleic acid is from a pathogen, e.g., the genome of a pathogen within the host cell. Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in culture.

[0172] Suitable host cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell of a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell of a mammal (e.g., a cell of a rodent; a cell of a human; a cell of a non- human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuña, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stemcell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

[0173] Suitable host cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single- cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell of a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell of a mammal (e.g., a cell of a rodent; a cell of a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuña, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4- cell, 8-cell, etc. stage zebrafish embryo; etc.).

[0174] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

[0175] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells,bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0176] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0177] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0178] Adult stem cells are resident in differentiated tissue, but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including 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.

[0179] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non- human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0180] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte 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 to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0181] In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (includingoligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0182] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No.5,736,396, which describes isolation of human MSC.

[0183] Promoters and translation control elements such as those described above can be used to achieve delivery of a subject Acr protein and / or a CRISPR nuclease at different levels relative to one another. For example, in some embodiments, the goal is to deliver the CRISPR nuclease and the Acr polypeptide at a 1:1 ratio. In some embodiments, the goal is to deliver the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) in a range of from 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, a subject system 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, e.g., 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.

[0184] In some embodiments, the goal is to deliver the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) in a range of from 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, a subject system 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, e.g., 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. “Protospacer adjacent motif” (PAM)

[0185] A wild type CRISPR nuclease normally has nuclease activity that cleaves a target nucleic acid (e.g., a double stranded DNA (dsDNA)) at a target site defined by (i) the region of complementarity between the guide sequence of the guide RNA and the target nucleic acid; and (ii) a short motif referred to as the “protospacer adjacent motif” (PAM) in the target nucleic acid.

[0186] For additional information related to CRISPR based programmable gene editing tools (e.g., CRISPR / Cas RNA-guided proteins, CRISPR / Cas guide RNAs, and PAMs)refer to, for example, to the following review papers 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. Vectors

[0187] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication and / or expression of the attached segment in a cell. An “expression cassette” comprises a DNA sequence (coding or non-coding) operably linked to a promoter. In some cases, a subject vector is a viral vector (e.g., AAV, lentivirus, adenovirus). In some cases, a subject vector includes an origin of replication (e.g., can be a plasmid).

[0188] In some cases, both an Acr protein and its target Cas protein (the protein that the Acr inhibits) is present in a single vector – which ensures that all cells receiving the Cas protein (e.g., an endonuclease such as Cas9, Cas12a, and the like) will also express the Acr “off-switch”. Whether or not both proteins (Acr and Cas) are present on the same nucleic acid, the translation of one or both proteins can be regulated by a translational control element in order to achieve a proper balance (expression level ratio) between the two proteins.

[0189] Vectors may be provided directly to a target host cell (target cell). In other words, the cells are contacted with vectors comprising the subject nucleic acids (e.g., recombinant expression vectors) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors (e.g., adeno-associated virus (AAV)).

[0190] In some embodiments, a subject vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No.7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.

[0191] Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591594, 1996; Srivastava in 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 (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0192] In some embodiments a subject vector is an AAV vector. By adeno-associated virus, or “AAV” it is meant the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise, for example, 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, a hybrid AAV (i.e., an AAV comprising a capsid protein of one AAV subtype and genomic material of another subtype), an AAV comprising a mutant AAV capsid protein or a chimeric AAV capsid (i.e. a capsid protein with regions or domains or individual amino acids that are derived from two or more different serotypes of AAV, e.g. AAV-DJ, AAV-LK3, AAV-LK19). “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

[0193] In some embodiments a subject vector is an integrative vector, e.g., integrates into the genome of a target cell.

[0194] By a “recombinant AAV vector”, or "rAAV vector" it is meant an AAV virus or AAV viral chromosomal material comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a nucleic acid sequence of interest to be integrated into the cell following the subject methods. In general, theheterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). In some instances, the recombinant viral vector also comprises viral genes important for the packaging of the recombinant viral vector material. By "packaging" it is meant a series of intracellular events that result in the assembly and encapsidation of a viral particle, e.g. an AAV viral particle. Examples of nucleic acid sequences important for AAV packaging (i.e., “packaging genes”) include the AAV "rep" and "cap" genes, which encode for replication and encapsidation proteins of adeno-associated virus, respectively. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0195] A “viral particle” refers to a single unit of virus comprising a capsid encapsidating a virus-based polynucleotide, e.g. the viral genome (as in a wild type virus), or, e.g., the subject targeting vector (as in a recombinant virus). An "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0196] A rAAV virion can be constructed using methods that are well known in the art. See, e.g., 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, the heterologous sequence(s) can be directly inserted into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published Jan.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.

[0197] In order to produce rAAV virions, an AAV expression vector can be introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., 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 micro-injection 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 transduction (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).

[0198] Suitable cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. For example, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol.36:59), and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a convenient platform in which to produce rAAV virions. Methods of producing an AAV virion in insect cells are known in the art, and can be used to produce a subject rAAV virion. See, e.g., U.S. Patent Publication No. 2009 / 0203071; U.S. Patent No.7,271,002; and Chen (2008) Mol. Ther.16:924.

[0199] AAV virus that is produced may be replication competent or replication- incompetent. A "replication-competent" virus (e.g. a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (e.g., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replicationcompetent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector.

[0200] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).

[0201] A detailed discussion of delivery methods and formulations is presented elsewhere herein.

[0202] As noted elsewhere herein, proteins may instead be provided to cells as RNA (e.g., an RNA comprising the translational control element as discussed elsewhere herein). Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA.

[0203] 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 (as opposed to a nucleic acid). For example, one protein coding sequence (such as an Acr protein coding sequence) can be introduced as nucleic acid (RNA or DNA) where the protein coding sequence is operably linked to a translational control element; and the other protein (e.g., a Cas effector) is introduced as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. Examples of linkers are discussedelsewhere herein in a different context, but such linkers can be used in any convenient context including this one.

[0204] In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. METHODS

[0205] The present disclosure provides a method for nucleic acid targeting (e.g., modifying a target nucleic acid, e.g., for cleaving DNA such as in genome editing applications), where the Acr polypeptide and the CRISPR nuclease are delivered to the target nucleic acid as a subject system (e.g., in nucleic acid or protein form). In some cases, the contact is in a cell-free environment in vitro. In some cases, the contacting occurs in a cell, which can be ex vivo, in vivo, or in vitro (e.g., a cell in culture). Thus, in some embodiments a subject method includes introducing an Acr protein (or a nucleic acid encoding it) and a CRISPR nuclease (or a nucleic acid encoding it) into a host cell, whereby the ratio of on-target to off-target nucleic acid activity (e.g., cleavage) that results from said introducing is increased relative to the ratio of on-target to off-target nucleic acid targeting that would result in the absence of the Acr protein. In some cases, the ratio of on-target to off-target nucleic acid targeting that results is caused by an increase in on-target activity. In some cases, the ratio of on-target to off-target nucleic acid targeting that results is caused by a decrease in off-target activity. In some cases, the ratio of on-target to off-target nucleic acid targeting that results is caused by both an increase in on-target activity and a decrease in off-target activity.

