Engineered Acr proteins for modulating CRISPR activity

Abstract

Compositions and methods are provided that include engineered anti-CRISPR (Acr) proteins that can achieve a balance in which the Cas protein retains activity to perform targeting functions at a target nucleic acid (e.g., DNA cleavage for gene editing applications), but is inhibited by the subject Acr protein to a degree to reduce off-target activity, such that the subject Acr protein may not inhibit off-target activity. Thus, the ratio of target to off-target nucleic acid targeting events is increased. In some cases, the subject compositions (e.g., subject systems) include a CRISPR nuclease or a nucleic acid encoding the same. Methods of using the subject Acr polypeptides with CRISPR nuclease enzymes to achieve target nucleic acid modification are provided.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,494, filed on June 15, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporated by Reference

[0004] Sequence Listing provided as a Sequence Listing XML file

[0005] The Sequence Listing is provided herein as a Sequence Listing XML, "ACRG-006WO_SEQ_LIST.xml," created on June 5, 2023 and 557,231 bytes in size. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. I. Background Technology

[0006] CRISPR (clustered, regularly spaced, short palindromic repeats)-Cas systems are found in different bacterial and archaeal species, acting as immune defense mechanisms 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 provided great therapeutic potential for the treatment of human diseases. However, in practice, CRISPR-Cas mediated genome editing may be associated with off-targeting, for example, introducing unexpected mutations, insertions or deletions, and performing DNA recombination at unexpected "off-target" sites. Off-target editing caused by CRISPR-Cas systems has been reported in various cell and animal models (including human cells), and with the extension of nuclease activity, such events may accumulate in vivo. Further, human gene variation and the uncertain Cas protein expression lifespan in vivo increase the unpredictability of off-target events, which is a problem to be solved for the safe clinical translation of CRISPR-Cas systems. Unintended editing events can cause genomic instability, disrupt gene functionality, and induce serious adverse events, including cell death and cancer.

[0007] There is a need for compositions and methods that modify Cas activity, such as reducing off-target events (e.g., relative to on-target events), resulting in enhanced nucleic acid targeting specificity. The present disclosure provides such compositions and methods. II. Summary of the Invention

[0008] Anti-CRISPR (Acr) proteins have been found in bacteriophages to bind and inhibit certain Cas proteins, thereby thwarting the CRISPR system's attempts to cut the invading phage DNA. Thus, Acr proteins are apparently used by phages to circumvent the CRISPR-Cas system.

[0009] The inventors have generated non-naturally occurring engineered Acr proteins (ErAcr) (see, e.g., SEQ ID NO: 126-132 and 264-267), and they can be used to achieve a balance in which the Cas protein retains sufficient activity to perform the desired on-target nucleic acid function (e.g., loading, complex assembly, binding and cutting), but is inhibited to the extent of reducing off-target activity. Therefore, many compositions and methods disclosed herein include anti-CRISPR (Acr) polypeptides (ErAcr), or nucleic acids encoding Acr polypeptides, wherein the Acr polypeptides 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, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the sequence shown in any one of SEQ ID No: 126-132. Likewise, many compositions and methods disclosed herein include anti-CRISPR (Acr) polypeptides (ErAcr), or nucleic acids encoding Acr polypeptides, wherein the Acr polypeptides 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, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the sequence shown in any one of SEQ ID No 264-267. In some cases, the subject Acr polypeptide has 90% or more sequence identity with the sequence shown in any one of SEQ ID No: 126-132. In some cases, the subject Acr polypeptide comprises a sequence shown in any one of SEQ ID No: 126-132. In some cases, the subject Acr polypeptide has 90% or more sequence identity with the sequence shown in any one of SEQ ID No: 264-267. In some cases, the subject Acr polypeptide comprises a sequence shown in any one of SEQ ID No: 264-267.

[0010] Typically, Acr polypeptides are capable of reducing the activity of CRISPR nucleases. For example, in some embodiments, the subject Acr polypeptide reduces the off-target CRISPR nuclease activity by 10% or more (e.g., 20% or more, 30% or more, 50% or more, etc.) compared to the off-target (off-target) CRISPR nuclease activity in the absence of Acr polypeptides. In some embodiments, the subject Acr polypeptide reduces the on-target CRISPR nuclease activity by no more than 40% (e.g., no more than 30%, no more than 20%, etc.) compared to the on-target (on-target) CRISPR nuclease activity in the absence of Acr polypeptides. In some embodiments, the subject Acr polypeptide increases the ratio of on-target to off-target CRISPR nuclease activity compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of Acr polypeptides (e.g., an increase of at least 1.25x, 1.5x, 2x, 3x, etc.).

[0011] In some embodiments, the CRISPR nuclease is a Cas12a protein. In some embodiments, the CRISPR nuclease is a NUX protein. For example, in some cases, the subject Acr polypeptide reduces (inhibits) the activity of the Cas12a nuclease (see, e.g., SEQ ID NO: 175 and 245-262). In some cases, the subject Acr polypeptide reduces the activity of the CRISPR nuclease, and the CRISPR nuclease has 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 shown in any one of SEQ ID NO: 175 and 245-262. In some cases, the subject Acr polypeptide reduces the activity of the CRISPR nuclease, and the CRISPR nuclease has 90% or more sequence identity (e.g., 95% or more, or 100% sequence identity) with the Cas12a nuclease amino acid sequence shown in any one of SEQ ID NO: 175 and 245-262. In some cases, the subject Acr polypeptide reduces the 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, the subject Acr polypeptide reduces the 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.

[0012] In some embodiments, the subject Acr polypeptide reduces (inhibits) the activity of a NUX protein (see, e.g., SEQ ID NOs: 1-86 and 176-244). In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having 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 shown in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having 90% or more sequence identity (e.g., 95% or more, or 100% sequence identity) with the NUX protein amino acid sequence shown in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the subject Acr polypeptide reduces the activity of a CRISPR nuclease having 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: 176-178. In some cases, the subject Acr polypeptide reduces the 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.

[0013] In some cases, the subject Acr polypeptide reduces the 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, the subject Acr polypeptide reduces the 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.

[0014] The subject Acr polypeptide can be fused to a fusion partner. In some such cases, the subject Acr polypeptide is fused to a nuclear localization signal (NLS).

[0015] Also provided herein are systems including a subject Acr polypeptide (e.g., ErAcr) or a nucleic acid encoding an Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding a CRISPR nuclease. In such systems, the Acr polypeptide can reduce (inhibit) the activity of the CRISPR nuclease. The Acr polypeptide of such a system can be any of those discussed above (e.g., with SEQ ID NO: 126-132 having 75% or more sequence identity, for example, with SEQ ID NO: 264-267 having 75% or more sequence identity), and the CRISPR nuclease can also be any of those discussed above (e.g., Cas12a nucleases, such as SEQ ID NO: 175 and 245-262, NUX proteins, such as SEQ ID NO: 1-86 and 176-244, etc. - see, for example, SEQ ID NO: 263 CRISPR nuclease). In some cases, the subject system will also include a guide RNA for targeting the target sequence of the CRISPR nuclease to the target nucleic acid.

[0016] In some embodiments, the Acr polypeptide of the subject system (or method) may 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 shown in any one of SEQ ID NOs: 165-169. In some embodiments, the Acr polypeptide of the subject system (or method) may 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 shown 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 exist together. In some cases, the Acr polypeptide of the subject system (or method) 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, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169, and the CRISPR nuclease comprises an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) to the NUX protein amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244 or to the Cas12a amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system (or method) comprises an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) to the Acr amino acid sequence set forth in any one of SEQ ID NOs: 165-169, and the CRISPR nuclease comprises an amino acid sequence having 75% or more sequence identity (e.g., 80% or more, 85% or more, 90% or more, 95% or more, or 100% sequence identity) to the amino acid sequence set forth in 263.

[0017] Acr polypeptide and CRISPR nuclease can be provided in the form of nucleic acid (DNA, RNA) encoding protein independently of each other, or can be provided in the form of protein.When both are provided as nucleic acid, Acr polypeptide and CRISPR nuclease can be encoded on separate nucleic acid (for example, provided as two separate vectors), or can be encoded on the same nucleic acid (for example, provided on the same vector).In either case, translation control elements (for example, IRES sequence, 2A peptide coding sequence, non-standard start codon or any combination thereof) can be operably connected to Acr polypeptide coding sequence and / or CRISPR nuclease coding sequence.In some cases, the first promoter is operably connected to the Acr polypeptide coding sequence, and the second promoter is operably connected to the CRISPR nuclease coding sequence.In some such embodiments, the first promoter is stronger than the second promoter, and in other embodiments, the second promoter is stronger than the first promoter.Suitable promoters include but are not limited to: CMV, miniCMV, EF1A, CAG, CBh, EFS, SV40 and hPGK. In some cases, the nucleic acid (encoding Acr polypeptide, CRISPR nuclease or both) is a viral vector, e.g., an AAV vector. Whether the Acr polypeptide and / or CRISPR nuclease is provided in nucleic acid form or in protein form, in some cases, it can be included in a lipid nanoparticle (LNP) formulation.

[0018] Also provided herein is a method for utilizing the above-mentioned composition / system. For example, a method for modifying a target nucleic acid is provided, wherein the method includes contacting the target nucleic acid with an Acr polypeptide and a CRISPR nuclease (see above), wherein the contact results in modification (e.g., substitution, insertion, deletion) of the nucleotide sequence of the target nucleic acid. In some cases, the contact includes delivering CRISPR nuclease and Acr polypeptide at a ratio of 1:1. In other cases, the contact includes delivering CRISPR nuclease and Acr polypeptide at a ratio (CRISPR:Acr) in the range of 1:1.25 to 1:10. In other cases, the contact includes delivering Acr polypeptide and CRISPR nuclease at a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10. Contact can occur in vitro, in vitro and in vivo, and contact can occur in cells, such as eukaryotic cells, animal cells, mammalian cells, human cells, etc. In some embodiments, the target nucleic acid encodes a gene product (e.g., non-coding RNA, mRNA / protein, etc.).

[0019] On-target / off-target CRISPR activities mentioned in the subject compositions, systems or methods may include editing of the target nucleic acid (e.g., via DNA cutting in the presence or absence of a donor polynucleotide), e.g., genome editing. In some cases, the editing efficiency of the on-target CRISPR nuclease activity is greater than the editing efficiency of the off-target CRISPR nuclease activity (e.g., at least 2 times, at least 4 times, at least 5 times, or at least 10 times). In some cases, the off-target CRISPR nuclease activity is at an off-target site, and the off-target site includes no more than 5 mismatches compared to the on-target site. In some cases, the off-target activity of the CRISPR nuclease in the presence of an Acr polypeptide is reduced (e.g., reduced by 10% or more, 20% or more, 30% or more, etc.) compared to the off-target activity of the CRISPR nuclease in the absence of an Acr polypeptide. In some cases, the on-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced (e.g., reduced by no more than 10%, reduced by no more than 20%, reduced by no more than 30%, reduced by no more than 40%, etc.), compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0020] Thus, in some cases, the subject methods include measuring the editing efficiency at a target site. In some cases, the subject methods include measuring the editing efficiency at one or more off-target sites. III. Description of the drawings

[0021] Figure 1 Depicted is the 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 not AsCas12a.

[0022] Figure 2 Depicted is the editing efficiency of NUX (SEQ ID NO: 176) in the presence of Acrs tested in HEK293T cells.

[0023] Figure 3 Depicted are the results of dosing experiments using the CRISPR nuclease AsCas12a, with increasing amounts of Acr Acx-175 (left to right), using a guide that matches DNMT1 (for on-target editing) and one that is mismatched at position 9 (for measuring off-target editing). Acx-105 (which does not inhibit AsCas12a) was added to keep the relative amount of Acr protein consistent across samples.

[0024] Figure 4Depicted are the results of a dosing experiment using CRISPR nuclease NUX (SEQ ID NO: 177) with Acr Acx-175 (SEQ ID NO: 165) to observe the 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).

[0025] Figure 5 Depicted are the results of using seven engineered Acrs (ErAcrs) (Table 4, Acx-305, -306, -307, -308, -310, -311) that were transferred into a CMV expression vector and tested for on-target editing with NUX (SEQ ID NO: 178) and DNMT1 in HEK293T cells, and for off-target editing with a guide RNA with a single base change at position 8 (MM8).

[0026] Figures 6A-6C Results using four engineered Acrs (Acx-306, Acx-308, Acx-310, and Acx-311) are depicted. 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.

[0027] Figure 7 Depicted are the results of comparing Acr (Acx-175, Acx-306, Acx-310) and negative control Acx-105 with nuclease NUX (SEQ ID NO: 178) in HEK293T cells using guide RNAs that either perfectly matched the target or had one base mismatch at different positions in the guide RNA.

[0028] Figures 8A-8B Depicted are the results of on-target and off-target editing of DNMT1 (using MM8 guide RNA) in HEK293T cells using Acr Acx-315 (SEQ ID NO: 132) and NUX (SEQ ID NO: 178) at a range of Nux:Acx ratios. Fig. 8A The results are expressed as % editing efficiency, and Figure 8B Results are expressed as the ratio of on-target editing to off-target editing.

[0029] Figures 9A-9DA schematic diagram depicting a non-limiting example of the arrangement of components in a subject nucleic acid is shown. "P1" and "P2" are promoters, "Cas" is a Cas coding sequence, "Acr" is an Acr coding sequence, "2A" is a 2A peptide coding sequence, and "X" is a "spacer" sequence.

[0030] Figures 10A-10F A schematic diagram depicting a non-limiting example of the arrangement of components in a subject nucleic acid is shown. "P1" and "P2" are promoters, "Cas" is a Cas coding sequence, "Acr" is an Acr coding sequence, "IRES" is an IRES sequence, and "X" is a "spacer" sequence.

[0031] Figures 11A-11B Schematic diagrams depicting two non-limiting examples of arrangement of components in subject nucleic acids. "P1" and "P2" are promoters, "Cas" is a Cas coding sequence, "Acr" is an Acr coding sequence, and the asterisk indicates a non-AUG start codon.

[0032] Fig.12 Depicted are the results of on-target versus off-target editing experiments using ErAcr3, ErAcr4, ErAcr13, and ErAcr20.

[0033] Fig.13 Depicted is the sequence alignment of ErAcr3, ErAcr4, ErAcr13 and ErAcr20 (SEQ ID Nos: 264-27, respectively).

[0034] IV. Definitions

[0035] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0036] "Hybridizable", "hybridization", "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to non-covalently bind (i.e., form Watson-Crick base pairs and / or G / U base pairs), "anneal" or "hybridize" with another nucleic acid in a sequence-specific antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate conditions of temperature and solution ionic strength in vitro and / or in vivo. Standard Watson-Crick base pairs include: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs 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 is base paired with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, in the case of tRNA anticodon base pairing with codons in mRNA, G / U base pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code. Therefore, in the context of the present disclosure, guanine (G) (e.g., guanine (G) of the dsRNA duplex of the guide RNA molecule; guanine (G) of the guide RNA that base pairs with the target nucleic acid, etc.) is considered to be complementary to both uracil (U) and adenine (A). For example, when a G / U base pair can be formed at a given nucleotide position of the dsRNA duplex of the guide RNA molecule, the position is not considered to be non-complementary, but rather complementary.

[0037] Hybridization requires that two nucleic acids contain complementary sequences, although there may be mismatches between bases. The conditions applicable to hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the nucleic acid hybrid with these sequences. For hybridization between nucleic acids with shorter complementary segments (for example, complementary on 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides), the position of mismatch may become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of hybridizable nucleic acids is 8 nucleotides or more (for example, 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, washing solution salt concentration and other conditions can be adjusted as needed according to factors such as the length and degree of complementarity of the complementary region.

[0038] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0039] As used herein, "binding" (e.g., with respect to a nucleic acid binding domain of a polypeptide, binding to a target nucleic acid, etc.) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid such as DNA or RNA). When in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner). Not all components of the binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some parts of the binding interaction may be sequence-specific. Binding interactions can typically be expressed by less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M or less than 10 -15 The dissociation constant (K D ) is used to characterize the binding affinity. “Affinity” refers to the strength of binding, and increased binding affinity is associated with a lower K D Related.

[0040] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region that is capable of binding RNA polymerase and initiating transcription of downstream (3' direction) coding or non-coding sequences. For the purposes of this disclosure, a promoter sequence is defined at its 3' end by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including constitutive promoters, tissue-specific promoters, and inducible promoters, can be used to drive expression of various vectors of this disclosure. The expression level of a given promoter can be described as weak, medium, or strong - and thus promoters can be classified as weak promoters, medium promoters, or strong promoters.

[0041] "Operably linked" refers to a juxtaposition in which the components are in a relationship that permits them to function in the intended manner. For example, if a promoter affects the transcription of the nucleotide sequence, then the promoter is operably linked to a nucleotide sequence (one can also say that the nucleotide sequence is operably linked to the promoter). As another example, if a translation control element affects the translation of a protein from a protein coding sequence, then the translation control element is operably linked to a protein coding sequence (one can also say that the protein coding sequence is operably linked to the translation control element).