[0206] The targeted cell (the host cell) can be any desired cell / cell type. Examples of suitable cells and promoters are described in detail elsewhere herein (see, e.g., 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 mammaliancell, 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 culture in vitro.

[0207] In some embodiments the nucleic acid targeted by the CRISPR complex (on- target events) is the host cell’s genome. In some embodiments the nucleic acid targeted by the CRISPR complex (on-target events) is the genome of a pathogen (e.g., a virus) – In some cases, the pathogen is in the host cell. In some embodiments the nucleic acid targeted by the CRISPR complex (on-target events) is the genome of a pathogen (virus, bacteria, and the like) – In some cases, the pathogen is in the host cell. In some embodiments the nucleic acid targeted by the CRISPR complex (on-target events) is and RNA molecule. In some cases, the on-target nucleic acid targeting alters expression of a protein within the host cell (e.g., via decreasing transcription of the mRNA). In some cases, the on-target nucleic acid targeting alters expression of an RNA (e.g., a noncoding RNA, an mRNA, a microRNA, and the like) within the host cell.

[0208] In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes gene editing (e.g., correction of a genetic mutation in the host cell genome). In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes alteration of a genetic site (editing) from a disease-associated sequence to a healthy-associated sequence – e.g., correction of the Huntington’s Disease (HD), Duchenne Muscular Dystrophy (DMD), or Alpha-1 antitrypsin Disease (AATD) disease-causing alleles into alleles not associated with (non-causative of) disease.

[0209] In some embodiments, a subject method includes a step of measuring editing efficiency at an ON target site. In some embodiments, a subject method includes a step of measuring editing efficiency at one or more OFF target sites.

[0210] As noted elsewhere herein, the location of an on-target (“ON target”) event (e.g., target DNA cleavage / editing) is in effect determined by the guide sequence of the guide RNA. CRISPR complex mediated events that take place at a location that is not a 100% match with the guide sequence are referred to herein as off-target (“OFF target”) events. Any convenient method can be used to measure on-target and off-target events and the selection of method will depend on the type of CRISPR complex used and desired outcome of the complex’s activity (e.g., when using a nickase protein, when performing double stranded target cleavage, when using a donor polynucleotide – which can edit the target by introducing known heterologous sequence; when not usinga donor polynucleotide which can lead to numerous 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 such as next generation sequencing, and the like (and any combination thereof). Sequencing assays or alternative expression assays such as qRT-PCR and / or microarray analysis can be used when the activity of the CRISPR complex results in an alteration of expression of a target sequence (e.g., when a promoter sequence is targeted, when a coding sequence is targeted and the new sequence is susceptible to nonsense-mediated decay, and the like). Various assays exist to test for on-target and off-target activities and any desired assay or combination of assays can be used. In some cases, a desirable outcome (e.g., a desirable balance between reduced ON target and OFF target CRISPR nuclease activity) can be achieved by using a particular Acr protein (e.g., see above for a description of subject Acr proteins). In some cases, the off-target sites are predicted and / or known sties, and In some cases, the off-target sites can be identified after the fact (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).

[0211] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein decreases OFF target CRISPR nuclease activity (i.e., OFF target activity of the CRISPR nuclease). For example, in some cases, the Acr polypeptide decreases 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) as compared with OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide decreases 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) as compared with OFF target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0212] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein decreases ON target CRISPR nuclease activity (i.e., ON target activity of the CRISPR nuclease) as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide. For example, in some cases, the Acr polypeptide decreases ON target CRISPR nuclease activity by no more than 50% (e.g.,by no more than 40%, 30%, 20%, 10%, 5%, 3%, 2%, or 1%) as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide decreases ON target CRISPR nuclease activity by no more than 40% (e.g., by no more than 40%, 30%, 20%, 10%, 5%, 3%, 2%, or 1%) as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0213] In some embodiments, the Acr polypeptide of the compositions, systems, or methods described herein increases the ratio of ON target to OFF target CRISPR nuclease activity as compared with the ratio of ON target to 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 to OFF target CRISPR nuclease activity by at least 1.25x (i.e., 1.25 times) (e.g., by at least 1.5x, 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x) as compared the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity by at least 1.5x (e.g., by at least 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x) as compared the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity by at least 2x (e.g., by at least 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x) as compared the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0214] In some embodiments, the editing efficiency of ON target CRISPR nuclease activity is at least 4 times (4x) greater than editing efficiency of OFF target CRISPR nuclease activity (e.g., at least 5x, 6x, 7x, 8x, 9x, 10x, 20x, 25x, 30x, 35x, 40x, 50x, or 100x). In some embodiments, the editing efficiency of ON target CRISPR nuclease activity is at least 6 times (6x) greater than editing efficiency of OFF target CRISPR nuclease activity (e.g., at least 7x, 8x, 9x, 10x, 20x, 25x, 30x, 35x, 40x, 50x, or 100x). In some embodiments, the editing efficiency of ON target CRISPR nuclease activity is at least 10 times (10x) greater than editing efficiency of OFF target CRISPR nuclease activity (e.g., at least 20x, 25x, 30x, 35x, 40x, 50x, or 100x).

[0215] In some embodiments, the OFF target CRISPR nuclease activity is at an off- target site that comprises no more than 5 mismatches (e.g., no more than 4, no more than 3, no more than 2 or no more than 1) as 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 comprises 5 or less mismatches (e.g., 4 or less, 3 or less, 2 or less or 1 mismatch) ascompared to the on-target site (i.e., mismatches between the intended target site and the off-target site). In some embodiments, the OFF target CRISPR nuclease activity is at an off-target site that comprises no more than 3 mismatches (e.g., more than 2 or no more than 1) as 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 comprises 3or less mismatches (e.g., 2 or less or 1 mismatch) as compared to the on-target site (i.e., mismatches between the intended target site and the off-target site).

[0216] In some cases, a desirable outcome is one in which the off-target rate is less than 100 off-target events detected per cell population (e.g., off-target cleavage events such as insertion / deletions (indels) detected per cell population). In some such cases, the number of cells in the cell population is in a range of from 104to 106(e.g., In some cases, the number of cells in the cell population is about 105cells). In some cases, a desirable outcome is one in which the off-target rate is less than 90 off-target events detected per cell population (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, a desirable outcome is one in which the off-target rate is less than 50 off-target events detected per cell population (e.g., less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell).

[0217] In some cases, a desirable outcome is one in which the off-target rate is less than 100 off-target events detected per 105cells. In some cases, a desirable outcome is one in which the off-target rate is less than 90 off-target events detected per 105cells (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, a desirable outcome is one in which the off-target rate is less than 50 off-target events detected per 105cells (e.g., less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell).