[0042] As used herein, "coordinated delivery system" refers to the coordinated delivery of Acr protein and Cas nuclease. The coordinated delivery system includes one or more nucleic acids (e.g., vectors) for expressing Acr protein and CRISPR nuclease, for example, in a host cell. In some cases, the coordinated delivery system provides a single nucleic acid (e.g., vector) for expressing Acr protein and CRISPR nuclease. In some cases, the coordinated delivery system provides more than one nucleic acid (e.g., vector) for expressing Acr protein and Cas nuclease, and the expression and / or function are coordinated, such as providing a fracture Cas from 2 vectors. In some cases, in the coordinated delivery system, the expression and / or function of Acr protein and Cas nuclease are connected (i.e., coordinated) by a translation control element selected to regulate the translation of Acr protein and / or CRISPR nuclease.

[0043] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms and / or therapeutic in terms of partially or completely curing a disease and / or side effects attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (e.g., a human) and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) arresting the disease, i.e., causing the disease to regress.

[0044] The terms "subject" and "host" used interchangeably herein refer to individual organisms expressing or intended to express the coordinated delivery system described herein and / or Cas nucleases and / or Acr proteins. Hosts include, but are not limited to, fungi (such as yeast), plants, algae, insects, animals, such as birds and mammals (e.g., mammals, including but not limited to rodents, apes, humans, mammal farm animals, mammal sports animals, and mammal pets). Hosts include, but are not limited to, microorganisms such as bacteria and fungi (such as yeast), plants, algae, insects, animals, such as birds and mammals (e.g., mammals, including but not limited to rodents, apes, humans, mammal farm animals, mammal sports animals, and mammal pets).

[0045] The terms "on target" (or "on target") and "off target" (or "off target") are used herein to refer to the position of CRISPR complex activity (e.g., target DNA cutting, DNA editing) within the target DNA. Both types of positions (on target and off target) are based on the guide sequence of the guide RNA. Events mediated by the CRISPR complex that occur at a position based on 100% matching with the guide sequence are considered to be "on target", while those events that occur at a (unwanted) position that is not based on 100% matching with the guide sequence are "off target". If the sequence of the target DNA is known (e.g., most of the target cell genome has been sequenced), possible off-target sites can be predicted for a given guide sequence. In general, off-target events are more likely to occur at sequences that are closer to 100% matching between the guide RNA and the target, rather than at sequences with a lower percentage identity between the guide RNA and the target. Therefore, most off-target events tend to occur at sequences with 50% or more (e.g., 75% or more) sequence identity to the expected target sequence. For example, a miss-target event may occur when there is 1 mispairing between the guide RNA and the target, 2 mispairings between the guide RNA and the target, or 3 mispairings between the guide RNA and the target. Therefore, the sequence analysis of the target DNA can provide a list of possible miss-target sites expected in the target DNA. The number of predicted miss-target sites will depend on the target DNA sequence, but in some cases, the number of predicted miss-target sites will be within the range of 10-200 predicted sites (e.g., 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. Specific Implementation Methods

[0046] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and therefore it may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0047] When providing a numerical range, it should be understood that each intervening value between the upper and lower limits of the range (unless the context clearly indicates otherwise, the intervening value is up to one-tenth of the unit of the lower limit) and any other intervening values ​​specified or in the specified range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included in the present invention, except for any limit that is specifically outside the specified range. When the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the present invention.

[0048] Certain ranges may be expressed herein as a numerical value preceded by the term "about." The term "approximately" is used herein to provide literal support for the exact number that follows it, as well as for a number that is close to or approximately the number that follows the term. In determining whether a number is close to or approximately an exact enumerated number, a number that is close to or approximately an unenumerated number may be a number that, in the context in which it is presented, provides an approximately equivalent exact enumerated number.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, representative illustrative methods and materials are now described.

[0050] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and are incorporated herein to disclose and describe methods and / or materials related to the content of the publication cited. 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 the publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0051] It should be 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 should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as "only", "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0052] It should be appreciated that certain features of the present invention described in the context of separate embodiments for the sake of clarity may also be provided in combination with a single embodiment. On the contrary, various features of the present invention described in the context of a single embodiment for the sake of simplicity may also be provided separately or in any suitable sub-combination form. For example, it will be apparent to those skilled in the art, after reading this disclosure, that each of the separate embodiments described and shown herein has discrete components and features that are easily separated or combined with the features of any other embodiments without departing from the scope or spirit of the present invention. All combinations belonging to embodiments of the present invention are specifically encompassed in the present invention and disclosed herein as each and every combination is individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed in the present invention and disclosed herein as each and every such sub-combination is individually and clearly disclosed herein.

[0053] As described above, the present disclosure provides Acr, including non-naturally occurring engineered Acr proteins (ErAcr), which can be used to achieve a balance in which the Cas protein retains sufficient activity to perform the desired function on the target nucleic acid (e.g., loading, complex assembly, binding and cutting), but is inhibited to the extent that off-target activity is reduced compared to the activity of the Cas protein in the absence of Acr (see, e.g., SEQ ID NO: 126-132). Therefore, many compositions and methods disclosed herein include anti-CRISPR (Acr) polypeptides or nucleic acids encoding Acr polypeptides, wherein 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 shown in any one of SEQ ID No: 126-132.

[0054] Also provided herein are systems and methods for modifying target nucleic acids using those systems, wherein the systems include a subject Acr polypeptide (e.g., ErAcr of SEQ ID NO: 126-132, and / or Acr of SEQ ID NO: 165-169) or a nucleic acid encoding an Acr polypeptide, and a CRISPR nuclease or a nucleic acid encoding a CRISPR nuclease. In some cases, the CRISPR nuclease and the Acr polypeptide of the system do not naturally exist together.

[0055] CRISPR complex

[0056] As used herein, the terms "CRISPR complex" and "effector complex" refer to a protein-RNA complex that is guided to a specific sequence within a target nucleic acid (e.g., target genomic DNA) by an RNA component - often referred to as a "guide RNA." In class 2 CRISPR systems, the function of the effector complex is performed by a single protein (which may be referred to as an "effector protein", also referred to herein as a "CRISPR nuclease" or simply a "Cas protein") - where the native 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. 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. 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. Microbiol. 2020 Feb;18(2):67-83).

[0057] Thus, 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 effector proteins from a Class 2 CRISPR system. Non-limiting examples of CRISPR nucleases include Cas12a proteins and NUX proteins (described elsewhere herein).

[0058] Acr protein

[0059] Acr proteins are proteins that inhibit Cas proteins (reduce the activity of Cas proteins) - thereby acting as negative regulators of CRISPR complexes. In some cases, the subject Acr proteins are inhibitors of Cas proteins of class 2 CRISPR complexes (e.g., class 2 effector proteins, such as Cas12 proteins, e.g., Cas12a - also known as Cpf1) - thereby directly regulating the effector proteins of the CRISPR complex.

[0060] The effector CRISPR-Cas nuclease compounded with gRNA is highly diverse and spread across 6 different types (types I-VI). So far, anti-CRISPR proteins (Acr) that inhibit CRISPR type I, type II, and type V systems have been found. SpCas9, a type II-A CRISPR-Cas ortholog from Streptococcus pyogenes (S.pyogenes), is the most widely used CRISPR-Cas enzyme for biotechnology applications, and has also been deployed in DNA binding applications. Acr proteins that work for type II-A systems have been found by bioinformatics methods, which investigate the self-targeting of bacterial genomes. Among the different Acr protein families found so far, specific inhibition mechanisms of some of them (e.g., AcrIE1, AcrIF1-3, AcrIF10, AcrIIA2, AcrIIA4, AcrIIC1-3, and AcrVA5) have been determined. The known mechanism is highly diverse, presenting a pool of off-switch modalities for reference. Acr proteins can act at three 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 so far is that anti-CRISPR proteins occupy the DNA binding site on the Cas protein, thereby mimicking DNA and inhibiting the protein's DNA binding and cleavage activities.

[0061] However, the mechanisms by which Acr blocks DNA binding may be different. For example, even though AcrIF1, AcrIF2, and AcrIF10 bind to different subunits of the cascade effector complex of the IF-type CRISPR-Cas system, they all prevent DNA from binding to the complex. AcrIIC3 also blocks DNA binding, but uses a fourth different mechanism - promoting dimerization of Cas9. For the most potent SpCas9 inhibitor, AcrIIA4, High-resolution cryo-electron structures revealed that the Cas9-sgRNA-AcrIIA4 complex has AcrIIA4 bound to the PAM interaction domain of Cas9, thereby preventing target DNA binding. Interestingly, AcrIIA4 only binds to assembled Cas9-sgRNA complexes, but not to Cas9 protein alone or to preformed Cas9-sgRNA-DNA complexes.

[0062] It should be understood that when discussing specific proteins such as Cas or Acr proteins (e.g., "Cas12a", "Nux", "ErAcr", "Acr") throughout this disclosure, and when such terms are presented in the claims, such terms are intended to cover modified / mutated forms of such proteins that maintain their intended functions. These terms are intended to cover embodiments in which Cas and / or Acr proteins are fused to one or more heterologous proteins (e.g., fluorescent proteins (such as GFP), one or more nuclear localization signals (NLS), and / or tags, such as MBP, CBP, strep tags, GST, HA, poly (His), Myc, V5, Spot, NE, AviTag, etc.).

[0063] Therefore, in some embodiments, the theme "Acr protein" comprises a wild-type (natural) sequence. In some cases, the theme Acr protein is a mutation. As described above, the inventors have generated non-naturally occurring engineered Acr proteins (ErAcr) (see, e.g., SEQ ID NO: 126-132), and it can be used to achieve a balance, wherein the Cas protein retains enough activity to perform the desired on-target nucleic acid function (e.g., loading, composite assembly, binding and cutting), but is suppressed to the extent of reducing off-target activity. Therefore, many compositions and methods disclosed herein include anti-CRISPR (Acr) polypeptides (ErAcr), or nucleic acids encoding Acr polypeptides.

[0064] Examples of engineered Acr proteins (ErAcr) provided herein include SEQ ID NOs: 126-132 (see, e.g., working examples). Additional examples of ErAcr provided herein include SEQ ID NOs: 264-267 (see, e.g., Example 7 below). Additional examples of Acr proteins provided herein include those shown in SEQ ID NOs. 165-169.

[0065] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the composition, system or method disclosed 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 shown 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 shown 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 shown in any one of SEQ ID NO: 126-132. 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 shown in any one of SEQ ID NO: 126-132. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in any one of SEQ ID NO: 126-132.

[0066] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 126. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 126.

[0067] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown in SEQ ID NO: 127. 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 shown in SEQ ID NO: 127. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 127.

[0068] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 128. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 128.

[0069] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown in SEQ ID NO: 129. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 129.

[0070] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems, or methods disclosed 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 shown 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 shown in SEQ ID NO: 130. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 130.

[0071] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 131. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 131.

[0072] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 132. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 132.

[0073] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the composition, system or method disclosed 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 shown 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 shown 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 shown in any one of SEQ ID NO: 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 shown in any one of SEQ ID NO: 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 shown in any one of SEQ ID NO: 126-132 and 264-267.

[0074] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the composition, system or method disclosed 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 shown 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 shown in any one of SEQ ID NOs: 264-267. In some cases, the subject Acr protein comprises an amino acid sequence with 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence shown in any one of SEQ ID NO:264-267. In some cases, the subject Acr protein comprises an amino acid sequence with 95% or more sequence identity (e.g., 97% or more, 98% or more, 99% or more, or 100% sequence identity) to the Acr protein amino acid sequence shown in any one of SEQ ID NO:264-267. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in any one of SEQ ID NO:264-267.

[0075] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 264. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 264.

[0076] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown in SEQ ID NO: 265. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 265.

[0077] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 266. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 266.

[0078] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 267. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 267.

[0079] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the composition, system or method disclosed 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 shown 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 shown 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 shown in any one of SEQ ID NO: 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 shown in any one of SEQ ID NO: 165-169. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in any one of SEQ ID NO: 165-169.

[0080] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown in SEQ ID NO: 165. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 165.

[0081] In some cases, the subject Acr protein comprises the Acr protein amino acid sequence shown in SEQ ID NO: 165, but has an amino acid variation at at least one position between amino acid 2 and amino acid 159 of SEQ ID NO: 165 (including amino acid 2 and amino acid 159) (see, e.g., Table 6). In some cases, the Acr polypeptide comprises one or more amino acid variations listed in Table 6. In some cases, the Acr polypeptide comprises one or more amino acid variations listed in Table 7.

[0082] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the composition, system or method disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 166. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 166.

[0083] In some cases, the subject Acr protein (ie, polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 167. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 167.

[0084] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown 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 the Acr protein amino acid sequence shown 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 shown in SEQ ID NO: 168. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 168.

[0085] In some cases, the subject Acr protein (i.e., polypeptide) (e.g., the Acr protein of the compositions, systems or methods disclosed 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 shown 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 shown in SEQ ID NO: 169. In some cases, the subject Acr protein comprises an Acr protein amino acid sequence shown in SEQ ID NO: 169.

[0086] CRISPR nucleases

[0087] As with the theme Acr polypeptide, in some cases, the theme CRISPR nuclease comprises a wild-type (natural) sequence. In some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease comprises an amino acid sequence with a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein) 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). In some cases, the CRISPR nuclease comprises an amino acid sequence with a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein) 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). 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., Cas12a or 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., Cas12a or Nux protein). In some cases, the CRISPR nuclease comprises an amino acid sequence of a wild-type CRISPR nuclease (e.g., Cas12a or Nux protein). In some cases, the subject CRISPR nuclease has been "evolved" so that it has a low overall sequence homology with a natural CRISPR nuclease, but retains the recognizable characteristic domains of the protein. In some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease is a Cas12a protein (see, e.g., SEQ ID NOs: 175 and 245-262). In some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease is a NUX protein (see, e.g., SEQ ID NOs: 1-86 and 176-244). In some cases, the CRISPR nuclease is SEQ ID NO: 263.

[0088] 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) to 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) to 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) to 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) to the amino acid sequence set forth in SEQ ID NO: 263. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 263.

[0089] Examples of CRISPR nucleases include those in Table 1.

[0090] Table 1. Examples of CRISPR nuclease proteins

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] As described above, in some cases, the Cas effector protein (i.e., CRISPR nuclease) is a variant (modified / mutated) (i.e., comprising one or more amino acid mutations, such as substitutions, insertions, deletions, relative to the wild-type Cas effector protein). For example, in some cases (e.g., in the systems and / or methods disclosed herein), the CRISPR nuclease has one or more amino acid mutations relative to the wild-type protein.

[0099] Cas12a protein

[0100] Thus, 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the CRISPR nuclease comprises an amino acid sequence set forth in any one of SEQ ID NOs: 175 and 245-262.

[0101] 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) to 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) to 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) to 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% sequence identity) to 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 set forth in any one of SEQ ID NOs: 245-262.

[0102] 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) to 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) to 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) to 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) to the amino acid sequence set forth in SEQ ID NO: 175. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 175.

[0103] NUX proteins

[0104] The term "NUX" protein is used herein to refer to a nuclease having Cas-like activity (RNA-guided CRISPR effector protein) and having an amino acid sequence of any one of SEQ ID NOs: 1-86 and 176-244, or an amino acid sequence of any one of SEQ ID NOs: 1-86 and 176-244 having at least 70% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, or 99% identity. 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 the Cas12a (Cpf1) protein. In some embodiments, the amino acid sequence of the NUX protein lacks identity or significant amino acid homology with 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% similarity to the Cas12a (Cpf1) protein, SaCas9, or SpyCas9 protein.

[0105] 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the CRISPR nuclease comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-86 and 176-244.

[0106] 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence having 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 176-178. In some cases, the CRISPR nuclease comprises an amino acid sequence set forth in any one of SEQ ID NOs: 176-178.

[0107] 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) to 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) to 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) to 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) to the amino acid sequence set forth in SEQ ID NO: 176. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 176.

[0108] 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) to 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) to 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) to 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) to the amino acid sequence set forth in SEQ ID NO: 177. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 177.

[0109] 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) to 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) to 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) to 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) to the amino acid sequence set forth in SEQ ID NO: 178. In some cases, the CRISPR nuclease comprises the amino acid sequence set forth in SEQ ID NO: 178.

[0110] Guide RNA

[0111] In some embodiments, the subject composition, system or method includes a guide RNA (or a nucleic acid encoding a guide RNA). For example, in some cases, the subject composition, system or method (e.g., a vector or vector system) includes an expression cassette comprising a promoter operably linked to a sequence encoding the guide RNA. In some such cases, the promoter is an RNA polymerase III promoter (e.g., U6, H1), which can be used to express non-coding RNA in eukaryotic cells.

[0112] A "guide RNA" is a nucleic acid that binds to a Cas protein (e.g., a class 2 CRISPR nuclease, such as Cas12a), thereby forming a CRISPR complex (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 understood that in some cases, a hybrid DNA / RNA can be prepared such that the guide RNA includes DNA bases in addition to RNA bases - but the term "guide RNA" is still used herein to cover such hybrid molecules.