[0218] In some cases, a desirable outcome is one in which less than 50% (e.g., less than 45%, less than 40%, or less than 35%) of the total measured nucleic acid targeting events (e.g., cleavage) are off-target events. In other words, In some cases, the ratio of on-target to off-target events (e.g., measured on-target to off-target events) 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 passaging the host cell (e.g., In some cases, for 10 or more generations) after the Acr and Cas proteins are introduced. Thus, In some cases, a desirable outcome is anoutcome in which, after passaging the host cell (e.g., for 10 or more generations) after the Acr and Cas proteins are introduced, less than 50% (e.g., less than 45%, less than 40%, or less than 35%) of the total measured nucleic acid targeting events (e.g., cleavage) are off-target events. In other words, in some such cases the ratio of on- target to off-target events (e.g., measured on-target to off-target events) 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).

[0219] As noted above, off-target sites can In some cases, be predicted. Generally, the rate (frequency) of off-target activity (e.g., cleavage / editing) will vary from site to site, e.g., when measuring rates of activity using a population of cells. As such, in some cases, a desirable outcome is one in which 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%) when compared to the off-target events measured (or expected) in the absence of the Acr protein. As an illustrative example, on can measure the frequency of off-target events at one particular predicted or known off-target site (or at any number of off-target sites – predicted / known or not predict / known) in the presence of the Acr protein (meaning – when the experiment is performed in the present of the Acr protein) and in the 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 evens when the Acr protein is absent. As an additional illustrative example of the above, if 100 total off-target events are measured when the method is performed in the presence of the Acr protein, but 200 such events are measured (or expected) in the absence of the Acr protein, then the outcome would be a measured frequency of off- target events in the presence of the Acr protein that is 50% when compared to the off- target events in the absence of the Acr protein. Delivery

[0220] As noted above, in some embodiments both the Cas protein and the Acr protein will be delivered to a host cell as DNA and in some such cases the sequence encoding the two proteins will be present on the same nucleic acid (e.g., DNA vector) or on separate nucleic acids. However, in some embodiments a subject protein (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 a host cell as RNA encoding the protein. In such cases the RNA encoding the two proteins can be delivered in an appropriate ratio to achieve the desired affect (i.e., increased ratio of on-target to off-target CRISPR complex activity) – e.g., by decreasing off-target activity while retaining desirable on-target activity, and one or more translational control elements can be present on the RNAs.

[0221] As another example, either protein (or both) can be introduced into a host cell directly as proteins. In some such cases (e.g., if the Cas protein is a class 2 effector protein) the Cas protein can be delivered as an RNP (ribonucleoprotein complex) in which it is already complexed with an appropriate guide RNA. In such cases the other protein (e.g., the Acr protein) can be delivered as DNA or RNA and its coding sequence can be operably linked to a subject translational control element.

[0222] Thus, the Cas protein and the Acr protein can be delivered in any desired format (DNA, RNA, protein). For example, if the Cas protein is delivered as DNA, the Acr protein can be delivered as DNA, RNA, or protein; if the Cas protein is delivered as RNA, the Acr protein can be delivered as DNA, RNA, or protein; and if the Cas protein is delivered as protein, the Acr protein can be delivered as DNA or RNA. Likewise, if the Acr protein is delivered as DNA, the Cas protein can be delivered as DNA, RNA, or protein; if the Acr protein is delivered as RNA, the Cas protein can be delivered as DNA, RNA, or protein; and if the Acr protein is delivered as protein, the Cas protein can be delivered as DNA or RNA.

[0223] As would be readily understood by one of ordinary skill in the art, subject nucleic acids (e.g., vectors) and proteins can be delivered to cells using any convenient method. Methods of introducing nucleic acids and / or proteins into a host cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, insect cell, mammalian cell, human cell, and the like) are known in the art, and any convenient method can be used. Suitable methods include, e.g., viral infection (e.g., AAV, adenovirus, lentiviral), 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 micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev.2012 Sep 13. pii: S0169- 409X(12)00283-9), and the like.

[0224] In some cases, a protein of the present disclosure (e.g., Cas protein, Acr protein) is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the protein. In some cases, a subject protein isprovided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e., as a ribonucleoprotein complex). A subject protein can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a subject protein can be injected directly into a cell. As another example, a subject protein can be introduced into a cell (e.g, eukaryotic cell) via nucleofection; via a protein transduction domain (PTD) conjugated to the protein, etc.

[0225] In some cases, a subject protein is delivered to a cell (e.g., a target host cell) in a particle, or associated with a particle. In some cases, a subject protein is delivered with a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl- sn-glycero-3-phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5).

[0226] A subject protein (as RNA or DNA or protein) may be delivered using particles or lipid envelopes. For example, a biodegradable core-shell structured nanoparticle with a poly (β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self assembling bioadhesive polymers are used; such particles / nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used.

[0227] Lipidoid compounds (e.g., as described in US patent application 20110293703) are also useful in the administration of polynucleotides, and can be used to deliver a subject protein or nucleic acid (RNA or DNA). In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. toform the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0228] In some cases, lipid nanoparticles (LNPs) are used to deliver a subject protein or nucleic acid to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Ionizable cationic lipids can include, but are not limited to: 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2- dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N- dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)- [1,3]-dioxolane (DLinKC2-DMA). An example of preparation of LNPs is described in, e.g., Rosin et al. (2011) Molecular Therapy 19:1286-2200). The cationic lipids 1,2- dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2''-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S- DMG), and R-3-[(.omega.-methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2- dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. A nucleic acid may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated. Suitable lipids include for LNPs include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a subject protein or nucleic acid using a spontaneous vesicle formation procedure. The component molar ratio may be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12- 200 / disteroylphosphatidyl choline / 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), the particles may be extruded up to three times through 80 nm membranes prior to adding, e.g., RNA such as guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50 / 10 / 38.5 / 1.5) which may be further optimized to enhance activity.

[0229] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver a subject protein or nucleic acid to a targetcell.. See, e.g., Cutler et al., J. Am. Chem. Soc.2011133:9254-9257, Hao et al., Small. 20117:3158-3162, Zhang et al., ACS Nano.20115:6962-6970, Cutler et al., J. Am. Chem. Soc.2012134:1376-1391, Young et al., Nano Lett.201212:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA.2012109:11975-80, Mirkin, Nanomedicine 20127:635- 638 Zhang et al., J. Am. Chem. Soc.2012134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA.2013110(19): 7625-7630, Jensen et al., Sci. Transl. Med.5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

[0230] Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).

[0231] In general, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a subject protein or nucleic acid to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering a a subject protein or nucleic acid to a target cell have a diameter of from 25 nm to 200 nm.

[0232] Nanoparticles suitable for use in delivering a subject protein or nucleic acid to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core- shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure.

[0233] Semi-solid and soft nanoparticles are also suitable for use in delivering a subject protein or nucleic acid to a target cell. A prototype nanoparticle of semi-solid nature is the liposome.

[0234] In some cases, a carrier / medium can include a microparticle. Microparticles can include, but are not limited to, liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. In some cases, microparticle can include one or more of the following: a poly(lactide-co-glycolide), aliphatic polyesters including, but notlimited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo- poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids – in any combination thereof.

[0235] In some cases, a carrier / medium can include a liposome, e.g., one that is capable of attaching and releasing therapeutic agents (e.g., the subject nucleic acids and / or proteins). Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid- soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, 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; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

[0236] In some embodiments, a cationic or anionic liposome is used as part of a subject composition or method, or liposomes having neutral lipids can also be used. Cationic liposomes can include negatively-charged materials by mixing the materialsand fatty acid liposomal components and allowing them to charge-associate. The choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include but are not limited to: lipofectin, lipofectamine, and lipofectace.

[0237] A subject protein or nucleic acid may be delivered encapsulated in PLGA microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279.

[0238] In some cases, an exosome is used to deliver a subject protein or nucleic acid to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.

[0239] A poly(beta-amino alcohol) (PBAA) can be used to deliver a subject protein or nucleic acid to a target cell. US Patent Publication No.20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization.

[0240] Sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver a subject protein or nucleic acid to a target cell.

[0241] Supercharged proteins can be used to deliver a subject protein or nucleic acid to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can facilitate the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.

[0242] Cell Penetrating Peptides (CPPs) can be used to deliver a subject protein or nucleic acid to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids.

[0243] Carriers or mediums contemplated include materials such as gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, 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(ortho esters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof. Donor Polynucleotide (donor template)

[0244] In some cases, a subject composition or method may include a donor polynucleotide. For example, in applications in which it is desirable to insert a polynucleotide sequence into the genome where a target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site targeted by the CRISPR complex (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). 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. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886- 889. A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor template can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV). Examples of Non-Limiting Aspects of the Disclosure