[0113] The guide RNA provides target specificity for the CRISPR complex by including a targeting segment, the targeting segment including a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the sequence of the target nucleic acid. Therefore, the subject guide RNA includes (i) a guide sequence (also referred to as a "protospacer" or "targeting sequence") that hybridizes with a target sequence (also referred to as a "protospacer") of a target nucleic acid (e.g., a target DNA); and (ii) a constant region (e.g., a region adjacent to the guide sequence and bound to the Cas protein). The "constant region" may also be referred to herein as a "protein binding segment" or "handle". Therefore, the position of the target event (e.g., target DNA cleavage, transcriptional regulation, DNA methylation, histone modification) is actually determined by the guide sequence of the guide RNA. CRISPR complex-mediated events that occur at positions that are not 100% matched to the guide sequence are referred to herein as off-target events.

[0114] A guide RNA may be represented by its corresponding protein. For example, when the guide RNA binds to and guides a Class 2 CRISPR / Cas effector protein, the guide RNA may 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 may be referred to as a "Cpf1 guide RNA" or a "Cas12a guide RNA".

[0115] In some embodiments, the guide RNA includes two separate nucleic acid molecules: an "activator" (e.g., tracrRNA) and a "targeting agent" (e.g., crRNA), and is referred to herein as a "dual guide RNA", "dual molecule guide RNA", "two molecule guide RNA" or "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., crRNA and tracrRNA) - and the two molecules (activator, e.g., tracrRNA, and targeting agent, e.g., crRNA) can be covalently linked to each other, for example, 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", "single molecule guide RNA", "one molecule guide RNA", or simply "sgRNA". "Guide RNA" (or "gRNA") is a general term covering dual-guide and single-guide forms.

[0116] The guide sequence has complementarity (hybridization) with a target sequence of a target nucleic acid (e.g., a target DNA). In some cases, the guide sequence has a length of 15-28 nucleotides (nt) (e.g., a length of 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 some cases, the guide sequence has a length of 18-24 nucleotides (nt). In some cases, the guide sequence has a length of 17-18 nucleotides (nt). 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.

[0117] 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.

[0118] Various modifications (e.g., by chemical modification, changes in spacer length, sequence modification in spacers or scaffolds, fusion with additional DNA or RNA components, replacement with DNA moieties, etc.) with increased efficiency relative to naturally occurring guide RNAs are known in the art and readily available to those of ordinary skill in the art. See, e.g., Moon et al., Trends Biotechnol. 2019 August; 37(8): 870-881, "Improving CRISPRGenome Editing by Engineering Guide RNAs". As used herein, the term "guide RNA" encompasses such modifications, and any convenient guide RNA can be used with the methods and compositions disclosed herein (e.g., as part of a subject system - e.g., as RNA or encoded by a subject nucleic acid).

[0119] Acr / CRISPR Nuclease Combination – “The System”

[0120] Typically, the term "system" is used herein to refer to a 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 described above, in some cases, the Acr polypeptide and the CRISPR nuclease do not naturally exist together. In some cases, the subject system also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0121] In some embodiments (e.g., in the systems and / or methods disclosed herein), the Acr polypeptide and the CRISPR nuclease do not naturally exist together (i.e., they are heterologous to each other). In some cases, the Acr polypeptide and / or the CRISPR nuclease include 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 a bacterial species carrying a CRISPR nuclease).

[0122] Since any protein can be provided as a nucleic acid (DNA or RNA) or in the form of a protein, the subject system may include any combination thereof. For example, in some embodiments, the subject system includes an Acr polypeptide or a nucleic acid (DNA or RNA) encoding an Acr polypeptide; and a CRISPR nuclease or a nucleic acid (DNA or RNA) encoding a CRISPR nuclease. In some cases, the subject system includes a CRISPR nuclease and a nucleic acid encoding an Acr polypeptide. In some cases, the subject system includes an Acr polypeptide and a nucleic acid encoding a CRISPR nuclease. In some cases, the subject system includes a nucleic acid encoding an Acr polypeptide and a nucleic acid encoding a CRISPR nuclease. In some such cases, the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the CRISPR nuclease are on the same nucleic acid (e.g., the same vector). In other cases, the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the CRISPR nuclease are on different nucleic acids (e.g., different vectors). For discussion of nucleic acids (e.g., vectors, promoters, translation control elements, etc.), see elsewhere herein.

[0123] The Acr polypeptides and CRISPR nucleases of the subject systems can be any of those described herein, in any desired combination.

[0124] Acr protein (SEQ ID NO: 165-169) plus CRISPR nuclease (Cas12a or NUX)

[0125] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-168; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the Acr polypeptide of the 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 shown in 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 shown in any one of SEQ ID NO: 1-86 and 175-262. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0126] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-168; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the 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 shown in 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 shown in any one of SEQ ID NO: 175 and 245-262. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0127] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-168; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 165; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 175. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0128] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-168; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the 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 shown in 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 shown in any one of SEQ ID NO: 1-86 and 176-244. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0129] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 165-168; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the Acr polypeptide of the 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 shown in 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 shown in any one of SEQ ID NO: 176-178. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0130] In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 176. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-167; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 176. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NO: 165 or 167; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 176.In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 165; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 176. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0131] In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 165; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with the amino acid sequence as shown in SEQ ID NO: 177. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0132] In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 165-169; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 178. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to the amino acid sequence as set forth in SEQ ID NO: 178. In some cases, the Acr polypeptide of the 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 shown in 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 shown in SEQ ID NO: 178. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0133] Acr (SEQ ID NO: 126-132) plus CRISPR nuclease

[0134] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-262. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0135] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having 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) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0136] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0137] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0138] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0139] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0140] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO: 263. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 126-132; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in SEQ ID NO: 263. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0141] Acr (SEQ ID NO: 264-267) plus CRISPR nuclease

[0142] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having 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) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 175-263. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0143] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 175 and 245-262. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0144] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in SEQ ID NO: 175. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0145] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-86 and 176-244. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0146] In some cases, the Acr polypeptide of the subject system comprises the amino acid sequence set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-86, 176-244, and 263. In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence having 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 an amino acid sequence as shown in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) with an amino acid sequence as shown in any one of SEQ ID NOs: 1-86, 176-244, and 263. In some cases, the system described in this paragraph also includes a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0147] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 176-178. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0148] In some cases, the Acr polypeptide of the subject system comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease comprises an amino acid sequence as set forth in SEQ ID NO: 263. In some cases, the Acr polypeptide of the subject system 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in any one of SEQ ID NOs: 264-267; and the CRISPR nuclease 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, 98% or more, 99% or more, 99.5% or more, or 100% sequence identity) to an amino acid sequence as set forth in SEQ ID NO: 263. In some cases, the systems described in this paragraph also include a guide RNA (which guides the CRISPR nuclease to the target sequence) or a nucleic acid encoding a guide RNA.

[0149] Fusion

[0150] 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, protein tags, etc.). 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 (NLS) for targeting to the nucleus, sequences for retaining the fusion protein outside the nucleus, e.g., nuclear export sequences (NES), sequences for retaining the fusion protein in the cytoplasm, mitochondrial localization signals for targeting to mitochondria, chloroplast localization signals for targeting to chloroplasts, ER retention signals, etc.); (ii) protein tags, e.g., to facilitate tracking and / or purification (e.g., fluorescent proteins, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc., MBP, CBP, strep tag, GST, HA, FLAG, poly(His), Myc, V5, Spot, NE, AviTag, etc.); and (iii) polypeptides that provide increased or decreased stability (e.g., degradation determinants, which are controllable in some cases, e.g., temperature-sensitive or drug-controllable degradation determinant sequences).

[0151] The subject protein (e.g., Acr protein and / or CRISPR nuclease, such as Cas12a or NUX protein) can have multiple (1 or more, 2 or more, 3 or more, etc.) fusion partners in any combination. As an illustrative example, the subject protein may have a fusion partner (e.g., GFP) that provides a label, and may also have a subcellular localization sequence (e.g., one or more NLS). In some cases, such fusion proteins may also have a tag that is convenient for tracking and / or purification. As another illustrative example, the subject protein (e.g., Acr protein, such as the subject engineered Acr protein) may have one or more NLS (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4 or 5 NLS). In some cases, the 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 the C-terminus.

[0152] Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, which has the amino acid sequence PKKKRKV (SEQ ID NO:87); an NLS from a nuclear plasmin (e.g., a nuclear plasmin bipartite NLS having the sequence KRPAATKKAGQAKKK (SEQ ID NO:88)); a c-myc NLS having the amino acid sequence PAAK RVKLD (SEQ ID NO:89) or RQRRNELKRSP (SEQ ID NO:90); an hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYF AKPRNQGGY (SEQ ID NO:91); the sequence RMRIZFKNKGKDTAELRRRRVEVSVEL RKAKKDEQILKRRNV (SEQ ID NO:92) from the IBB domain of importin-α; the sequence VSR KRPRP (SEQ ID NO:93) of the myoma T protein. NO:93) and PPKKARED (SEQ ID NO:94); the sequence of human p53 PQPKKKPL (SEQ ID NO:95); the sequence of mouse c-abl IV SAL IKKKKKMAP (SEQ ID NO:96); the sequences DRLRR (SEQ ID NO:97) and PKQKKRK (SEQ ID NO:98) of influenza virus NS1; the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO:99); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:100); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:101); and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:102). In some cases, the NLS has the sequence GRSSDDEATADSQHAAPP KKKRKV (SEQ ID NO: 125). Typically, the NLS (or multiple NLSs) have sufficient strength to drive the Cas protein to accumulate in the nucleus of a eukaryotic cell in a detectable amount.

[0153] In some cases, the fusion partner includes a "protein transduction domain" or PTD (also known as CPP-cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversal of a lipid bilayer, a micelle, a cell membrane, an organelle membrane, or a vesicle membrane. The PTD attached to another molecule (the molecule may range from a small polar molecule to a macromolecule and / or a nanoparticle) facilitates the molecule to traverse the membrane, for example, from the extracellular space into the intracellular space or the cytosol into the organelle. In some embodiments, the PTD is covalently linked to the amino terminus of the polypeptide, and in some embodiments, the PTD is covalently linked to the carboxyl terminus of the polypeptide. In some cases, the PTD is inserted internally at a suitable insertion site. In some cases, the subject Cas protein includes (conjugated to, 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 sufficient number of arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) to direct entry into a cell; a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a 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); a polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLM KR (SEQ ID NO:104); transporter GWTLNSAGYLLGKINLKALAALA KKIL (SEQ ID NO:105); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:106); and RQIKIWFQNRRMKWKK (SEQ ID NO:105); NO:107).Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 108), RKKRRQRRR (SEQ ID NO: 109); arginine homopolymers of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 110); RKKRRQRR (SEQ ID NO: 111); YARAAARQARA (SEQ ID NO: 112); THRLPRRRRRR (SEQ ID NO: 113); and GGRRARRRRRR (SEQ ID NO: 114). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPP comprises a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero and thereby inhibits binding and uptake into cells. When the linker is cleaved, the polyanion is released, locally exposing polyarginine and its inherent adhesive properties, thereby "activating" the ACPP to traverse the membrane.

[0154] Linkers (e.g., for fusion partners)

[0155] In some embodiments, the subject Cas protein can be fused to the 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. The protein may be connected by a spacer peptide that is generally flexible, but other chemical connections are not excluded. Suitable linkers include polypeptides having a length between 4 amino acids and 40 amino acids, or a length between 4 amino acids and 25 amino acids. These linkers can be produced by coupling proteins using synthetic oligonucleotides encoding linkers, or can be encoded by a nucleic acid sequence encoding a fusion protein. Peptide linkers with certain flexibility can be used. The connecting peptide can actually have any amino acid sequence, remember: the preferred linker will have a sequence that produces a general flexible peptide. The use of small amino acids such as glycine and alanine is useful in producing flexible peptides. The generation of such sequences is conventional for those skilled in the art. Various different linkers are commercially available and are considered suitable for use.

[0156] Examples of linker polypeptides include glycine polymers (G) n ; Glycine-serine polymers (including, for example, (GS) n 、GSGGS n (SEQ ID NO: 115), GGSGGS n(SEQ ID NO: 116) and GGGS n (SEQ ID NO: 117, wherein n is an integer of at least one); glycine-alanine polymer; alanine-serine polymer. Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 118), GGSGG (SEQ ID NO: 119), GSGSG (SEQ ID NO: 120), GSGGG (SEQ ID NO: 121), GGGSG (SEQ ID NO: 122), GSSSG (SEQ ID NO: 123), etc. One of ordinary skill will recognize that the design of a peptide conjugated to any desired element may include a fully or partially flexible linker, such that the linker may include a flexible linker and one or more portions that confer a less flexible structure.

[0157] Translational control elements

[0158] The present disclosure provides compositions, systems and methods including one or more nucleic acids, wherein the one or more nucleic acids encode Acr proteins and CRISPR nucleases-wherein the Acr proteins are inhibitors of Cas proteins (e.g., Cas effector proteins / CRISPR nucleases), such as preferentially inhibiting off-target editing compared to 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).

[0159] The subject nucleic acid may include a translation control element operably connected to an Acr protein or a Cas protein (e.g., a CRISPR nuclease, such as Cas12a, a NUX protein, or a variant thereof) or both - so as to achieve a desired balance (ratio of expression levels) between the two proteins. For example, in some cases, the subject nucleic acid includes a translation control element operably connected to a sequence encoding an Acr protein (and thus regulating / regulating its translation). In some cases, the subject nucleic acid includes a translation control element operably connected to a sequence encoding a Cas protein (and thus regulating / regulating its translation). In some cases, the sequence encoding an Acr protein and the sequence encoding a Cas protein are both operably connected to a translation control element, for example, independently connected (in this case, the two control elements may be the same or different).

[0160] In some cases, the sequence encoding the Acr protein and the sequence encoding the Cas protein are both operably linked to the same translation control element (e.g., IRES element, 2A peptide coding sequence), so that the sequence is part of a polycistronic transcript. Therefore, in some cases, the subject translation control element is a polycistronic linker. In other words, in some cases, the translation control element promotes (causes) the production of independent gene products (e.g., Acr protein and CRISPR nuclease) from the same transcript.

[0161] Thus, in some cases, the translation control element connects a first protein coding sequence (e.g., a sequence encoding an Acr protein) to a second protein coding sequence (e.g., a sequence encoding a CRISPR nuclease) such that the first and second proteins (e.g., an Acr protein and a CRISPR nuclease) are encoded by a polycistronic sequence. Thus, in such cases, both protein sequences are operably linked to the same promoter, and the RNA transcribed therefrom includes two protein coding sequences in addition to the sequence encoded by the translation control element.

[0162] As described in more detail below, in some cases, more than one translation control element (e.g., IRES element, 2A peptide, non-AUG start codon) is used to control the expression of a given protein (e.g., Acr protein, CRISPR nuclease (such as Cas12a or Nux protein)). Any convenient combination of translation control elements can be used.

[0163] 2A peptide

[0164] One non-limiting example of a translational control element, which can function as a polycistronic linker and facilitate the production of separate protein products (eg, two separate proteins) from the same single RNA transcript, is a 2A peptide sequence.

[0165] "2A peptide" refers to a small peptide sequence (usually 18-25 amino acids, although several such sequences may be placed in tandem) that allows the expression (translation) of discrete protein products from a single RNA transcript (e.g., by a self-"cleavage" event often referred to as "ribosome jumping" - although the disclosure herein is not dependent on and is not bound by a mechanism of action), even if the separate proteins are encoded as part of the same open reading frame (ORF). 2A peptides are readily identified by their consensus motif (DXEXNPGP, sometimes described as DVEXNPGP) and their ability to promote protein cleavage / jumping. Any convenient 2A peptide sequence may be used for the subject nucleic acid. Examples of 2A peptides include, but are not limited to, 2A peptides from viruses, such as foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine Teschovirus-1 (P2A), or Trichogramma virus (T2A). See, e.g., Szymczak-Workman, A. et al. "Design and Construction of 2A Peptide-Linked Multicistronic Vectors". Cold Spring Harb Protoc. Feb. 1, 2012; 2012(2):199-204; Liu et al., Sci Rep. 2017; 7:2193; Kim et al., PLOS One 6:e18556, 2011; and U.S. Pat. Nos. 10,738,325; 9,655,956; 10,577,417; the disclosures relating to 2A peptides are incorporated herein by reference.

[0166] Typically, the subject 2A peptide coding sequence will be positioned so as to regulate the expression (translation) of the subject protein (e.g., Acr protein or Cas protein) - and will therefore be positioned 5' (usually immediately 5') and in frame with the protein coding sequence it regulates. Figure 9 provides non-limiting illustrative examples of embodiments in which the 2A peptide coding sequence is positioned in different ways. In some cases, the 2A peptide sequence is the 5' position (and usually immediately 5') of the Cas protein coding sequence. In some cases, the 2A peptide sequence is the 5' position (and usually immediately 5') of the Acr protein coding sequence.