[0245] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below (see Set A and Set B). As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numberedaspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: Set A 1. An anti-CRISPR (Acr) polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having 75% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.: 126-132. 2. The Acr polypeptide or nucleic acid of 1, wherein said amino acid sequence has 90% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.126-132. 3. The Acr polypeptide or nucleic acid of 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID Nos.126-132. 4. The Acr polypeptide or nucleic acid of any one of 1-3, wherein the Acr polypeptide decreases OFF target CRISPR nuclease activity by 10% or more as compared with OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. 5. The Acr polypeptide or nucleic acid of any one of 1-4, wherein the Acr polypeptide decreases ON target CRISPR nuclease activity by no more than 40% as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide. 6. The Acr polypeptide or nucleic acid of any one of 1-5, wherein the Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity by at least 1.25 times as compared with the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. 7. The Acr polypeptide or nucleic acid of any one of 4-6, wherein the CRISPR nuclease is a Cas12a nuclease. 8. The Acr polypeptide or nucleic acid of any one of 4-6, wherein the CRISPR nuclease comprises an amino acid sequence having 80% or more sequence identity with 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 of any one of 4-6, wherein the CRISPR nuclease comprises an amino acid sequence having 80% or more sequence identity with 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 of any one of 1-9, wherein the Acr polypeptide is fused to a nuclear localization signal (NLS). 11. A system comprising: (i) an Acr polypeptide or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide of any one of 1-10, or comprises an amino acid sequence having 80% or more sequence identity with the CRISPR nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 165-169; and (ii) a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease. 12. The system of 11, wherein the CRISPR nuclease is a Cas12a nuclease. 13. The system of 11, wherein the CRISPR nuclease comprises an amino acid sequence having 80% or more sequence identity with the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 175 and 245-262. 14. The system of 11, wherein the CRISPR nuclease comprises an amino acid sequence having 80% or more sequence identity with the NUX protein amino acid sequence set forth in any one of SEQ ID NOs.: 1-86 and 176-244. 15. The system of any one of 11-14, further comprising a guide RNA or a nucleic acid encoding the guide RNA. 16. The system of 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 of 16, wherein the system comprises a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease. 18. The system of 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 that encodes both the Acr polypeptide and the CRISPR nuclease. 19. The system of any one of 16-18, wherein a translation control element is operably linked to the Acr polypeptide coding sequence or to the CRISPR nuclease coding sequence. 20. The system of 19, wherein the translation control element is selected from the group consisting of an IRES sequence, a 2A peptide encoding sequence, a non- canonical start codon or any combination thereof.21. The system of 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 of 21, wherein the first promoter is a stronger promoter than the second promoter. 23. The system of 21, wherein the second promoter is a stronger promoter than the first promoter. 24. The system of 21 or 22 wherein the first promoter is selected from the group consisting of CMV, miniCMV, EF1A, CAG, or CBh. 25. The system of any one of 21-24, wherein the second promoter is selected from the group consisting of CMV, miniCMV, EF1A, CAG, and CBh. 26. The system of 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 of any one of 16-26, wherein one or more of said nucleic acids is a viral vector. 28. The system of 27, wherein the viral vector is an AAV vector. 29. The system of any one of 16-26, wherein one or more of said nucleic acids is comprised in a lipid nanoparticle (LNP). 30. The system of any one of 11-15, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form. 31. The system of 30, wherein the Acr polypeptide and / or the CRISPR nuclease is comprised in a lipid nanoparticle (LNP). 32. The system of any one of 11-31, wherein the ratio of ON target to OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 33. The system of 32, wherein the ratio of ON target to 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 ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 34. The system of any one of 11-33, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or more when compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide.35. The system of any one of 11-34, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with 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 of any one of 11-35, wherein the contacting results in a modification to the nucleotide sequence of the target nucleic acid. 37. The method of 36, wherein the modification results from deletion of one or more nucleotides. 38. The method of any one of 36 or 37, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide at a 1:1 ratio. 39. The method of any one of 36 or 37, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) in a range of from 1:1.25 to 1:10. 40. The method of any one of 36 or 37, wherein the contacting step delivers the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) in a range of from 1:1.25 to 1:10. 41. The method of any one of 36-40, wherein said contacting occurs in vivo. 42. The method of any one of 36-40, wherein said contacting occurs in a eukaryotic cell. 43. The method of 42, wherein the eukaryotic cell is an animal cell. 44. The method of 43, wherein the animal cell is a human cell. 45. The method of 42, wherein the eukaryotic cell is a stem cell. 46. The method of any of 36-45, wherein the target nucleic acid encodes a gene product. 47. The method of any one of 36-46, wherein editing efficiency of ON target CRISPR nuclease activity is at least 4 times greater than editing efficiency of OFF target CRISPR nuclease activity. 48. The method of 47, wherein said OFF target CRISPR nuclease activity is at an off- target site that comprises no more than 5 mismatches as compared to the on- target site. 49. The method of any one of 36-48, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or morewhen compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 50. The method of any one of 36-49, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with the ON target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 51. The method of any one of 36-50, wherein the method further comprises measuring editing efficiency. 52. The method of any one of 36-51, wherein the method further comprises measuring 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 75% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.: 126-132 and 264-267. 2. The Acr polypeptide or nucleic acid of 1, wherein said amino acid sequence has 90% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.126-132 and 264-267. 3. The Acr polypeptide or nucleic acid of 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID Nos.126-132. 4. The Acr polypeptide or nucleic acid of 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in SEQ ID NO:165 with an amino acid variation at least one position between and including amino acid 2 and amino acid 159 of SEQ ID NO:165. 5. The Acr polypeptide or nucleic acid of 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID Nos.264-267. 6. The Acr polypeptide or nucleic acid of 1 or 4, wherein the Acr polypeptide comprises one or more amino acid variations set forth in Table 6. 7. The Acr polypeptide or nucleic acid of 1 for 4, wherein the Acr polypeptide comprises one or more amino acid variations set forth in Table 7. 8. The Acr polypeptide or nucleic acid of any one of 1-7, wherein the Acr polypeptide decreases OFF target CRISPR nuclease activity by 10% or more as compared with OFF target CRISPR nuclease activity in the absence of the Acr polypeptide.9. The Acr polypeptide or nucleic acid of any one of 1-8, wherein the Acr polypeptide decreases ON target CRISPR nuclease activity by no more than 40% as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide. 10. The Acr polypeptide or nucleic acid of any one of 1-9, wherein the Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity by at least 1.25 times as compared with the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide. 11. The Acr polypeptide or nucleic acid of any one of 8-10, wherein the CRISPR nuclease is a Cas12a nuclease. 12. The polypeptide or nucleic acid of any one of 8-10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 175 and 245-262. 13. The 13. The Acr polypeptide or nucleic acid of any one of 8-10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with 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 of any one of 1-13, wherein the Acr polypeptide is fused to a nuclear localization signal (NLS). 15. A system comprising: (i) an Acr polypeptide or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide of any one of claims 1- 14, comprises an amino acid as set forth in any one of SEQ ID NOs.: 165-169, or comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with the Acr amino acid sequence set forth in any one of SEQ ID NOs.: 165-169; and (ii) a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease. 16. The system of 15, wherein the CRISPR nuclease is a Cas12a nuclease. 17. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with the Cas12a nuclease amino acid sequence set forth in any one of SEQ ID NOs.: 175 and 245-262. 18. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO.263 or an amino acid sequence having80%, 85%, 90% or 95% or more sequence identity with the CRISPR nuclease amino acid sequence set forth in SEQ ID NO.263 19. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence as 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 with the NUX protein amino acid sequence set forth in any one of SEQ ID NOs.: 1-86 and 176-244. 20. The system of any one of 15-19, further comprising a guide RNA or a nucleic acid encoding the guide RNA. 21. The system of any one of 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 of 21, wherein the system comprises a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease. 23. The system of 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 that encodes both the Acr polypeptide and the CRISPR nuclease. 24. The system of any one of 21-23, wherein a translation control element is operably linked to the Acr polypeptide coding sequence or to the CRISPR nuclease coding sequence. 25. The system of 24, wherein the translation control element is selected from the group consisting of an IRES sequence, a 2A peptide encoding sequence, a non- canonical start codon or any combination thereof. 26. The system of any one of 21-23, 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 of 26, wherein the first promoter is a stronger promoter than the second promoter. 28. The system of 26, wherein the second promoter is a stronger promoter than the first promoter. 29. The system of any one of 21-28, wherein one or more of said nucleic acids is a viral vector. 30. The system of 29, wherein the viral vector is an AAV vector.31. The system of any one of 21-30, wherein one or more of said nucleic acids is comprised in a lipid nanoparticle (LNP). 32. The system of any one of 15-20, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form. 33. The system of 32, wherein the Acr polypeptide and / or the CRISPR nuclease is comprised in a lipid nanoparticle (LNP). 34. The system of any one of 15-33, wherein the ratio of ON target to OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 35. The system of 34, wherein the ratio of ON target to 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 ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 36. The system of any one of 15-35, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or more when compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 37. The system of any one of 15-36, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with 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 of any one of 15-37, wherein the contacting results in a modification to the nucleotide sequence of the target nucleic acid. 39. The method of 38, wherein the modification results from deletion of one or more nucleotides. 40. The method of any one of 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide at a 1:1 ratio. 41. The method of any one of 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide at a ratio (CRISPR:Acr) in a range of from 1:1.25 to 1:10.42. The method of any one of 38 or 39, wherein the contacting step delivers the Acr polypeptide and the CRISPR nuclease at a ratio (Acr:CRISPR) in a range of from 1:1.25 to 1:10. 43. The method of any one of 38-42, wherein said contacting occurs in vivo. 44. The method of any one of 38-42, wherein said contacting occurs in a eukaryotic cell. 45. The method of 44, wherein the eukaryotic cell is an animal cell. 46. The method of 45, wherein the animal cell is a human cell. 47. The method of 44, wherein the eukaryotic cell is a stem cell. 48. The method of any one of 38-47, wherein the target nucleic acid encodes a gene product. 49. The method of any one of 38-47, wherein editing efficiency of ON target CRISPR nuclease activity is at least 4 times greater than editing efficiency of OFF target CRISPR nuclease activity. 50. The method of 49, wherein said OFF target CRISPR nuclease activity is at an off- target site that comprises no more than 5 mismatches as compared to the on- target site. 51. The method of any one of 38-50, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or more when compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 52. The method of any one of 38-51, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with the ON target activity of the CRISPR nuclease in the absence of the Acr polypeptide. 53. The method of any one of 38-52, wherein the method further comprises measuring editing efficiency. 54. The method of any one of 38-53, wherein the method further comprises measuring editing efficiency at one or more off-target sites. VI. EXAMPLES

[0246] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the presentinvention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0247] General methods in molecular and cellular biochemistry can be found in such standard textbooks 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 incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like. Example 1 Vector Construction Nuclease expression vectors

[0248] 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. Vector included an in-frame nuclear localization signal OPT NLS (GRSSDDEATADSQHAAPPKKKRKV) (SEQ ID NO: 125) followed by the linker (GGSGGSGGSGGSGGSGGSGGSGGS) (SEQ ID NO: 124) and followed by a 3x HA tag. Guide vectors

[0249] To make guide vectors, direct repeat (DR) sequences for 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 is NUX (SEQ ID NO: 176),NUX (SEQ ID NO: 177) and NUX (SEQ ID NO: 178) direct repeat and DR18s is AsCas12a direct repeat. A spacer target sequence was placed downstream of the DR sequence. The spacer sequence used for DNMT was CTGATGGTCCATGTCTGTTA (SEQ ID NO: 172) for which the PAM was TTTC. The spacer sequence used for FANCF1 was GGCGGGGTCCAGTTCCGGGA (SEQ ID NO: 162) for which the PAM was TTTG. Mismatched guides were made by changing a single nucleotide on a given position into its counterpart such as A->T and G->C. Table 2: Direct repeat sequences used in the guide vectors Direct Repeat Sequence SEQ ID NO: DR19s AATTTCTACTGTGTGTAGAT 163 DR18s AATTTCTACTCTTGTAGAT 164 Acr Vectors

[0250] Potential Anti-CRISPR (Acr) sequences for use with nucleases were identified in EBI and NCBI databases (Table 3). Codon-optimized (human) genes encoding the Acrs from Table 3 were synthesized and cloned into the same CMV expression vector as the nucleases. Table 3: Identified Acrs SEQ Acx Amino Acid Sequence ID 165 175 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIHTS WESLWNYESGDYDTLWGYIDYGINEMLVEANLPTIDFNYESGDIP TEADYLDLTEEEREEYGNDSWTWREESGAFDEFTDFLNEVNNKI EDYLMKVDKEHGTHYAPTGFERLK 166 176 MDERYEAKRRYVEKIMQENAEVETLTEKQHEALQELARIRHKIHT SWEVMWNYEASEYKKLWDYIEYDIDEMLTEAELPAIEFNYDPCDV PTSADFEIFLTEEEQAKYDNDSWKWREESGAFDEFADFLSSVNN KIEDYLTNIDKKHGTHYAPTGFARLK 167 177 MDKWEAKRRYVERKNRENEKIKTLTPEQHDVIQELCAKRHEFHC SFESMWNRNAADLLNDFSDCNDYNMSYKLKEAGLPELKLLISCD MPGEEDYYELLSDEEKEEWERKADKFNEENPKAFFPHSGYTLW KEESWEYDTFLKICEKQNDIIEKYLREIDEKYGTEYCPTGIARFEG VI 168 178 MAILDKEAYWGKMMYAEKRNAENREKGTENGLTDEQADALESM ARARHNMHTNIDSLVSSSENRDCERDLIRANAKLREAGLPTVPSI PSYEGDYIDIDSFDIIMYDTDIAKEEYGMPDKYENEEAWQEWYDKMYSRLYDKWEAINDKIENYLGEIDEKYGTSYKPTGITRI 169 179 MSYLSKEDYDCQTKAAAKKMERNSLIETLTPEQHKALRELCRAR HFIHSNMKSIFNDESSEAETFLNYFVEGGTSNVLDNLTAAGLPKLD LSVDIIMDMPTLSTYNEIDDMTLEDALDLHFELLERINTEIENYLGAI DEEHGTKYKPTGALRARGL Example 2 Acr activity

[0251] Editing efficiency of AsCas12a (SEQ ID NO: 175) in the presence of Acrs was tested in HEK293T cells. The vectors constructed in Example 1 were transfected into cells using Mirus Transit X2 reagent, MirusBio CAT MIR6003. Tests were performed in 96 well plates transfected with 100 ng of nuclease expression vector, 100 ng of Acr expression vector and 50 ng of targeting guide vector following the Mirus Transit X2 transfection recommendations. Samples were incubated for 72h and harvested with Quick Extract. Genomic DNA was amplified using genomic region-specific primers. Target FWD Primer REV Primer DNMT1 CCAGAATGCACAAAGTACTGCAC GCCAAAGCCCGAGAGAGTGCC (SEQ ID NO: 170) (SEQ ID NO: 171)

[0252] Samples were checked on a 2% agarose gel for purity and sequenced by Sanger sequencing. TIDE analysis was performed following the method of Brinkman et al., 2014 as well as TIDE’s website (https: / / tide.nki.nl / ) recommendations. TIDE output data on editing efficiency was plotted using Prism software. The Acrs were compared to AcrVA1 (ACX-137) (SEQ ID NO: 67) and AcrIIA4 (ACX-105) (SEQ ID NO: 35). Results are shown in Figure 1.

[0253] The Acrs were also tested for activity by co-transfecting with a second nuclease, NUX (SEQ ID NO: 176). Expression vectors were used to transiently deliver NUX (SEQ ID NO: 176) and the Acrs into HEK293T cells following the same protocol above. Editing efficiency was measured as above. Results are shown in Figure 2.

[0254] The identified Acrs, Acx-175, Acx-176, Acx-177, and Acx-178 (SEQ ID NOs: 165-168, respecrtively) showed the ability to inhibit the editing efficiency of Cas12a. The Acrs demonstrated a different pattern of inhibition when tested with NUX (SEQ ID NO: 176). Acx-178 showed little inhibition and the inhibition activity of Acx-176 was less effective as compared to Acx-175, whereas Acx-176 was a stronger inhibitor than Acx- 175 with Cas12a.Example 3 Dosage dependent nuclease inhibiting properties of Acx-175

[0255] In a series of dosing experiments, Acx-175 showed an intrinsic ErAcr property that inhibits off- target editing while leaving on-target activity substantially unchanged.

[0256] The AsCas12a endonuclease was co-transfected with decreasing doses of Acx- 175 into HEK293T cells to observe editing activity targeting DNMT1. Transfection, incubation and analysis including measurements of editing efficiency were performed as described in Example 2. Off target editing was measured using a guide RNA with a single base change at position 9 (MM9, CTGATGGTGCATGTCTGTTA: SEQ ID NO: 173). Results are shown in Figure 3.

[0257] The NUX (SEQ ID NO: 177) endonuclease was co-transfected with decreasing doses of Acx-175 to observe editing activity targeting DNMT1. Transfection, incubation and analysis protocols were performed as Example 2. Off target editing was measured using a guide RNA with a single base change at position 9 (MM9) and a guide RNA with a single base change at position 5 (MM5, CTGAAGGTCCATGTCTGTTA: SEQ ID NO: 174). Results are shown in Figure 4. Example 4: Generation of engineered Acrs

[0258] Strong 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 screen approach as described by Huimin Zhao in 2005; https: / / academic.oup.com / nar / article / 33 / 18 / e154 / 2401371.

[0259] 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 a guide RNA with a single base change at position 8 (MM8, CTGATGGaCCATGTCTGTTA: SEQ ID NO: 161) was used to measure off-target editing.

[0260] The first round of the screen resulted in six Acx-175 variants (Table 4, except for Acx-315) that were transferred into a CMV expressing vector and tested in HEK293T cells with NUX (SEQ ID NO: 178) and DNMT1 for validation following the methods in Example 2. Results are shown in Figure 5. Table 4: Engineered Acrs SEQ ID Acx- Amino Acid Sequence 126 305 MEKRYDSWTWREESGAFDEFTDFLNEVNNKIEDYLMKVDKEH GTHYAPTGFERLK127 306 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIH TSWESLWNYESGDYDTLWGYIDYGINEMLVEANLPTIDFNYES GDIPTEADYLDLTEEEREEYGNDSWTWREESGAFDEFTDFLN EVNNKIEDYLMKVDKEHGTHYAPTGFKRLK 128 307 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIH TSWESLWNYESGDYDTLWGYIDYDINEMLVEANLPTIDFNYES GDIPTEADYLDLTEEEREEYGNDSWTWREESGAFDEFTDFLN EVNNKIEDYLMKVDKEHGTHYAPTGFERLK 129 308 MEKRYEAGHGGKSLVPSMNLLTF 130 310 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIH TSWESLWNYESGDYDTLWGYIDYGINEMLVEANLPTIDFNYES GDIPTEADYLDLTEEEWEEYGNDSWTWREESGAFDEFTDFLN EVNNKIEDYLMKVDKEHGTNYAPTGFERLK 131 311 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIH TSWESLWNYESGDYDTLWGYIDYGINEMLVEANLPTIDFNYES GDIPTEADYLDLTEEEREEYGNDSWTWRESLVPSMNLLTF 132 315 MEKRYEAKRRYVEKIMRENAEIETLTEEQHNALQKLASIRHKIH TSWESLWNYESGDYDTLWGYIDYGINEMLVEANLPTIDFNYES GDIPTEADYLDLTEEEWEEYGNDSWTWREESGAFDEFTDFLN EVNNKIEDYLMKVDKEHGTNYAPTGFKRLK Example 5: Dosage dependent nuclease inhibition with engineered Acrs