[0167] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to the same promoter (encoded by the polycistronic sequence) and positioned in tandem, with the 2A peptide sequence positioned between them (see, e.g., Fig. 9A and 9B). In some such cases, the Cas protein coding sequence is located 5' of the Acr coding sequence, and thus the 2A peptide coding sequence is 3' of the Cas sequence and 5' of the Acr sequence. In other such cases, the Acr protein coding sequence is located 5' of the Cas coding sequence, and thus the 2A peptide coding sequence is 3' of the Acr sequence and 5' of the Cas sequence.

[0168] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to a first promoter and a second promoter, respectively, such that they are transcribed as separate transcripts (see, e.g., Fig. 9C and 9D ). The first promoter and the second promoter (labeled "P1" and "P2" in the figure) can be different from each other or can be the same (i.e., can be copies of the same promoter). In some such cases, the 2A peptide sequence regulates the Acr sequence (and is therefore located 5' to the Acr sequence). In other cases, the 2A peptide sequence regulates the Cas sequence (and is therefore located 5' to the Cas sequence). In some cases where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, each is regulated by the 2A peptide sequence (and therefore each is located 3' to the 2A peptide sequence).

[0169] In some embodiments where the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, a "spacer" protein coding sequence is used 5' to the 2A peptide sequence so that the "spacer" sequence is transcribed as part of a polycistronic sequence in which the protein sequence is regulated. Fig. 9C In the , the Cas protein coding sequence and the Acr protein coding sequence are operably linked to different promoters (P1 and P2). The spacer sequence (marked as "X" in the figure) is positioned 5' to the 2A peptide sequence, which is 5' to the Acr coding sequence - and thus the spacer sequence and the Acr sequence are transcribed as part of the same RNA. However, the presence of the 2A peptide sequence causes the Acr protein to be produced as a separate protein. Similarly, in Fig.9D In the example, the Cas protein coding sequence and the Acr protein coding sequence are again operably connected to different promoters (P1 and P2). In this example, the spacer sequence (marked as "X" in the figure) is located at the 5' of the 2A peptide sequence, which is the 5' of the Cas coding sequence-and therefore the spacer sequence and the Cas sequence are transcribed as part of the same RNA. However, the presence of the 2A peptide sequence in this RNA causes the Cas protein to be produced as a separate protein.

[0170] "Spacer" protein can be any desired sequence-because its purpose is to simply provide a sequence to be translated, the sequence is the 5' (N-terminus) of the 2A peptide sequence. The spacer sequence can be any convenient length, from very short to encoding a complete 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 1 to 100 amino acids (e.g., 1 to 80, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 100, 2 to 80, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 100, 5 to 80, 5 to 50, 5 to 40, 5 to 30, 5 to 20, or 5 to 10 amino acids). Examples of spacer sequences include, but are not limited to, linker sequences, repeated single amino acids (e.g., AAAA), random sequences, protein fragments, and marker proteins (e.g., fluorescent proteins (such as GFP, YFP, CFP, RFP, etc.), drug-selective protein markers, enzymes (such as β-galactosidase, etc.)).

[0171] Examples of 2A peptide sequences include, but are not limited to, those shown as SEQ ID NOs: 133-138.

[0172] 2A peptide sequences can be used in series, 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, the 2A peptide sequence is selected from the group consisting of: P2A, F2A, E2A, T2A, and any combination thereof. In some embodiments, the translation control element encodes 2 or more 2A peptides (e.g., 3 or more, 4 or more, or 5 or more) in series. In some embodiments, the translation control element encodes 2, 3, 4, or 5 2A peptides in series. In some embodiments, the translation control element encodes one 2A peptide.

[0173] In some cases, the 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) to the amino acid sequence set forth in any one of SEQ ID NOs: 133-138.

[0174] IRES

[0175] In some embodiments herein, the translation control element is an internal ribosome entry site (IRES) sequence. "Internal ribosome entry site" or "IRES" refers to a nucleotide sequence that allows protein translation to be initiated inside a messenger RNA (mRNA) sequence (i.e., downstream of the first start codon). For example, when an IRES segment is between two open reading frames in a bicistronic eukaryotic mRNA molecule, it can drive the translation of the downstream protein coding region, regardless 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 setting, both proteins are produced in cells. The protein located in the first cistron is synthesized by a cap-dependent initiation mechanism, and the translation initiation of the second protein is guided by the IRES segment in the intercistronic spacer located between the two protein coding regions. IRES has been separated from viral genomes and cell genomes. Artificially engineered IRES is also known in the art. One of ordinary skill in the art will recognize that a sequence described herein as an IRES sequence (which functions as part of an RNA molecule) will have a related sequence in an encoding DNA molecule, e.g., an RNA sequence 5'-uuacuggc-3' will correspond to a DNA sequence 5'-ttactggc-3', and vice versa. The term "IRES sequence" or simply "IRES" is used herein to refer to any sequence.

[0176] Any convenient IRES can be used in the subject compositions, systems and methods. Examples of IRES sequences include, but are not limited to, Fig.13 A-13D (SEQ ID NO: 139-159). One of ordinary skill in the art will recognize that when the subject system is used to express Cas and Acr proteins in non-animal cells (e.g., plants / plant cells), they should select a convenient IRES sequence suitable for the desired cell type (e.g., an IRES from wheat mosaic virus (TriMV)). See, e.g., Urwin et al., Plant J. 2000 December; 24(5): 583-9 and U.S. Pat. Nos. 8,772,465; 9,879,271, each of which is incorporated by reference for teachings on the use of IRES sequences in plants.

[0177] In some cases, the IRES sequence comprises a nucleotide 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) to the nucleotide sequence set forth in any one of SEQ ID NOs: 139-159.

[0178] In some cases, the IRES sequence is selected from the group consisting of the following IRES sequences: EMCV, BIP, CAT-1, c-myc, HCV, VCIP, Apaf-1, mEMCV-1, mEMCV-2, HRV, NRF, FGF-1, KMI1, KMI2, (GAAA)16, (PPT19)4, EMCV 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) [see also, e.g., International Patent Publication No. WO2022072673].

[0179] Typically, the subject IRES sequence will be positioned in the subject nucleic acid to regulate the expression (translation) of the subject protein (e.g., Acr protein or Cas protein) - and will therefore be positioned 5' (usually immediately 5') of the protein coding sequence it regulates. Figure 10 provides non-limiting illustrative examples of embodiments in which the IRES sequence is positioned in different ways. In some cases, the IRES sequence is positioned at the 5' position (and usually immediately 5') of the Cas protein coding sequence. In some cases, the IRES sequence is positioned at the 5' position (and usually immediately 5') of the Acr protein coding sequence.

[0180] In some cases, the Cas coding sequence and the Acr coding sequence are operably connected to the same promoter (encoded by a polycistronic sequence) and positioned in series, wherein the IRES sequence is positioned between them (see, e.g., Figure 10, first and second embodiments). In some such cases, the Cas protein coding sequence is positioned at 5' of the Acr coding sequence, and therefore the IRES sequence is 3' of the Cas sequence and 5' of the Acr sequence. In some such cases, the Acr protein coding sequence is positioned at 5' of the Cas coding sequence, and therefore the IRES sequence is 3' of the Acr sequence and 5' of the Cas sequence.

[0181] In some cases, the Cas coding sequence and the Acr coding sequence are operably connected to the first promoter and the second promoter, respectively, so that they are transcribed as separate transcripts (see, e.g., Figure 10, CF). The first promoter and the second promoter (labeled "P1" and "P2" in the figure) may be different from each other or may be identical (i.e., may be copies of the same promoter). In some such cases, the IRES sequence regulates the Acr sequence (and is therefore positioned at 5' of the Acr sequence). In other cases, the IRES sequence regulates the Cas sequence (and is therefore positioned at 5' of the Cas sequence). In some cases in which the Cas coding sequence and the Acr coding sequence are transcribed as separate sequences, each is regulated by the IRES sequence (and therefore each is positioned at 3' of the IRES sequence).

[0182] In some embodiments in which the Cas encoding sequence and the Acr encoding sequence are transcribed as separate sequences, a "spacer" protein encoding sequence is used 5' to the IRES sequence, such that the "spacer" sequence is transcribed as part of a polycistronic sequence in which the protein sequence is regulated (see, e.g., Fig.10E and F). For example, in Fig. 10E In , the Cas protein coding sequence and the Acr protein coding sequence are operably linked to different promoters (P1 and P2). The spacer sequence (marked as "X" in the figure) is positioned 5' to the IRES sequence, which is 5' to the Acr coding sequence - and thus the spacer sequence and the Acr sequence are transcribed as part of the same RNA, but the presence of the IRES sequence results in the Acr protein being produced as a separate protein. Similarly, in Fig.10F In the example, the Cas protein coding sequence and the Acr protein coding sequence are again operably linked to different promoters (P1 and P2). In this example, the spacer sequence (marked as "X" in the figure) is positioned 5' of the IRES sequence, which is 5' of the Cas coding sequence - and thus 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.

[0183] "Spacer" protein can be any desired sequence-because its purpose is to simply provide a sequence to be translated, the sequence is 5' of the IRES sequence. Spacer sequences can be any convenient length, from very short to encoding a complete 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 1 to 100 amino acids (e.g., 1 to 80, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 2 to 100, 2 to 80, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 100, 5 to 80, 5 to 50, 5 to 40, 5 to 30, 5 to 20, or 5 to 10 amino acids). Examples of spacer sequences include, but are not limited to, linker sequences, repeated single amino acids (e.g., AAAA), random sequences, protein fragments, and marker proteins (e.g., fluorescent proteins (such as GFP, YFP, CFP, RFP, etc.), drug-selective protein markers, enzymes (such as β-galactosidase, etc.)).

[0184] In some cases where the Acr coding sequence and the Cas coding sequence are operably linked to separate promoters, no spacer sequence is used (see, e.g., Fig. 10C and D).

[0185] Start codon

[0186] In some embodiments herein, the translation control element is a non-AUG start codon (also referred to as a non-AUG start codon). The term "non-AUG start codon" or "non-AUG start codon" refers to any non-AUG polynucleotide (usually a triplet) that acts as a start site for translation initiation, and its efficiency is reduced relative to the efficiency of the AUG start codon. Examples of naturally occurring alternative start codon usage are described in, for example, Kozak (1991) J. Cell Biol. 115 (4): 887-903; Mehdi et al. (1990) Gene 91: 173-178; Kozak (1989) Mol. Cell. Biol. 9 (11): 5073-5080. Generally, non-AUG start codons have reduced translation efficiency compared to AUG start codons.

[0187] In some cases, the non-AUG start codon is used as the start codon of the sequence encoding the Acr protein. In some cases, the non-AUG start codon is used as the start codon of the sequence encoding the Cas protein (e.g., CRISPR nuclease). In some cases, the non-AUG start codon (used with the Acr sequence or with the Cas sequence) is any one of the following: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC or AUU. In some cases, the non-AUG start codon (used with the Acr sequence or with the Cas sequence) is any one of the following: CUG, GUG, ACG, AUA or UUG. For example, in some cases, the non-AUG start codon used with the Acr sequence is any one of the following: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC or AUU. In some cases, the non-AUG start codon used with the Acr sequence is any one of the following: CUG, GUG, ACG, AUA or UUG. As another example, in some cases, the non-AUG start codon used with the Cas sequence is any one of the following: CUG, GUG, ACG, AUA, UUG, GCG, AGG, AAG, AUC or AUU. In some cases, the non-AUG start codon used with the Cas sequence is any one of the following: CUG, GUG, ACG, AUA or UUG. In some cases, the non-AUG start codon used with the Acr sequence is CUG. In some cases, the non-AUG start codon used with the Acr sequence is GUG. In some cases, the non-AUG start codon used with the Acr sequence is ACG. In some cases, the non-AUG start codon used with the Cas sequence is CUG. In some cases, the non-AUG start codon used with the Cas sequence is GUG. In some cases, the non-AUG start codon used with the Cas sequence is ACG.

[0188] The translation efficiency of non-AUG start codons may also be affected by their sequence context; for example, in eukaryotic cells, it is reported that the optimal Kozak consensus sequence has 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), in which the start codon ATG (AUG in RNA) is just before the last "G", the A of the ATG start codon is designated as the +1 position, and the "R" at the -3 position is a purine (A or G). The two most highly conserved positions are purines, usually A at the -3 position and G at the +4 position (Kozak (1991) J Cell Biol 115 (4): 887-903). In some cases, a subject non-AUG start codon (eg, any of those discussed above) is coupled to a compromised Kozak sequence (ie, does not conform to a consensus Kozak sequence).

[0189] For the above examples, see, e.g., Kearse and Wilusz, Genes Dev. Sep 1, 2017; 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; their disclosures are incorporated herein by reference as they relate to Kozak sequences and non-AUG start codons (and assays related thereto). One skilled in the art will recognize that sequences described herein as DNA will have associated sequences as RNA molecules, e.g., a DNA sequence ATG will correspond to an RNA sequence AUG, and vice versa.

[0190] Typically, the subject non-AUG start codon will be positioned in the subject nucleic acid to regulate the expression (translation initiation) of the subject protein (e.g., Acr protein or Cas protein)-and will therefore be positioned 5' (usually immediately 5') of the protein coding sequence it regulates and in frame with it. In some cases (e.g., when the non-AUG start codon is used as the start codon of the Acr coding sequence), the sequence encoding the Acr protein does not include its natural 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 the non-AUG start codon is used as the start codon of the Cas coding sequence), the sequence encoding the CRISPR nuclease does not include its natural 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 start codon of a Cas coding sequence or an Acr coding sequence), the sequence encoding the subject protein (i.e., CRISPR nuclease or Acr protein) is codon-optimized in whole or in part to avoid having an out-of-frame AUG that may direct the translation machinery to an incorrect reading frame (i.e., a reading frame that does not encode the subject protein) contained in the nucleic acid encoding the Cas or Acr protein. In some such cases, the first 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50 codons starting from the start of the subject protein are optimized so as not to include an out-of-frame AUG.

[0191] In some cases, the Cas coding sequence and the Acr coding sequence are operably linked to a first promoter and a second promoter, respectively, so that they are transcribed as separate transcripts (see, e.g., FIG. 11 ). The first promoter and the second promoter (labeled “P1” and “P2” in the figure) may be different from each other or may be identical (i.e., may be copies of the same promoter).

[0192] In some embodiments, more than one translation control element can be used to control the 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) translation control elements are used to control the expression of Acr protein. In some cases, more than one (e.g., two, two or more, three) translation control elements are used to control the expression of CRISPR nuclease. Therefore, in some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRES, non-AUG start codons) are used to control the expression of Acr protein and / or CRISPR nuclease. In some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRES, non-AUG start codons) are used to control the expression of Acr protein. In some cases, one or more (e.g., two, two or more, three) translation control elements (e.g., 2A peptides, IRES, non-AUG start codons) are used to control the expression of CRISPR nuclease.

[0193] Promoter

[0194] The compositions, methods and systems of ACRs and nucleases provided herein include an expression cassette, such as an expression cassette on one or more vectors, comprising a promoter to drive expression of a gene encoding a Cas protein (e.g., a class 2 effector protein / CRISPR nuclease) and / or an Acr protein and / or a guide RNA.

[0195] In some cases, the Acr coding sequence and the CRISPR nuclease sequence are operably connected to the same promoter and are therefore transcribed as part of the same RNA. In other cases, the Acr coding sequence and the CRISPR nuclease sequence are operably connected to different promoters. For example, in some cases, the Acr coding sequence is operably connected to the first promoter, and the CRISPR nuclease sequence is operably connected to the second promoter. In some such cases, the first promoter and the second promoter are identical-so that 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 controls the expression of a protein, and the other controls the expression of another protein). In other such cases, the first promoter and the second promoter are different promoters.

[0196] Promoter type

[0197] A variety of promoter types can be used, for example, to control the expression of Acr proteins and / or CRISPR nucleases (e.g., Cas12a or NUX proteins). The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), it can 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 specific temperature, compound, or protein), it can be a spatially restricted promoter (i.e., a transcriptional control element, an enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and / or it can be a temporally restricted promoter, for example, during a specific stage of embryonic development or during a specific stage of a biological process, the promoter is in an "ON" state or an "OFF" state.

[0198] Suitable promoters may be derived from viruses and may therefore be referred to as viral promoters, or they may be derived from any convenient organism. Suitable promoters may be derived from viruses and may therefore be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Exemplary promoters include, but are not limited to, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), human H1 promoter (H1), etc. Pol III promoters (such as U6, enhanced U6, and H1) are often used to express non-coding RNAs, such as guide RNAs.

[0199] Examples of inducible promoters include, but are not limited to, heat shock promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, estrogen receptor-regulated promoters, etc. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline; estrogen receptor; estrogen receptor fusions; estrogen analogs; IPTG; etc.

[0200] Inducible promoters suitable for use include any inducible promoter described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically-regulated and physically-regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and chelate 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).