[0261] Expression vectors were constructed using NUX (SEQ ID NO: 178) and four of the 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. Results for editing efficiency of Nux:Acx 2:1 are shown in Figure 6A. Results for editing efficiency of Nux:Acx 1:1 are shown in Figure 6B. Results for editing efficiency of Nux:Acx 1:2 are shown in Figure 6C. Example 6: Measured off target inhibition of Acrs

[0262] NUX (SEQ ID NO: 178) was co-transfected into HEK293T cells with either Acx- 306 or Acx-310. Editing efficiency was measured for the on-target and off-target for single mismatches at positions 1, 9, 19 and 20. Editing efficiency was compared to Acx- 105 as a negative control and wild-type Acx-175 as a positive control. 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 the on-target and off-target for singlemismatches at positions 8 of DNMT1. NUX (SEQ ID NO: 178) and Acx-315 were tested over a range of Nux:Acx ratios. Results are shown in Figure 8A for on-target and off-target editing efficiencies and as a ratio of on targeting:off targeting in both Figure 8B and Table 5. Table 5 Acx:NUX ON:OFF NT 1.13 100% 3.47 50% 5.08 25% 6.95 10% 4.83 5% 3.05 1% 2.30 No Acr 1.36 Example 7: Variant Acr activity

[0263] Acr Acx-175 (SEQ ID NO: 165) was subjected to random mutagenesis at codon positions 2 through 159 using error prone PCR (Genemorph II, Agilent Biosciences). The library of resulting variant Acrs was screened using the methods described in Example 4. Sequencing of the variants yielded a collection of amino acid substitutions at each position as shown in Table 6.

[0264] Table 6 CDS Codon No. CDS Codon No. WT (amino acid WT AA Variants (amino acid ition) position)AVariantsposA2 E V, A, D, K, G 81 I F, T, V, N, M 3K M, R, T, E, N 82 DG, N, V, H, E,Y, A4 R Q, W, L, P, G 83 FL, S, I, L, V, S,Y5 Y S, C, N, F, H 84 NK, D, S, I, Y, K,T6 EG, V, D, K, D,A85 Y F, H, S, N, C7 A T, V, S, D, P 86 EK, D, G, D, V,Q8 KR, M, E, N, Q,T, L, N 87 S F, T, P, C, Y, A9 RC, S, H, L, G,P, T 88 G D, V, C, S, A10 R K, G, S, I, T, S 89 DV, N, H, E, G,Y, A, E11 Y C, N, F, H 90 I F, T, V, N, L, S12 VI, A, D, L, G,A, F, T 91 P Q, S, T, L, A13 EV, G, D, K, Q,D92 T S, A, I, P, K, R14 KR, E, M, N, Q,D, V, D, G, K, N, T 93 EM15 IF, N, V, T, R,F, L 94 A S, E, T, V, G16 MI, L, I, T, V, K,95N, Y, E, E, H, R, L, I DV, G, F 17 R W, Q, L, G, P 96 Y N, F, H, C, H18 EV, G, D, Q, D,M, S, V, W, F, K, A, R 97 LF19 NS, Y, I, K, D,G, V, Y, E, N, H, K 98 DA, H, E20 AV, S, E, T, P,G, T 99 L I, H, F, P, V21 EV, K, D, G, D,M, A 100 T S, I, A, S, N22 IN, F, T, V, M,L, L 101 E D, K, D, V, G, A23 EV, G, D, A, D,K, Q, V, V 102 E K, D, V, G, A24 T S, A, I, N, S 103 ED, V, G, D, Q,K, A25 L I, F, S, V 104 R Q, L, W, G26 TA, S, A, P, I,D, V, G, A, K, K, R 105 EQ, D27 EV, D, K, G, Q,A, D 106 E D, V, Q, G28 EV, G, D, K, D,Q107 Y C, N, H, D29 QH, R, L, K, P,W, E, H 108 G D, S, CY, L, R, QS, I, D, K, K, H, 30 H, D,C, N, Q, Y, P109 N T31 ND, Y, I, S, Y,N, Y, E, V, G, T, K, H, K 110 DA, E32 AT, V, G, D, P,111 SN, G, C, R, R, I, S, VR 33 L P, Q, M, R 112 W R, L, R, G, C, S 34 Q L, H, R, K, P 113 T I, S, A, K, R35 KR, M, N, Q, T,E, N, Q 114 W R, R, L, C36 L H, I, P, F 115 R Q, W, L, G37 AT, V, S, P, G,D116 E D, G, V, D, K38 ST, Y, F, P, I, A,V, G, D, K, D, C, L 117 EA, V39 IN, F, T, V, F,S, M 118 S P, Y, T, F, C, A40 RS, H, C, G, L,P119 G S, D, R, C, A, V41 HL, Y, Q, N, R,Q, P, D 120 A V, D, S, G, P, T42 K M, T, N, E, R, 121 F Y, I, L, S, L, V,N L, C43 IN, T, V, F, L,12G, V, E, N, A, S2 DY, H, N, A44 HL, P, R, N, Q,D, G, V, K, A, Q, D 123 ED, G45 TI, S, A, P, A,I, S, Y, L, C, T, S, N 124 FL, V, S46 S T, A, L, P 125 TN, S, I, S, P, P,A47 WR, C, R, C, G,N, V, Y, G, E, L, L 126 DE, H, A48 EV, D, G, K, A,I, L, S, L, Y, C, D, Q 127 FV 49 S T, Y, P, F, P 128 L P, Q, M, R, V50 L H, P, I, F, R, Y 129 NY, I, D, S, K, T,K, F51 WR, R, G, C, S,K, G, V, D, D, L, C 130 EN, A 52 NS, D, Y, I, H,K, C, K, T, G131 V I, E, A, L, L53 YC, N, F, H, S,I, D, T, S, K, Y, D132 NK, H54 EG, D, K, V, A,K, I, S, D, Y, H, G, Q 133 NT55 SN, C, I, G, C,R, R, T 134 K I, T, R, N, E, N56 GD, S, C, A, V,N, T, V, F, L, S, V, R 135 IT, M57 DV, G, N, H, E,D, K, D, G, V, Y, G, A 136 EQ, N58 YH, C, F, N, S,Y, E, N, V, E, C, D 137 DG, H, G59 DE, V, G, N, Y,G138 Y N, H, F, C, D, L60 T A, N, S, I, S, P 139 LI, V, H, P, R, F,V, Q61 L Q, P, M, R 140 MT, V, K, L, R, I,I, L62 WR, C, R, S, G,N, M, E, R, N, L, C 141 KT, Q63 GD, C, S, V, A,R, E 142 V A, M, E, G, L, L64 Y H, N, F, C, S 143 DG, E, V, Y, E,N, A, C65 I F, N, T, V, L 144 K M, N, T, E, R, E66 DG, N, V, E, Y,G, D, K, V, G, E145 ED, A67 YF, H, C, N, S,Y, D, L, R, N, H146 HQ, P, Q, L68 G S, D, V, C, A 147 GW, V, R, E, A,R69 IF, V, T, N, M,I, N, S, A, P, S, L148 TR, R70 ND, I, S, T, H,N, R, Y, Q, L, Y, K, K 149 HQ, R, N, P, L 71 E G, V, D, D, A, 150 Y H, C, N, D, S, FK, Q 72 MK, L, I, V, T, I,L, R 151 A T, V, G, E, SR L,