[0201] Examples of suitable promoters include, but are not limited to, the following:

[0202] Mammalian (Pol II) promoter (for nucleases and Acr)

[0203] Retrovirus Rous sarcoma virus (RSV)

[0204] LTR promoter (optionally with RSV enhancer),

[0205] Cytomegalovirus (CMV) promoter (optionally with CMV enhancer)

[0206] SV40 promoter

[0207] Dihydrofolate reductase promoter,

[0208] β-actin promoter,

[0209] Phosphoglycerate kinase (PGK) promoter,

[0210] EF1.alpha.(EF1a) promoter

[0211] MMLV LTR promoter

[0212] HIV LTR promoter, MCMV LTR promoter,

[0213] MND,

[0214] UBc,

[0215] CAG,

[0216] ·HSV TK promoter,

[0217] fos promoter,

[0218] E2F promoter

[0219] Polyomavirus

[0220] Adenovirus, fowlpox virus

[0221] ·Bovine papillomavirus

[0222] Avian sarcoma virus

[0223] Eukaryotic tissue specificity

[0224] Bowman et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12115-12119 describe a brain-specific transferrin promoter;

[0225] The synapsin I promoter is neuron-specific (Schoch et al., 1996 J. Biol. Chem. 271, 3317-3323);

[0226] The necdin promoter is specific for postmitotic neurons (Uetsuki et al., 1996 J. Biol. Chem. 271, 918-924);

[0227] The neurofilament light promoter is neuron-specific (Charron et al., 1995 J. Biol. Chem. 270, 30604-30610);

[0228] The acetylcholine receptor promoter is neuron-specific (Wood et al., 1995 J. Biol. Chem. 270, 30933-30940);

[0229] Potassium channel promoters are specific for high-frequency firing neurons (Gan et al., 1996 J. Biol. Chem 271, 5859-5865);

[0230] Chromogranin A promoter is specific for neuroendocrine cells (Wu et al., 1995 A. J. Clin. Invest. 96, 568-578);

[0231] The von Willebrand factor promoter is specific for brain endothelium (Aird et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4567-4571);

[0232] The flt-1 promoter is endothelial-specific (Morishita et al., 1995 J. Biol. Chem. 270, 27948-27953);

[0233] The preproendothelin-1 promoter is endothelial, epithelial and muscle specific (Harats et al., 1995 J. Clin. Invest. 95, 1335-1344);

[0234] The GLUT4 promoter is specific for skeletal muscle (Olson and Pessin, 1995 J. Biol. Chem. 270, 23491-23495);

[0235] The slow / fast troponin promoter is specific for slow / fast twitch muscle fibers (Corin et al., 1995 Proc. Natl. Acad. Sci. USA 92, 6185-6189);

[0236] The actin promoter is specific for smooth muscle (Shimizu et al., 1995 J. Biol. Chem. 270, 7631-7643);

[0237] The myosin heavy chain promoter is specific for smooth muscle (Kallmeier et al., 1995 J. Biol. Chem. 270, 30949-30957);

[0238] The E-cadherin promoter is epithelial-specific (Hennig et al., 1996 J. Biol. Chem. 271, 595-602);

[0239] The cytokeratin promoter is specific for keratinocytes (Alexander et al., 1995 B. Hum. Mol. Genet. 4, 993-999);

[0240] The transglutaminase 3 promoter is keratinocyte-specific (J. Lee et al., 1996 J. Biol. Chem. 271, 4561-4568);

[0241] The bullous pemphigoid antigen promoter is specific for basal keratinocytes (Tamai et al., 1995 J. Biol. Chem. 270, 7609-7614);

[0242] The keratin 6 promoter is specific for proliferating epidermis (Ramirez et al., 1995 Proc. Natl. Acad. Sci. USA 92, 4783-4787);

[0243] The collagen 1 promoter is specific for hepatic stellate cells and skin / tendon fibroblasts (Houglum et al., 1995 J. Clin. Invest. 96, 2269-2276);

[0244] The type X collagen promoter is specific for hypertrophic chondrocytes (Long and Linsenmayer, 1995 Hum. Gene Ther. 6, 419-428);

[0245] The Factor VII promoter is liver-specific (Greenberg et al., 1995 Proc. Natl. Acad. Sci. USA 92, 12347-1235);

[0246] The fatty acid synthase promoter is specific for liver and adipose tissue (Soncini et al., 1995 J. Biol. Chem. 270, 30339-3034);

[0247] The carbamoyl phosphate synthetase I promoter is specific for periportal hepatocytes and small intestine (Christoffels et al., 1995 J. Biol. Chem. 270, 24932-24940);

[0248] The Na--K--Cl transporter promoter is kidney (loop of Henle) specific (Igarashi et al., 1996 J. Biol. Chem. 271, 9666-9674);

[0249] The scavenger receptor A promoter is specific for macrophages and foam cells (Horvai et al., 1995 Proc. Natl. Acad. Sci. USA 92, 5391-5395);

[0250] The glycoprotein IIb promoter is specific for megakaryocytes and platelets (Block and Poncz, 1995 Stem Cells 13, 135-145);

[0251] The yc chain promoter is specific for hematopoietic cells (Markiewicz et al., 1996 J. Biol. Chem. 271, 14849-14855);

[0252] • The CD11b promoter is specific for mature myeloid cells (Dziennis et al., 1995 Blood 85, 31 9-329).

[0253] Cell Type

[0254] Host cells (also referred to as "target cells") can be cultured in vitro (e.g., fresh isolates-early passages), in vivo or in vitro (e.g., immortalized cell lines). In some cases, the targeting nucleic acid is chromosomal (e.g., the genome of the host cell), and in some cases, the targeting nucleic acid is from a pathogen, e.g., the genome of a pathogen within the host cell. Cells can be from established cell lines or they can be primary cells, wherein "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein, referring to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages (i.e., the culture is split). For example, a primary culture is a culture that can be passaged 0, 1, 2, 4, 5, 10, or 15 times but has not been passaged enough times to pass through the transition period. Typically, primary cell lines are maintained in culture for less than 10 generations.

[0255] Suitable host cells (which may contain target nucleic acids, such as genomic DNA) include, but are not limited to, cells of unicellular eukaryotic organisms; plant cells; algal cells, such as Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa ( Chlorella pyrenoidosa ), Sargassum patens C. agardh, etc.; fungal cells (e.g., yeast cells); animal cells; cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.); cells of insects (e.g., mosquitoes; bees; agricultural pests; etc.); cells of arachnids (e.g., spiders; ticks; etc.); cells of vertebrates (e.g., fish, amphibians, reptiles, birds, mammals); cells of mammals (e.g., rodent cells; human cells; cells of non-human mammals; cells of rodents (e.g., mice, rats); cells of lagomorphs (e.g., rabbits); cells of ungulates (e.g., cattle, horses, camels, llamas, llamas, cells of mammals (e.g., sheep, goats, etc.); cells of marine mammals (e.g., whales, seals, elephant seals, dolphins, sea lions; etc.); cells of any type of cell may be of interest (e.g., stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, eggs, spermatogonia, etc.), adult stem cells, somatic cells, such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells; in vitro or in vivo embryonic cells at any stage of embryos, such as zebrafish embryos at 1-cell, 2-cell, 4-cell, 8-cell, etc. stages; etc.).

[0256] Suitable host cells (which may contain target nucleic acids, such as genomic DNA) include, but are not limited to, bacterial cells; archaeal cells; cells of unicellular eukaryotic organisms; plant cells; algal cells, such as Botrytis cinerea, Chlamydomonas reinhardtii, Nannochloropsis spp., Chlorella pyrenoidosa , Sargassum spathulatum, etc.; fungal cells (e.g., yeast cells); animal cells; cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.); cells of insects (e.g., mosquitoes; bees; agricultural pests; etc.); cells of arachnids (e.g., spiders; ticks; etc.); cells of vertebrates (e.g., fish, amphibians, reptiles, birds, mammals); cells of mammals (e.g., rodents; human cells; cells of non-human mammals; cells of rodents (e.g., mice, rats); cells of lagomorphs (e.g., rabbits); cells of ungulates (e.g., cattle, horses, camels, llamas, vicunas, cells of mammals (e.g., sheep, goats, etc.); cells of marine mammals (e.g., whales, seals, elephant seals, dolphins, sea lions; etc.); cells of any type of cell may be of interest (e.g., stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells (e.g., oocytes, sperm, eggs, spermatogonia, etc.), adult stem cells, somatic cells, such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells; in vitro or in vivo embryonic cells at any stage of embryos, such as zebrafish embryos at 1-cell, 2-cell, 4-cell, 8-cell, etc. stages; etc.).

[0257] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells; germ cells (e.g., oocytes, sperm, eggs, spermatogonia, etc.); somatic cells, such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, etc.

[0258] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous transplant 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, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitors, unipotent progenitors, monocytes, cardiomyocytes, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells and postpartum stem cells.

[0259] In some cases, the cell is an immune cell, a neuron, an epithelial cell and an 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).

[0260] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also called somatic stem cells.

[0261] Adult stem cells reside in differentiated tissues but retain the property of self-renewal and the ability to generate a variety of cell types, often typical of the tissue in which the stem cells are found. Many examples of somatic stem cells are known to those skilled 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; mesoderm stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0262] The stem cells of interest include mammalian stem cells, wherein the term "mammal" refers to any animal classified as a mammal, including humans; non-human primates; domestic animals and farm animals; and zoo animals, laboratory animals, sports animals or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cells are human stem cells. In some cases, the stem cells are rodent (e.g., mouse; rat) stem cells. In some cases, the stem cells are non-human primate stem cells.

[0263] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesodermal cells that can be isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac. HSCs are characterized by CD34 + and CD3 - HSC can repopulate the erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lines in vivo. In vitro, HSC can be induced to undergo at least some self-renewal cell divisions, and can be induced to differentiate into the same lineages as seen in vivo. Thus, HSC can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphocytes.

[0264] In other embodiments, stem cell is neural stem cell (NSC).Neural stem cell (NSC) can differentiate into neurons and glial cells (including oligodendrocytes and astrocytes).Neural stem cell is a multipotent stem cell, which can divide multiple times, and can produce daughter cells as neural stem cells under specific conditions, or can be a neural progenitor cell of neuroblast or glial cell, for example, dedicated to becoming one or more types of neurons and glial cells respectively.The method of obtaining NSC is known in the art.

[0265] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs, originally derived from embryonic mesoderm and isolated from adult bone marrow, can differentiate into muscle, bone, cartilage, fat, bone marrow stroma, and tendon. Methods for isolating MSCs are known in the art; and any known method can be used to obtain MSCs. See, e.g., U.S. Pat. No. 5,736,396, which describes the isolation of human MSCs.

[0266] Promoters and translation control elements (such as those described above) can be used to achieve delivery of subject Acr proteins and / or CRISPR nucleases at levels different from each other. For example, in some embodiments, the goal is to deliver CRISPR nucleases and Acr polypeptides at a ratio of 1: 1. In some embodiments, the goal is to deliver CRISPR nucleases and Acr polypeptides at a ratio (CRISPR: Acr) within the range of 1: 1.25 to 1: 10 (e.g., 1: 2 to 1: 10, 1: 4 to 1: 10, 1: 5 to 1: 10, 1: 1.25 to 1: 5 or 1: 2 to 1: 5). For example, the subject system can be configured to (e.g., for use in a method) deliver CRISPR nucleases and Acr polypeptides at a ratio (CRISPR: Acr) of, for example, 1: 1.25, 1: 1.5, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10.

[0267] In some embodiments, the goal is to deliver the Acr polypeptide and CRISPR nuclease at a ratio (Acr:CRISPR) in the range of 1:1.25 to 1:10 (e.g., 1:2 to 1:10, 1:4 to 1:10, 1:5 to 1:10, 1:1.25 to 1:5, or 1:2 to 1:5). For example, the subject system can be configured (e.g., for use in the methods) to deliver the Acr polypeptide and CRISPR nuclease at a ratio (Acr:CRISPR) of, for example, 1:1.25, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0268] "Protospacer Adjacent Motif" (PAM)

[0269] Wild-type CRISPR nucleases typically have nuclease activity that cleaves a target nucleic acid (e.g., double-stranded DNA (dsDNA)) at a target site defined by (i) a region of complementarity between the guide sequence of the guide RNA and the target nucleic acid; and (ii) a short motif in the target nucleic acid called a "protospacer adjacent motif" (PAM).

[0270] For additional information related to CRISPR-based programmable gene editing tools (e.g., CRISPR / Cas RNA-guided proteins, CRISPR / Cas RNA guide RNAs, and PAMs), see, e.g., the following review papers and 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. Microbiol. 2020 Feb;18(2):67-83; all of which are incorporated herein by reference in their entirety.

[0271] Carrier

[0272] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, an "insert," can be attached so as to cause 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, the subject vector is a viral vector (e.g., AAV, lentivirus, adenovirus). In some cases, the subject vector includes an origin of replication (e.g., can be a plasmid).

[0273] In some cases, both the Acr protein and its target Cas protein (the protein that the Acr inhibits) are present in a single vector – this ensures that all cells that receive the Cas protein (e.g., an endonuclease such as Cas9, Cas12a, etc.) will also express the Acr “off switch.” Regardless of whether both proteins (Acr and Cas) are present on the same nucleic acid, translation of one or both proteins can be regulated by translational control elements in order to achieve an appropriate balance (ratio of expression levels) between the two proteins.

[0274] The vector can be directly provided to the target host cell (target cell). In other words, the cell is contacted with a vector (e.g., a recombinant expression vector) comprising the subject nucleic acid so that the vector is absorbed by the cell. The method for contacting the cell with a nucleic acid vector as a plasmid includes electroporation, calcium chloride transfection, microinjection, and lipofection, and these methods are well known in the art. For viral vector delivery, the cell can be contacted with a viral particle comprising a subject viral expression vector (e.g., adeno-associated virus (AAV)).

[0275] In some embodiments, a subject vector is a viral construct, e.g., a recombinant adeno-associated viral construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, and the like.

[0276] Suitable expression vectors include, but are not limited to, viral vectors (e.g., those based on vaccinia virus; polio virus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543-2549, 1994; Borras et al., Gene Ther 6:515-524, 1999; Li and Davidson, PNAS 92:7700-7704, 1995; Saka moto et al., H Gene Ther 5:1088-1097, 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 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava, in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelsohn ... n 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: 1031923, 1997; Takahashi et al., J Virol 73: 7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0277] In some embodiments, the subject vector is an AAV vector. Adeno-associated virus or "AAV" refers to the virus itself or its derivatives. Unless otherwise required, the term encompasses all subtypes and naturally occurring and recombinant forms, 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-12), AAV type 13 (AAV-14), AAV type 15 (AAV-16), AAV type 17 (AAV-18), AAV type 19 (AAV-20), AAV type 21 (AAV-21), AAV type 22 (AAV-23), AAV type 24 (AAV-25), AAV type 25 (AAV-26), AAV type 27 (AAV-27), AAV type 28 (AAV-29), AAV type 30 (AAV-31), AAV type 31 (AAV-32), AAV type 32 (AAV-33), AAV type 33 (AAV-34), AAV type 34 (AAV-35), AAV type 35 (AAV-36), AAV type 37 (AAV-37), AAV type 38 (AAV-39), AAV type 39 (AAV-40), AAV type 30 (AAV-41), AAV type 31 (AAV-42), AAV type 32 (AAV-43), AAV type 33 (AAV-44), AAV type 34 (AAV-45), AAV type 35 (AAV-46), A AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11, AAV-11

[0278] In some embodiments, a subject vector is an integrating vector, eg, integrates into the genome of a target cell.

[0279] "Recombinant AAV vector" or "rAAV vector" refers to AAV virus or AAV viral chromosome material, which includes a polynucleotide sequence that is not derived from AAV (i.e., a polynucleotide heterologous to AAV), typically a nucleic acid sequence of interest to be integrated into a cell according to the subject method. Typically, the flanks of the heterologous polynucleotide are at least one and typically two AAV inverted terminal repeats (ITRs). In some cases, the recombinant viral vector also includes viral genes important for the packaging of the recombinant viral vector material. "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of viral particles (e.g., AAV viral particles). Examples of nucleic acid sequences important for AAV packaging (i.e., "packaging genes") include AAV "rep" and "cap" genes, which encode replication and encapsidation proteins of adeno-associated viruses, respectively. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0280] "Viral particle" refers to a single viral unit comprising a capsid that encapsulates a virus-based polynucleotide, such as a viral genome (as in a wild-type virus), or, for example, a subject targeting vector (as in a recombinant virus). "AAV viral particle" refers to a viral particle consisting of at least one AAV capsid protein (usually consisting of all capsid proteins of a wild-type AAV) and an encapsidated polynucleotide AAV vector. If the particle includes 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), the particle is typically referred to as a "rAAV vector particle" or simply "rAAV vector". Therefore, the production of rAAV particles necessarily includes the production of rAAV vectors, because such vectors are contained within rAAV particles.

[0281] rAAV virions can be constructed using methods well known in the art. See, for example, Koerber et al. (2009) Mol. Ther. 17: 2088; Koerber et al. (2008) Mol Ther. 16: 1703–1709; U.S. Patent Nos. 7,439,065, 6,951,758, and 6,491,907. For example, heterologous sequences can be directly inserted into the AAV genome from which the main AAV open reading frame ("ORF") has been excised. As long as enough ITR portions are retained to allow replication and packaging functions, other portions of the AAV genome may also be missing. 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 Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Curr. Topics Microbiol. Immunol. 158:97-129; Kotin, RM (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.

[0282] To produce rAAV virions, the AAV expression vector can be introduced into a suitable host cell using known techniques (such as by transfection). Many transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al. (1973) Virol. 52:456-467), direct microinjection into cultured cells (Capecchi, MR (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-speed microbullet shooting (Klein et al. (1987) Nature 327:70-73).

[0283] Suitable cells for producing rAAV virions include microorganisms, yeast cells, insect cells and mammalian cells, which can or have been used as receptors for heterologous DNA molecules. Cells from a stable human cell line 293 (easily obtained through, for example, the American Type Culture Collection, with a deposit number of 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 DNA fragments of type 5 (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses adenovirus E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is easy to transfect and provides a convenient platform for producing rAAV virions therein. Methods for producing AAV virions in insect cells are known in the art and can be used to produce subject rAAV virions. See, e.g., U.S. Patent Publication No. 2009 / 0203071; U.S. Patent No. 7,271,002; and Chen (2008) Mol. Ther. 16:924.

[0284] The AAV virus produced can be replication-competent or non-replication-competent. "Replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypic wild-type virus that is infective and can also replicate in infected cells (e.g., in the presence of a helper virus or helper virus function). In the case of AAV, replication ability generally requires the presence of functional AAV packaging genes. Typically, due to the lack of one or more AAV packaging genes, rAAV vectors as described herein cannot be replicated in mammalian cells (particularly in human cells). Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of producing replication-competent AAV by recombination between AAV packaging genes and the rAAV vectors entering.

[0285] Retroviruses, such as lentiviruses, are suitable for use in the methods disclosed herein. Commonly used retroviral vectors are "defective", i.e., they cannot produce the viral proteins required for productive infection. Moreover, vector replication requires growth in a packaging cell line. In order to produce viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins (tropic, amphophilic or heterophilic) to be incorporated into the capsid, which determine the specificity of the viral particles to the cell (tropic to mice and rats; amphophilic to most mammalian cell types including humans, dogs and mice; and heterophilic to most mammalian cell types except mouse cells). Appropriate packaging cell lines can be used to ensure that cells are targeted by packaged viral particles. Methods for introducing the subject vector expression vector into a packaging cell line and collecting viral particles generated by the packaging cell line are well known in the art. Nucleic acids can also be introduced by direct microinjection (e.g., injection of RNA).

[0286] A detailed discussion of delivery methods and formulations is provided elsewhere herein.

[0287] As described elsewhere herein, the protein can be provided to the cell as RNA (e.g., RNA comprising translational control elements as discussed elsewhere herein). Methods for introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method for introducing DNA.

[0288] In some cases, one or more proteins (e.g., CRISPR nucleases and / or Acr polypeptides) can be introduced into cells as proteins (rather than nucleic acids). For example, a protein coding sequence (such as an Acr protein coding sequence) can be introduced as a nucleic acid (RNA or DNA), wherein the protein coding sequence is operably linked to a translation control element; and other proteins (e.g., Cas effectors) are introduced as polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases the solubility of the product. The domain may be connected to the polypeptide via a defined protease cleavage site, such as a TEV sequence cleaved by a TEV protease. The linker may also include one or more flexible sequences, such as 1 to 10 glycine residues. Examples of linkers are discussed elsewhere in this article in different contexts, but such linkers may be used in any convenient context, including this context.

[0289] In some embodiments, the cleavage of the fusion protein is carried out in a buffer that maintains the solubility of the product, such as in the presence of 0.5M to 2M urea, in the presence of a polypeptide and / or a polynucleotide that increases solubility, etc. The domains of interest include endosomal lysis domains, such as influenza HA domains; and other polypeptides that assist in production, such as IF2 domains, GST domains, GRPE domains, etc. The polypeptide can be formulated for improved stability. For example, the peptide can be PEGylated, wherein the polyethyleneoxy group provides an enhanced life span in the bloodstream.

[0290] method

[0291] The present disclosure provides a method for nucleic acid targeting (e.g., modifying a target nucleic acid, e.g., for cutting DNA, such as in genome editing applications), wherein an Acr polypeptide and a CRISPR nuclease are delivered to a target nucleic acid as a subject system (e.g., in the form of nucleic acid or protein). In some cases, contact is performed in a cell-free environment in vitro. In some cases, contact occurs in a cell, which may be ex vivo, in vivo or in vitro (e.g., a cell in culture). Therefore, in some embodiments, the 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, thereby increasing the ratio of on-target to off-target nucleic acid targeting caused by the introduction relative to the ratio of on-target to off-target nucleic acid targeting caused in the absence of an Acr protein. In some cases, the ratio of on-target to off-target nucleic acid targeting caused by the introduction is caused by an increase in on-target activity. In some cases, the ratio of on-target to off-target nucleic acid targeting caused by the reduction in off-target activity. In some cases, the ratio of on-target to off-target nucleic acid targeting caused by the reduction in on-target activity caused by the reduction in off-target activity.

[0292] The target cell (host cell) can be any desired cell / cell type. Examples of suitable cells and promoters are described in detail elsewhere herein (see, e.g., "Promoter" section). For example, in some cases, the cell is a prokaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. For example, in some cases, the cell is a eukaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. In some cases, the cell is isolated. In some cases, the cell is in vivo. In some cases, the cell is cultured in vitro.

[0293] In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of a pathogen (e.g., a virus) - in some cases, the pathogen is in the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is the genome of a pathogen (virus, bacteria, etc.) - in some cases, the pathogen is in the host cell. In some embodiments, the nucleic acid targeted by the CRISPR complex (on-target event) is and RNA molecules. In some cases, the on-target nucleic acid targeting changes the expression of proteins in the host cell (e.g., by reducing the transcription of mRNA). In some cases, the on-target nucleic acid targeting changes the expression of RNA (e.g., non-coding RNA, mRNA, microRNA, etc.) in the host cell.

[0294] In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes gene editing (e.g., correction of a gene mutation in the host cell genome). In some cases, the on-target nucleic acid targeting activity of the CRISPR complex causes the change (editing) of a gene locus from a disease-associated sequence to a healthy-associated sequence - for example, correcting a pathogenic allele of Huntington's disease (HD), Duchenne muscular dystrophy (DMD), or alpha-1 antitrypsin disease (AATD) to an allele that is not related to the disease (non-pathogenic).

[0295] In some embodiments, the subject methods include the step of measuring the editing efficiency at the target site. In some embodiments, the subject methods include the step of measuring the editing efficiency at one or more off-target sites.

[0296] As described elsewhere herein, the location of an on-target ("on-target") event (e.g., target DNA cleavage / editing) is actually determined by the guide sequence of the guide RNA. CRISPR complex-mediated events that occur at positions that are not 100% matched to 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 choice of method will depend on the type of CRISPR complex used and the desired outcome of the activity of the complex (e.g., when using a nickase protein, when performing double-stranded target cleavage, when using a donor polynucleotide - this can be edited by introducing a known heterologous sequence; when not using a donor polynucleotide - it can result in many different insertions and deletions, etc.), examples of suitable assays include, but are not limited to: mismatch cleavage assays (e.g., surveyor assays, T7E1 mismatch assays), PCR assays; PCR / sequencing assays, direct sequencing assays such as next-generation sequencing, etc. (and any combination thereof). When the activity of the CRISPR complex results in a change in the expression of the target sequence (e.g., when the promoter sequence is targeted, when the coding sequence is targeted and the new sequence is susceptible to nonsense-mediated decay, etc.), sequencing assays or alternative expression assays, such as qRT-PCR and / or microarray analysis, can be used. There are various assays to test on-target activity and off-target activity, and any desired assay or combination of assays can be used. In some cases, the desired result (e.g., reduced on-target and off-target CRISPR nuclease activity) can be achieved by using a specific Acr protein (e.g., see the description of the subject Acr protein above). In some cases, off-target sites are predicted and / or known states, and in some cases, off-target sites can be identified after the fact (e.g., based on a full genome search, such as can be achieved using a high-throughput / next generation sequencing method (such as RNA or DNA sequencing method)).

[0297] In some embodiments, the Acr polypeptides of the compositions, systems, or methods described herein reduce off-target CRISPR nuclease activity (i.e., off-target activity of CRISPR nuclease). For example, in some cases, the Acr polypeptide reduces off-target CRISPR nuclease activity by 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) compared to off-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide reduces off-target CRISPR nuclease activity by 20% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) compared to off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0298] In some embodiments, the Acr polypeptide of the composition, system, or method described herein reduces the on-target CRISPR nuclease activity (i.e., the on-target activity of the CRISPR nuclease) compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide. For example, in some cases, the Acr polypeptide reduces the on-target CRISPR nuclease activity by no more than 50% (e.g., a reduction of no more than 40%, 30%, 20%, 10%, 5%, 3%, 2% or 1%) compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide. In some cases, the Acr polypeptide reduces the on-target CRISPR nuclease activity by no more than 40% (e.g., a reduction of no more than 40%, 30%, 20%, 10%, 5%, 3%, 2% or 1%) compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0299] In some embodiments, the Acr polypeptides of the compositions, systems or methods described herein increase the ratio of on-target to off-target CRISPR nuclease activity compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of Acr polypeptides. 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., an increase of at least 1.5x, 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or 10x) compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of Acr polypeptides. 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., an increase of at least 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or 10x) compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of Acr polypeptides. 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) compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0300] In some embodiments, the editing efficiency of the on-target CRISPR nuclease activity is at least 4 times (4x) (e.g., at least 5x, 6x, 7x, 8x, 9x, 10x, 20x, 25x, 30x, 35x, 40x, 50x, or 100x) greater than the editing efficiency of the off-target CRISPR nuclease activity. In some embodiments, the editing efficiency of the on-target CRISPR nuclease activity is at least 6 times (6x) (e.g., at least 7x, 8x, 9x, 10x, 20x, 25x, 30x, 35x, 40x, 50x, or 100x) greater than the editing efficiency of the off-target CRISPR nuclease activity. In some embodiments, the editing efficiency of the on-target CRISPR nuclease activity is at least 10 times (10x) (e.g., at least 20x, 25x, 30x, 35x, 40x, 50x, or 100x) greater than the editing efficiency of the off-target CRISPR nuclease activity.

[0301] In some embodiments, the off-target CRISPR nuclease activity is at an off-target site that contains no more than 5 mismatches (e.g., no more than 4, no more than 3, no more than 2, or no more than 1) compared to the on-target site (i.e., mismatches between the intended target site and the off-target site). In other words, in some cases, the off-target CRISPR nuclease activity is at an off-target site that contains 5 or fewer mismatches (e.g., 4 or fewer, 3 or fewer, 2 or fewer, or 1 mismatch) compared 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 contains no more than 3 mismatches (e.g., no more than 2 or no more than 1) 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, off-target CRISPR nuclease activity is at an off-target site that comprises 3 or fewer mismatches (e.g., 2 or fewer or 1 mismatch) compared to the on-target site (i.e., mismatches between the intended target site and the off-target site).

[0302] In some cases, the desired result 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 insertions / deletions (indels) detected per cell population). In some such cases, the number of cells in the cell population is less than 10 4 Up to 10 6 In some cases, the number of cells in the cell population is about 10 5 In some cases, the desired result is a result 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 population). In some cases, the desired result is a result 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 population).

[0303] In some cases, the desired outcome is one in which the off-target rate is less than 10 5 In some cases, the desired outcome is one in which the off-target rate is less than one in 10 5 90 off-target events (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) are detected per cell. In some cases, the desired result is one in which the off-target rate is less than 10 per 105 The results of detecting 50 off-target events per cell (e.g., less than 40, less than 30, less than 20, less than 10, or less than 5 off-target events per cell) are presented.

[0304] In some cases, the desired result is that less than 50% (e.g., less than 45%, less than 40%, or less than 35%) of the total measured nucleic acid targeting events (e.g., cutting) are the result of off-target events. In other words, in some cases, the ratio of on-target events to off-target events (e.g., measured on-target events 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 event can be measured after the host cell is passaged (e.g., in some cases, passaged for 10 generations or more) after the introduction of Acr and Cas proteins. Therefore, in some cases, the desired result is such a result, wherein after the host cell is passaged (e.g., passaged for 10 generations or more) after the introduction of Acr and Cas proteins, 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., cutting) are the result of off-target events. In other words, in some such cases, the ratio of on-target events to off-target events (e.g., measured on-target events 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).

[0305] As mentioned above, in some cases, it is possible to predict off-target sites. Typically, the rate (frequency) of off-target activity (e.g., cutting / editing) will vary with the site, for example, when using a cell colony to measure the rate of activity. Therefore, in some cases, the desired result is such a result, wherein when compared with the off-target event measured (or expected) in the absence of Acr protein, the frequency of the off-target event measured 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%). As an illustrative example, the frequency of off-target events at a specific predicted or known off-target site (or at any number of off-target sites - predicted / known or unpredicted / unknown) can be measured in the presence of Acr protein (meaning - when the experiment is performed in the presence of Acr protein) and in the absence of Acr protein - and compared to the number of off-target events in the absence of Acr protein, the number of off-target events in the presence of Acr protein 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%). As another illustrative example of the above, if 100 total off-target events are measured when the method is performed in the presence of Acr protein, but 200 such events are measured (or expected) in the absence of Acr protein, the result will be that the frequency of off-target events measured in the presence of Acr protein is 50% when compared to the off-target events in the absence of Acr protein.

[0306] deliver

[0307] As described above, in some embodiments, both Cas protein and Acr protein will be delivered to the host cell as DNA, and in some such cases, the sequences 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, the subject protein (e.g., Cas protein and / or Acr protein) is not provided as a DNA vector. For example, any protein (or both) can be introduced into the host cell as RNA encoding the protein. In such cases, the RNA encoding the two proteins can be delivered at an appropriate ratio to achieve the desired effect (i.e., the ratio of the on-target to off-target CRISPR complex activity)-for example, by reducing off-target activity while retaining the desired on-target activity, and one or more translation control elements can be present on the RNA.

[0308] As another example, any protein (or both) can be directly introduced into a host cell as a protein. 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), wherein the Cas protein has been compounded with an appropriate guide RNA. In such cases, other proteins (e.g., Acr protein) can be delivered as DNA or RNA, and their coding sequence can be operably connected to a subject translation control element.

[0309] Therefore, Cas protein and Acr protein can be delivered in any desired form (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. Similarly, 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.

[0310] As will be readily understood by those of ordinary skill in the art, any convenient method can be used to deliver subject nucleic acid (e.g., vector) and protein to cells. Methods for introducing nucleic acid and / or protein into host cells (e.g., prokaryotic cells, eukaryotic cells, plant cells, animal cells, insect cells, mammalian cells, human cells, etc.) are known in the art, and any convenient method can be used. Suitable methods include, for example, viral infection (e.g., AAV, adenovirus, slow virus), transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, transfection mediated by polyethyleneimine (PEI), transfection mediated by DEAE-dextran, transfection mediated by liposomes, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, microflow (see, e.g., Panyam et al., Adv Drug Deliv Rev. September 13, 2012. pii: S0169-409X (12) 00283-9) etc.

[0311] In some cases, the proteins of the present disclosure (e.g., Cas proteins, Acr proteins) are provided as nucleic acids encoding proteins (e.g., mRNA, DNA, plasmids, expression vectors, viral vectors, etc.). In some cases, the subject protein is provided directly as a protein (e.g., without an associated guide RNA or with an associated guide RNA, i.e., as a ribonucleoprotein complex). The subject protein can be introduced into a cell (provided to a cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, the subject protein can be injected directly into a cell. As another example, the subject protein can be introduced into a cell (e.g., a eukaryotic cell) by nuclear transfection; by a protein transduction domain (PTD) conjugated to a protein, etc.

[0312] In some cases, the subject protein is delivered to a cell (e.g., a target host cell) in the form of particles (or associated with particles). In some cases, the subject protein is delivered with a cationic lipid and a hydrophilic polymer, e.g., wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particles further comprise cholesterol (e.g., particles 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).

[0313] Particles or lipid envelopes can be used to deliver the subject protein (as RNA or DNA or protein). For example, the biodegradable core-shell structured nanoparticles with poly (β-amino ester) (PBAE) cores encapsulated by phospholipid bilayer shells can be used. In some cases, particles / nanoparticles based on self-assembly bioadhesive polymers are used; such particles / nanoparticles can be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, for example, delivered to the brain. Other embodiments are also contemplated, such as oral absorption and ocular delivery of hydrophobic drugs. Molecular envelope technology can be used, which relates to an engineered polymer envelope that is protected and delivered to a disease site.

[0314] Lipidoid compounds (e.g., as described in U.S. Patent Application 20110293703) can also be used for administering polynucleotides, and can be used for delivering subject proteins or nucleic acids (RNA or DNA). In one aspect, amino alcohol lipidoid compounds are combined with a medicament to be delivered to a cell or subject to form microparticles, nanoparticles, liposomes or micelles. Amino alcohol lipidoid compounds can be combined with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. Then, these particles can be optionally combined with pharmaceutical excipients to form a pharmaceutical composition.