[0265] y g g t editing of a CRISPR nuclease (SEQ ID NO: 263) targeting DNMT1 (On-target) and DNMT1 mismatch 8 (Off-target) as described in Example 4. Results are shown in Figure 12 and the variants tested are listed in Table 7. The results show that both inhibition level and on-target to off-target activity ratios can be modulated through variations in the Acr. Figure 13 shows an amino acid sequence alignment of the ErAcrs shown in Figure 12.

[0266] Table 7. Variants tested Variant Amino acid substitutions SEQ ID (variants) NO: ErAcr3 V74E, L97V, S111G 264 ErAcr4 N52I 265 ErAcr13 K3N, T80P 266 ErAcr20 T80I, Y107N 267

[0267] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0268] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, 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. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0269] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

[0270] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. An anti-CRISPR (Acr) polypeptide, or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having 75% or more sequence identity with the sequence set forth in any one of SEQ ID Nos.: 126-132 and 264-267.

2. The Acr polypeptide or nucleic acid of claim 1, wherein said amino acid sequence has 90% or more sequence identity with the sequence set forth in any one of SEQ ID Nos. 126-132 and 264-267.

3. The Acr polypeptide or nucleic acid of claim 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID Nos.126-132.

4. The Acr polypeptide or nucleic acid of claim 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in SEQ ID NO:165 with an amino acid variation at least one position between and including amino acid 2 and amino acid 159 of SEQ ID NO:

165.

5. The Acr polypeptide or nucleic acid of claim 1, wherein the Acr polypeptide comprises the amino acid sequence set forth in any one of SEQ ID Nos.264-267.

6. The Acr polypeptide or nucleic acid of claim 1 or claim 4, wherein the Acr polypeptide comprises one or more amino acid variations set forth in Table 6.

7. The Acr polypeptide or nucleic acid of claim 1 or claim 4, wherein the Acr polypeptide comprises one or more amino acid variations set forth in Table 7.

8. The Acr polypeptide or nucleic acid of any one of claims 1-7, wherein the Acr polypeptide decreases OFF target CRISPR nuclease activity by 10% or more as compared with OFF target CRISPR nuclease activity in the absence of the Acr polypeptide.

9. The Acr polypeptide or nucleic acid of any one of claims 1-8, wherein the Acr polypeptide decreases ON target CRISPR nuclease activity by no more than 40% as compared with ON target CRISPR nuclease activity in the absence of the Acr polypeptide.

10. The Acr polypeptide or nucleic acid of any one of claims 1-9, wherein the Acr polypeptide increases the ratio of ON target to OFF target CRISPR nuclease activity by at least 1.25 times as compared with the ratio of ON target to OFF target CRISPR nuclease activity in the absence of the Acr polypeptide.

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

12. The Acr polypeptide or nucleic acid of any one of claims 8-10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with 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 of any one of claims 8-10, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with 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 of any one of claims 1-13, wherein the Acr polypeptide is fused to a nuclear localization signal (NLS).

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

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

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

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

263.

19. The system of claim 15, wherein the CRISPR nuclease comprises an amino acid sequence as 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 with the NUX protein amino acid sequence set forth in any one of SEQ ID NOs.: 1-86 and 176-244.

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

21. The system of 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 of claim 21, wherein the system comprises a first nucleic acid encoding the Acr polypeptide and a second nucleic acid encoding the CRISPR nuclease.

23. The system of 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 that encodes both the Acr polypeptide and the CRISPR nuclease.

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

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

26. The system of any one of claims 21-23, 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 of claim 26, wherein the first promoter is a stronger promoter than the second promoter.

28. The system of claim 26, wherein the second promoter is a stronger promoter than the first promoter.

29. The system of any one of claims 21-28, wherein one or more of said nucleic acids is a viral vector.

30. The system of claim 29, wherein the viral vector is an AAV vector.

31. The system of any one of claims 21-30, wherein one or more of said nucleic acids is comprised in a lipid nanoparticle (LNP).

32. The system of any one of claims 15-20, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form.

33. The system of claim 32, wherein the Acr polypeptide and / or the CRISPR nuclease is comprised in a lipid nanoparticle (LNP).

34. The system of any one of claims 15-33, wherein the ratio of ON target to OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is greater than the ratio of ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

35. The system of claim 34, wherein the ratio of ON target to OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is at least 1.25 times greaterthan the ratio of ON target to OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

36. The system of any one of claims 15-35, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or more when compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

37. The system of any one of claims 15-36, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with 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 of any one of claims 15-37, wherein the contacting results in a modification to the nucleotide sequence of the target nucleic acid.

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

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

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

10.

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

10.

43. The method of any one of claims 38-42, wherein said contacting occurs in vivo.

44. The method of any one of claims 38-42, wherein said contacting occurs in a eukaryotic cell.

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

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

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

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

49. The method of any one of claims 38-48, wherein editing efficiency of ON target CRISPR nuclease activity is at least 4 times greater than editing efficiency of OFF target CRISPR nuclease activity.

50. The method of claim 49, wherein said OFF target CRISPR nuclease activity is at an off-target site that comprises no more than 5 mismatches as compared to the on- target site.

51. The method of any one of claims 38-50, wherein the OFF target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by 10% or more when compared with the OFF target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

52. The method of any one of claims 38-51, wherein the ON target activity of the CRISPR nuclease in the presence of the Acr polypeptide is decreased by no more than 40% as compared with the ON target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

53. The method of any one of claims 38-52, wherein the method further comprises measuring editing efficiency.

54. The method of any one of claims 38-53, wherein the method further comprises measuring editing efficiency at one or more off-target sites.

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