[0315] In some cases, lipid nanoparticles (LNP) are used to deliver the subject protein or nucleic acid to target cells. Negatively charged polymers (such as RNA) can be loaded into LNP at low pH values ​​(e.g., pH 4), when ionizable lipids show positive charges. However, at physiological pH values, LNPs show low surface charges compatible with longer circulation times. Ionizable cationic lipids can include but are not limited to: 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyloxy-3-N, N-dimethylammonium propane (DLinDMA), 1,2-dilinoleoyloxy-ketone-N, N-dimethyl-3-aminopropane (DLinKDMA) and 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Examples of preparing LNPs are described, for example, in Rosin et al. (2011) Molecular Therapy 19: 1286-2200). Cationic lipids 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyloxy-3-N,N-dimethylammonium propane (DLinDMA), 1,2-dilinoleoyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2"-(methoxypolyethylene glycol 2000) succinyl]-1,2-dimyristoyl -sn-diol (PEG-S-DMG) and R-3-[(ω-methoxy-poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-C-DOMG). Nucleic acids can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipids: DSPC:CHOL:PEGS-DMG or PEG-C-DOMG, molar ratio of 40:10:40:10). In some cases, 0.2% SP-DiOC18. For LNP, suitable lipids include but are not limited to DLin-KC2-DMA4, C12-200 and auxiliary lipids distearoylphosphatidylcholine, cholesterol and PEG-DMG, which can be prepared with the subject protein or nucleic acid using a spontaneous vesicle formation procedure. The component molar ratio can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG).To ensure a narrow size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles can be extruded through an 80 nm membrane up to three times before adding, for example, RNA (such as guide RNA). Particles containing the highly potent amino lipid 16 can be used, where the molar ratios of the four lipid components 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance activity.

[0316] Spherical nucleic acids (SNA) can be used TM ) constructs and other nanoparticles (particularly gold nanoparticles) deliver the subject protein or nucleic acid to target cells. See, e.g., Cutler et al., J.Am.Chem.Soc.2011 133:9254-9257, Hao et al., Small.2011 7:3158-3162, Zhang et al., ACS Nano.2011 5:6962-6970, Cutler et al., J.Am.Chem.Soc.2012 134:1376-1391, Young et al., Nano Lett.2012 12:3867-71, Zheng et al., Proc.Natl.Acad.Sci.USA.2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin et al., Small, 10:186-192.

[0317] Self-assembled nanoparticles with RNA can be constructed with polyethyleneimine (PEI) PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG).

[0318] Typically, "nanoparticle" refers to any particle with a diameter less than 1000nm. In some cases, the nanoparticle suitable for delivering a subject protein or nucleic acid to a target cell has a diameter of 500nm or less, for example, 25nm to 35nm, 35nm to 50nm, 50nm to 75nm, 75nm to 100nm, 100nm to 150nm, 150nm to 200nm, 200nm to 300nm, 300nm to 400nm or 400nm to 500nm. In some cases, the nanoparticle suitable for delivering a subject protein or nucleic acid to a target cell has a diameter of 25nm to 200nm.

[0319] Nanoparticles suitable for delivering a subject protein or nucleic acid to a target cell can be provided in different forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles) can be prepared. If the nanoparticles made of semiconductor materials are small enough (usually less than 10 nm) so that quantization of the electronic energy levels occurs, they can also be labeled as quantum dots. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications, and can be suitable for similar purposes in the present disclosure.

[0320] Semisolid and soft nanoparticles are also suitable for delivering subject proteins or nucleic acids to target cells. The prototypical nanoparticle of semisolid nature is the liposome.

[0321] In some cases, the carrier / medium may include microparticles. Microparticles may include, but are not limited to, liposomes, nanoparticles, microspheres, nanospheres, microcapsules and nanocapsules. In some cases, microparticles may include one or more of the following: poly (lactide-co-glycolide), aliphatic polyesters (including but not limited to polyglycolic acid and polylactic acid), hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethane, polyacrylic acid, pseudo-poly (amino acid), polyhydroxybutyrate related copolymers, polyanhydrides, polymethyl methacrylate, poly (ethylene oxide), lecithin and phospholipids - in any combination thereof.

[0322] In some cases, the carrier / medium may include liposomes, for example, liposomes capable of attaching and releasing therapeutic agents (e.g., subject nucleic acids and / or proteins). Liposomes are microscopic spherical lipid bilayers around an aqueous core made of amphiphilic molecules such as phospholipids. For example, liposomes can capture therapeutic agents between the hydrophobic tails of phospholipid micelles. Water-soluble agents can be embedded in the core, while fat-soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special feature, that is, water-soluble and water-insoluble chemicals can be used simultaneously in the medium without the use of surfactants or other emulsifiers. Liposomes can be spontaneously formed by vigorously mixing phospholipids in an aqueous medium. Water-soluble compounds are dissolved in an aqueous solution that can hydrate phospholipids. Therefore, when liposomes are formed, these compounds are trapped in the center of the aqueous liposome. The liposome wall, which is a phospholipid membrane, accommodates fat-soluble materials, such as oil. 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 of several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although the formation of liposomes is spontaneous when the lipid film is mixed with an aqueous solution, the formation of liposomes can also be accelerated by applying force in the form of shaking using a homogenizer, a sonicator, or an extrusion device. Several other additives can be added to liposomes to change their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of cargo inside the liposome. Liposome preparations can be mainly composed of natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside.

[0323] In some embodiments, cationic or anionic liposomes are used as part of the subject composition or method, or liposomes with neutral lipids can also be used. Cationic liposomes can include negatively charged materials by mixing the materials with fatty acid liposome components and allowing them to charge associate. The selection of cationic or anionic liposomes depends on the desired pH of the final liposome mixture. Examples of cationic liposomes include, but are not limited to, lipofectin, lipofectamine, and lipofectace.

[0324] The subject proteins or nucleic acids can be encapsulated in PLGA microspheres for delivery, as further described in U.S. Published Applications 20130252281, 20130245107, and 20130244279.

[0325] In some cases, exosomes are used to deliver a subject protein or nucleic acid to a target cell. Exosomes are endogenous nanovesicles that transport RNA and proteins, and they can deliver RNA to the brain and other target organs.

[0326] Poly(β-amino alcohols) (PBAAs) can be used to deliver subject proteins or nucleic acids to target cells. US Patent Publication No. 20130302401 relates to a class of poly(β-amino alcohols) (PBAAs) prepared using combinatorial polymerization.

[0327] Sugar-based particles (eg, GalNAc, as described in reference WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136(49), 16958-16961)) can be used to deliver the subject protein or nucleic acid to target cells.

[0328] Supercharged proteins can be used to deliver subject proteins or nucleic acids to target cells. Supercharged proteins are a class of engineered or naturally occurring proteins with abnormally high positive or negative net theoretical charges. Both supernegatively charged proteins and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. The association of cargo with these proteins (such as plasmid DNA, RNA or other proteins) can facilitate the functional delivery of these macromolecules to mammalian cells in vitro and in vivo.

[0329] 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 contains relatively high abundance of positively charged amino acids, such as lysine or arginine, or a sequence that contains an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids.

[0330] Contemplated carriers or vehicles include materials such as gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, carbohydrates, 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(orthoesters), cyanoacrylates, gelatin-resorcinol-aldehyde type bioadhesives, polyacrylic acid and its copolymers and block copolymers.

[0331] Donor polynucleotide (donor template)

[0332] In some cases, the subject composition or method may include a donor polynucleotide. For example, in applications where a polynucleotide sequence needs to be inserted into a genome where a target sequence is cut, a donor polynucleotide (a nucleic acid comprising a donor sequence) may also be provided to a cell. "Donor sequence" or "donor polynucleotide" or "donor template" refers to a nucleic acid sequence inserted at a site targeted by a CRISPR complex (e.g., after dsDNA cutting, after cutting the target DNA, after double cutting the target DNA, etc.). In some cases, the donor sequence is provided to the cell as a single-stranded DNA. In some cases, the donor template is provided to the cell as a double-stranded DNA. It can be introduced into the cell in a linear or circular form. If introduced in a linear form, the end of the donor sequence can be protected by any convenient method (e.g., from exonucleolytic degradation), and such methods are known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of a linear molecule and / or a self-complementary oligonucleotide can be connected to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. The donor template can be introduced into the cell as part of a vector molecule having additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. In addition, the donor template can be introduced as naked nucleic acid, as a nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by a virus (e.g., adenovirus, AAV).

[0333] Examples of non-limiting aspects of the present disclosure

[0334] The various aspects of the subject matter described above (including embodiments) 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 skilled in the art, upon reading this disclosure, each individually numbered aspect may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of the aspects and is not limited to the combinations of aspects explicitly provided below:

[0335] Set A

[0336] 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 shown in any one of SEQ ID Nos: 126-132.

[0337] 2. The Acr polypeptide or nucleic acid as described in 1, wherein the amino acid sequence has 90% or more sequence identity with the sequence shown in any one of SEQ ID No.126-132.

[0338] 3. The Acr polypeptide or nucleic acid as described in 1, wherein the Acr polypeptide comprises the amino acid sequence shown in any one of SEQ ID No.126-132.

[0339] 4. The Acr polypeptide or nucleic acid of any one of 1-3, wherein the Acr polypeptide reduces off-target CRISPR nuclease activity by 10% or more compared to the off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0340] 5. The Acr polypeptide or nucleic acid of any one of 1-4, wherein the Acr polypeptide reduces the on-target CRISPR nuclease activity by no more than 40% compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0341] 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-fold compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0342] 7. An Acr polypeptide or nucleic acid as described in any one of 4-6, wherein the CRISPR nuclease is a Cas12a nuclease.

[0343] 8. An Acr polypeptide or nucleic acid as described in 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 shown in any one of SEQ ID NOs: 175 and 245-262.

[0344] 9. An Acr polypeptide or nucleic acid as described in 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 shown in any one of SEQ ID NOs: 1-86 and 176-244.

[0345] 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).

[0346] 11. A system comprising:

[0347] (i) an Acr polypeptide or a nucleic acid encoding the Acr polypeptide,

[0348] wherein the Acr polypeptide is an Acr polypeptide as described in any one of 1-10, or comprises an amino acid sequence having 80% or more sequence identity with the CRISPR nuclease amino acid sequence shown in any one of SEQ ID NOs: 165-169; and

[0349] (ii) a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease.

[0350] 12. The system of 11, wherein the CRISPR nuclease is a Cas12a nuclease.

[0351] 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 shown in any one of SEQ ID NOs: 175 and 245-262.

[0352] 14. The system of 11, wherein the CRISPR nuclease comprises an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the NUX protein shown in any one of SEQ ID NOs: 1-86 and 176-244.

[0353] 15. The system of any one of 11-14, further comprising a guide RNA or a nucleic acid encoding the guide RNA.

[0354] 16. The system of any one of 11-15, wherein the system comprises a nucleic acid encoding the Acr polypeptide and / or a nucleic acid encoding the CRISPR nuclease.

[0355] 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.

[0356] 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 nucleic acids encoding both the Acr polypeptide and the CRISPR nuclease.

[0357] 19. The system of any one of 16-18, wherein the translation control element is operably linked to an Acr polypeptide coding sequence or a CRISPR nuclease coding sequence.

[0358] 20. The system of 19, wherein the translation control element is selected from the group consisting of an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or any combination thereof.

[0359] 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.

[0360] 22. The system of 21, wherein the first promoter is a stronger promoter than the second promoter.

[0361] 23. The system of 21, wherein the second promoter is a stronger promoter than the first promoter.

[0362] 24. The system of 21 or 22, wherein the first promoter is selected from the group consisting of: CMV, miniCMV, EF1A, CAG or CBh.

[0363] 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.

[0364] 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.

[0365] 27. The system of any one of 16-26, wherein one or more of the nucleic acids is a viral vector.

[0366] 28. The system of 27, wherein the viral vector is an AAV vector.

[0367] 29. The system of any one of 16-26, wherein one or more of the nucleic acids are contained in a lipid nanoparticle (LNP).

[0368] 30. The system of any one of 11-15, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form.

[0369] 31. The system of 30, wherein the Acr polypeptide and / or the CRISPR nuclease is contained in a lipid nanoparticle (LNP).

[0370] 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.

[0371] 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 the ratio of on-target to off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0372] 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 reduced by 10% or more when compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0373] 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 reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0374] 36. A method for modifying a target nucleic acid, the method 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 a system as described in any one of 11-35, wherein the contacting results in modification of the nucleotide sequence of the target nucleic acid.

[0375] 37. The method of 36, wherein the modification is caused by the deletion of one or more nucleotides.

[0376] 38. The method of any one of 36 or 37, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio of 1:1.

[0377] 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) ranging from 1:1.25 to 1:10.

[0378] 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) ranging from 1:1.25 to 1:10.

[0379] 41. The method of any one of 36-40, wherein the contacting occurs in vivo.

[0380] 42. The method of any one of 36-40, wherein the contacting occurs in a eukaryotic cell.

[0381] 43. The method of 42, wherein the eukaryotic cell is an animal cell.

[0382] 44. The method of 43, wherein the animal cell is a human cell.

[0383] 45. The method of 42, wherein the eukaryotic cell is a stem cell.

[0384] 46. ​​The method of any one of 36-45, wherein the target nucleic acid encodes a gene product.

[0385] 47. The method of any one of 36-46, wherein the editing efficiency of on-target CRISPR nuclease activity is at least 4 times the editing efficiency of off-target CRISPR nuclease activity.

[0386] 48. A method as described in 47, wherein the off-target CRISPR nuclease activity is at an off-target site, and the off-target site contains no more than 5 mismatches compared to the on-target site.

[0387] 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 reduced by 10% or more when compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0388] 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 reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0389] 51. The method of any one of 36-50, wherein the method further comprises measuring editing efficiency.

[0390] 52. The method of any one of 36-51, wherein the method further comprises measuring the editing efficiency at one or more off-target sites.

[0391] Set B

[0392] 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 shown in any one of SEQ ID Nos: 126-132 and 264-267.

[0393] 2. The Acr polypeptide or nucleic acid as described in 1, wherein the amino acid sequence has 90% or more sequence identity with the sequence shown in any one of SEQ ID Nos. 126-132 and 264-267.

[0394] 3. The Acr polypeptide or nucleic acid as described in 1, wherein the Acr polypeptide comprises the amino acid sequence shown in any one of SEQ ID No.126-132.

[0395] 4. The Acr polypeptide or nucleic acid of 1, wherein the Acr polypeptide comprises the amino acid sequence shown in SEQ ID NO: 165, and the amino acid sequence has an amino acid variation at at least one position between amino acid 2 and amino acid 159 (including amino acid 2 and amino acid 159) of SEQ ID NO: 165.

[0396] 5. The Acr polypeptide or nucleic acid as described in 1, wherein the Acr polypeptide comprises the amino acid sequence shown in any one of SEQ ID No. 264-267.

[0397] 6. The Acr polypeptide or nucleic acid of 1 or 4, wherein the Acr polypeptide comprises one or more amino acid variations shown in Table 6.

[0398] 7. The Acr polypeptide or nucleic acid of 1 or 4, wherein the Acr polypeptide comprises one or more amino acid variations shown in Table 7.

[0399] 8. The Acr polypeptide or nucleic acid of any one of 1-7, wherein the Acr polypeptide reduces off-target CRISPR nuclease activity by 10% or more compared to the off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0400] 9. The Acr polypeptide or nucleic acid of any one of 1-8, wherein the Acr polypeptide reduces the on-target CRISPR nuclease activity by no more than 40% compared to the on-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0401] 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-fold compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of the Acr polypeptide.

[0402] 11. An Acr polypeptide or nucleic acid as described in any one of 8-10, wherein the CRISPR nuclease is a Cas12a nuclease.

[0403] 12. A polypeptide or nucleic acid as described in 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 shown in any one of SEQ ID NOs: 175 and 245-262.

[0404] 13.13. An Acr polypeptide or nucleic acid as described in any 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 shown in any one of SEQ ID NOs: 1-86 and 176-244.

[0405] 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).

[0406] 15. A system comprising:

[0407] (i) an Acr polypeptide or a nucleic acid encoding the Acr polypeptide,

[0408] wherein the Acr polypeptide is an Acr polypeptide as described in any one of claims 1 to 14, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 165 to 169, or comprising an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity with an Acr amino acid sequence as shown in any one of SEQ ID NOs: 165 to 169; and

[0409] (ii) a CRISPR nuclease or a nucleic acid encoding the CRISPR nuclease.

[0410] 16. The system of 15, wherein the CRISPR nuclease is a Cas12a nuclease.

[0411] 17. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence having 80%, 85%, 90% or 95% or more sequence identity to the Cas12a nuclease amino acid sequence shown in any one of SEQ ID NOs: 175 and 245-262.

[0412] 18. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence as shown 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 as shown in SEQ ID NO.263.

[0413] 19. The system of 15, wherein the CRISPR nuclease comprises an amino acid sequence as shown 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 shown in any one of SEQ ID NOs: 1-86 and 176-244.

[0414] 20. The system of any one of 15-19, further comprising a guide RNA or a nucleic acid encoding the guide RNA.

[0415] 21. The system of any one of 15-20, wherein the system comprises a nucleic acid encoding the Acr polypeptide and / or a nucleic acid encoding the CRISPR nuclease.

[0416] 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.

[0417] 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 nucleic acids encoding both the Acr polypeptide and the CRISPR nuclease.

[0418] 24. The system of any one of 21-23, wherein the translation control element is operably linked to an Acr polypeptide coding sequence or a CRISPR nuclease coding sequence.

[0419] 25. The system of 24, wherein the translation control element is selected from the group consisting of an IRES sequence, a 2A peptide coding sequence, a non-canonical start codon, or any combination thereof.

[0420] 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.

[0421] 27. The system of 26, wherein the first promoter is a stronger promoter than the second promoter.

[0422] 28. The system of 26, wherein the second promoter is a stronger promoter than the first promoter.

[0423] 29. The system of any one of 21-28, wherein one or more of the nucleic acids is a viral vector.

[0424] 30. The system of 29, wherein the viral vector is an AAV vector.

[0425] 31. The system of any one of 21-30, wherein one or more of the nucleic acids are contained in a lipid nanoparticle (LNP).

[0426] 32. The system of any one of 15-20, wherein the system comprises the Acr polypeptide and / or the CRISPR nuclease in protein form.

[0427] 33. The system of 32, wherein the Acr polypeptide and / or the CRISPR nuclease is contained in a lipid nanoparticle (LNP).

[0428] 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.

[0429] 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 the ratio of on-target to off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0430] 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 reduced by 10% or more when compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0431] 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 reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0432] 38. A method for modifying a target nucleic acid, the method 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 a system as described in any one of 15-37, wherein the contacting results in modification of the nucleotide sequence of the target nucleic acid.

[0433] 39. The method of 38, wherein the modification is caused by the deletion of one or more nucleotides.

[0434] 40. The method of any one of 38 or 39, wherein the contacting step delivers the CRISPR nuclease and the Acr polypeptide in a ratio of 1:1.

[0435] 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) ranging from 1:1.25 to 1:10.

[0436] 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) ranging from 1:1.25 to 1:10.

[0437] 43. The method of any one of 38-42, wherein the contacting occurs in vivo.

[0438] 44. The method of any one of 38-42, wherein the contacting occurs in a eukaryotic cell.

[0439] 45. The method of 44, wherein the eukaryotic cell is an animal cell.

[0440] 46. ​​The method of 45, wherein the animal cell is a human cell.

[0441] 47. The method of 44, wherein the eukaryotic cell is a stem cell.

[0442] 48. The method of any one of 38-47, wherein the target nucleic acid encodes a gene product.

[0443] 49. The method of any one of 38-47, wherein the editing efficiency of on-target CRISPR nuclease activity is at least 4 times the editing efficiency of off-target CRISPR nuclease activity.

[0444] 50. A method as described in 49, wherein the off-target CRISPR nuclease activity is at an off-target site, and the off-target site contains no more than 5 mismatches compared to the on-target site.

[0445] 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 reduced by 10% or more when compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0446] 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 reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

[0447] 53. The method of any one of 38-52, wherein the method further comprises measuring editing efficiency.

[0448] 54. The method of any one of 38-53, wherein the method further comprises measuring the editing efficiency at one or more off-target sites.

[0449] VI. Examples

[0450] The following examples are presented to provide a complete disclosure and description of how to carry out and use the present invention for those of ordinary skill in the art, and are not intended to limit the scope of the invention as considered by the inventors, nor are the following experiments represented to be all or only experiments carried out. Efforts have been made to ensure the correctness (e.g., amount, temperature, etc.) of the numerals used, but some experimental errors and deviations should be considered. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0451] General methods of molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (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 and Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle and Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for use in the methods referred to in or related to the present disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as Addgene, Inc. and the American Type Culture Collection (ATCC).

[0452] Example 1 Vector Construction

[0453] Nuclease expression vector

[0454] Codon-optimized genes encoding AsCas12a, NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 177), and NUX (SEQ ID NO: 178) were synthesized and cloned into a mammalian expression vector under the CMV promoter. The vector included an in-frame nuclear localization signal OPT NLS (GRSSDDEATADSQHAAPPKKKRKV) (SEQ ID NO: 125), followed by a linker (GGSGGSGGSGGSGGSGGSGGSGGS) (SEQ ID NO: 124), followed by a 3x HA tag.

[0455] Guidance carrier

[0456] To prepare the guide vector, the direct repeat (DR) sequences of nucleases NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 178) and AsCas12a (Table 2) were placed downstream of the U6 promoter with a start G. DR19s are NUX (SEQ ID NO: 176), NUX (SEQ ID NO: 177) and NUX (SEQ ID NO: 178) direct repeat sequences and DR18s are AsCas12a direct repeat sequences. The spacer target sequence is placed downstream of the DR sequence. The spacer sequence for DNMT is CTGATGGTCCATGTCTGTT A (SEQ ID NO: 172), and its PAM is TTTC. The spacer sequence for FANCF1 is GGCGGGGTCCAGTTCCGGGA (SEQ ID NO: 162), and its PAM is TTTG. By changing a single nucleotide at a given position to its counterpart (such as A->T and G->C), a mismatched guide is prepared.

[0457] Table 2: Direct repeat sequences used in guide vectors

[0458] Direct repeat sequence SEQ ID NO: DR19s AATTTCTACTGTGTGTAGAT 163 DR18s AATTTCTACTCTTGTAGAT 164

[0459] Acr vector

[0460] Potential anti-CRISPR (Acr) sequences for use with nucleases were identified in the 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.

[0461] Table 3: Identified Acr

[0462]

[0463] Example 2 Acr activity

[0464] The editing efficiency of AsCas12a (SEQ ID NO: 175) in the presence of Acr was tested in HEK293T cells. The vector constructed in Example 1 was transfected into cells using Mirus Transit X2 reagent MirusBio CAT MIR6003. According to the Mirus Transit X2 transfection suggestion, the test was performed in a 96-well plate transfected with 100ng nuclease expression vector, 100ng Acr expression vector and 50ng targeting guidance vector. The samples were incubated for 72h and harvested with a quick extraction method (QuickExtract). Genomic DNA was amplified using genomic region-specific primers.

[0465]

[0466] The samples were checked for purity on a 2% agarose gel and sequenced by Sanger sequencing. TIDE analysis was performed according to the method of Brinkman et al., 2014 and the recommendations of the TIDE website (https: / / tide.nki.nl / ). TIDE output data on editing efficiency were plotted using Prism software. Acr was compared with AcrVA1 (ACX-137) (SEQ ID NO: 67) and AcrIIA4 (ACX-105) (SEQ ID NO: 35). The results are shown in Figure 1 middle.

[0467] The activity of Acr was also tested by co-transfection with a second nuclease, NUX (SEQ ID NO: 176). Following the same protocol as above, NUX (SEQ ID NO: 176) and Acr were transiently delivered into HEK293T cells using expression vectors. The editing efficiency was measured as above. The results are shown in Figure 2 middle.

[0468] The identified Acr, Acx-175, Acx-176, Acx-177, and Acx-178 (SEQ ID NOs: 165-168, respectively) showed the ability to inhibit the editing efficiency of Cas12a. When tested with NUX (SEQ ID NO: 176), Acr showed different inhibition patterns. Compared with Acx-175, Acx-178 showed little inhibition, and the inhibitory activity of Acx-176 was less effective, while Acx-176 was a stronger inhibitor compared to Acx-175 with Cas12a.

[0469] Example 3 Dose-dependent nuclease inhibition properties of Acx-175

[0470] In a series of dosing experiments, Acx-175 demonstrated the intrinsic ErAcr property of inhibiting off-target editing while leaving on-target activity largely unchanged.

[0471] AsCas12a endonuclease was co-transfected into HEK293T cells with reduced doses of Acx-175 to observe editing activity targeting DNMT1. Transfection, incubation, and analysis were performed as described in Example 2, including measuring editing efficiency. Off-target editing was measured using a guide RNA with a single base change at position 9 (MM9, CTGATGGTGCATGTCTGTTA: SEQ ID NO: 173). The results are shown in Figure 3 middle.

[0472] NUX (SEQ ID NO: 177) endonuclease was co-transfected with reduced doses of Acx-175 to observe editing activity targeting DNMT1. The transfection, incubation, and analysis protocols 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) and a guide RNA with a single base change at position 5 (MM5, CTGAAGGTCCATGTCTGTTA: SEQ ID NO: 174). The results are shown in Figure 4 middle.

[0473] Example 4: Generation of engineered Acr

[0474] The potent nuclease inhibitor Acx-175 (SEQ ID NO: 165) was mutagenized using a random mutagenesis kit (Genemorph II). Mutants were selected using the DEAD / ALIVE bacterial screening method as described by Huimin Zhao in 2005; https: / / academic.oup.com / nar / article / 33 / 18 / e154 / 2401371.

[0475] Expression vectors were constructed using NUX (SEQ ID NO: 178) and a mutagenized Acx-175 library as described in Example 1. The target was DNMT1, and off-target editing was measured using a guide RNA with a single base change at position 8 (MM8, CTGATGGaCCATGTCTGTTA: SEQ ID NO: 161).

[0476] The first round of screening generated six Acx-175 variants (Table 4, except Acx-315), which were transferred into CMV expression vectors and tested with NUX (SEQ ID NO: 178) and DNMT1 in HEK293T cells according to the method in Example 2 for validation. The results are shown in Figure 5 middle.

[0477] Table 4: Engineered Acr

[0478]

[0479] Example 5: Dose-dependent nuclease inhibition using engineered Acr

[0480] An expression vector was constructed using NUX (SEQ ID NO: 178) and four engineered Acrs (Acx-306, Acx-308, Acx-310, and Acx-311). Three different ratios of Nux:Acx (2:1, 1:1, and 1:2) were transfected into HEK293T cells. The target was DNMT1. Transfection, incubation, and analysis were performed as described in Example 2. The results of the editing efficiency of Nux:Acx 2:1 are shown in Fig. 6A The results of the editing efficiency of Nux:Acx 1:1 are shown in Figure 6B The results of the editing efficiency of Nux:Acx1:2 are shown in Figure 6C middle.

[0481] Example 6: Off-target inhibition of Acr measured

[0482] NUX (SEQ ID NO: 178) was co-transfected into HEK293T cells with Acx-306 or Acx-310. On-target and off-target editing efficiencies were measured for single mismatches at positions 1, 9, 19, and 20. The editing efficiency was compared with Acx-105 as a negative control and wild-type Acx-175 as a positive control. The results are shown in Figure 7 middle.

[0483] NUX (SEQ ID NO: 178) was co-transfected with Acx-315 (SEQ ID NO: 132) into HEK293T cells. On-target and off-target editing efficiencies were measured for a single mismatch at position 8 of DNMT1. NUX (SEQ ID NO: 178) and Acx-315 were tested at a range of Nux:Acx ratios. The results are Fig. 8A The on-target and off-target editing efficiencies are shown in Figure 8B and shown in Table 5 as on-target:off-target ratios.

[0484] Table 5

[0485] Acx:NUX On Target: Off Target 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

[0486] Example 7: Variant Acr Activity

[0487] Acr Acx-175 (SEQ ID NO: 165) was subjected to random mutagenesis at codon positions 2 to 159 using error-prone PCR (Genemorph II, Agilent Biosciences). The resulting library of variant Acrs was screened using the methods described in Example 4. Sequencing of the variants generated a collection of amino acid substitutions at each position as shown in Table 6.

[0488] Table 6

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496] As described in Example 4, the ability of the variants to modulate on-target and off-target editing of the CRISPR nuclease targeting DNMT1 (on-target) and DNMT1 mismatch 8 (off-target) (SEQ ID NO: 263) was assessed. The results are shown in Fig.12 The tested variants are listed in Table 7. The results showed that both the inhibition level and the on-target to off-target activity ratio can be modulated by the variation of Acr. Fig.13 Shows Fig.12 Amino acid sequence alignment of ErAcr shown.

[0497] Table 7. Tested variants

[0498] Variants Amino Acid Substitutions (Variants) SEQ ID NO: ErAcr3 V74E, L97V, S111G 264 ErAcr4 N52I 265 ErAcr13 K3N, T80P 266 ErAcr20 T80I, Y107N 267

[0499] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of this invention that changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0500] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to envision various arrangements, although these arrangements are not explicitly described or shown in this article, but they embody the principle of the present invention and are included in its spirit and scope.In addition, all embodiments and conditional language cited herein are mainly intended to help readers understand the principle of the present invention and the conception of promoting technology provided by the inventor, and should be interpreted as not limiting such particularly cited embodiments and conditions.In addition, all statements citing the principles, aspects and embodiments of the present invention and its specific embodiments herein are intended to cover their structural equivalents and functional equivalents.In addition, it is hoped that such equivalents include currently known equivalents and equivalents to be developed in the future, that is, regardless of the structure, any element developed to perform the same function.In addition, any content disclosed herein is not intended to contribute to the public, regardless of whether such disclosure is clearly described in the claims.

[0501] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are 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 cited only for the limitation in the claims when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of the limitation in the claims; if such exact phrase is not used in the limitation in the claims, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is not cited.

[0502] Although the present invention has been described with reference to its specific embodiments, it will be appreciated 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 present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, process steps or multiple steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

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

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

3. The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises any of the amino acid sequences shown in SEQ ID Nos: 126-132.

4. The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises the amino acid sequence shown in SEQ ID NO: 165, and said amino acid sequence has an amino acid variation at at least one position between amino acid 2 and amino acid 159 (including amino acid 2 and amino acid 159) of SEQ ID NO:

165.

5. The Acr polypeptide or nucleic acid according to claim 1, wherein said Acr polypeptide comprises any of the amino acid sequences shown in SEQ ID Nos: 264-267.

6. The Acr polypeptide or nucleic acid according to claim 1 or claim 4, wherein said Acr polypeptide comprises one or more amino acid variations shown in Table 6.

7. The Acr polypeptide or nucleic acid according to claim 1 or claim 4, wherein said Acr polypeptide comprises one or more amino acid variations shown in Table 7.

8. The Acr polypeptide or nucleic acid according to any one of claims 1-7, wherein said Acr polypeptide reduces off-target CRISPR nuclease activity by 10% or more compared to off-target CRISPR nuclease activity in the absence of said Acr polypeptide.

9. The Acr polypeptide or nucleic acid according to any one of claims 1-8, wherein said Acr polypeptide reduces on-target CRISPR nuclease activity by no more than 40% compared to on-target CRISPR nuclease activity in the absence of said Acr polypeptide.

10. The Acr polypeptide or nucleic acid according to any one of claims 1-9, wherein said Acr polypeptide increases the ratio of on-target to off-target CRISPR nuclease activity by at least 1.25-fold compared to the ratio of on-target to off-target CRISPR nuclease activity in the absence of said Acr polypeptide.

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

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

13. The Acr polypeptide or nucleic acid according to 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 amino acid sequence of the NUX protein shown in any one of SEQ ID NOs: 1-86 and 176-244.

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

15. A system, which comprises: (i) An Acr polypeptide or a nucleic acid encoding the Acr polypeptide, wherein the Acr polypeptide is the Acr polypeptide according to any one of claims 1-14, comprises the amino acid sequence shown 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 shown 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 according to claim 15, wherein the CRISPR nuclease is a Cas12a nuclease.

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

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

263.

19. The system according to claim 15, wherein the CRISPR nuclease comprises the amino acid sequence shown 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 amino acid sequence of the NUX protein shown in any one of SEQ ID NOs: 1-86 and 176-244.

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

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

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

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

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

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

26. The system according to 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 according to claim 26, wherein the first promoter is a stronger promoter than the second promoter.

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

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

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

31. The system according to any one of claims 21-30, wherein one or more of the nucleic acids are contained in a lipid nanoparticle (LNP).

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

33. The system according to claim 32, wherein the Acr polypeptide and / or the CRISPR nuclease are contained in a lipid nanoparticle (LNP).

34. The system according to 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 according to 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 the ratio of on-target to off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

36. The system according to any one of claims 15-35, wherein the off-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

37. The system according to any one of claims 15-36, wherein the on-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

38. A method of modifying a target nucleic acid, which comprises: 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 a system according to any one of claims 15-37, and wherein the contacting results in modification of the nucleotide sequence of the target nucleic acid.

39. The method according to claim 38, wherein the modification is caused by deletion of one or more nucleotides.

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

1.

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

10.

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

10.

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

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

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

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

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

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

49. The method according to any one of claims 38-48, wherein the editing efficiency of on-target CRISPR nuclease activity is at least 4 times that of off-target CRISPR nuclease activity.

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

51. The method according to any one of claims 38-50, wherein the off-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by 10% or more compared to the off-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

52. The method according to any one of claims 38-51, wherein the on-target activity of the CRISPR nuclease in the presence of the Acr polypeptide is reduced by no more than 40% compared to the on-target activity of the CRISPR nuclease in the absence of the Acr polypeptide.

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

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

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