WO2021108442A2 - Modulators of cas9 polypeptide activity and methods of use thereof - Google Patents

Modulators of cas9 polypeptide activity and methods of use thereof Download PDF

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WO2021108442A2
WO2021108442A2 PCT/US2020/062073 US2020062073W WO2021108442A2 WO 2021108442 A2 WO2021108442 A2 WO 2021108442A2 US 2020062073 W US2020062073 W US 2020062073W WO 2021108442 A2 WO2021108442 A2 WO 2021108442A2
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cell
amino acid
polypeptide
acr
acid sequence
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PCT/US2020/062073
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French (fr)
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WO2021108442A3 (en
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Jennifer A. Doudna
Gavin KNOTT
Fayyaz-ul-Amir Afsar MINHAS
Simon EITZINGER
Kyle E. WATTERS
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The Regents Of The University Of California
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Publication of WO2021108442A2 publication Critical patent/WO2021108442A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • Cas9 Clustered Regularly Interspaced Short Palindromic Repeat
  • RNA- guided Cas9 has proven to be a versatile tool for genome engineering in multiple cell types and organisms.
  • the present disclosure provides polypeptides that inhibit activity of a CRISPR/Cas effector polypeptide, nucleic acids encoding the polypeptides, and systems comprising the polypeptides and/or nucleic acids encoding the polypeptides.
  • FIG.1 presents Table 1, which provides a list of Acrs used for training and cross-validation of the AcRanker model.
  • FIG.2 presents Table 2, which provides a list of Acrs used for independent testing of AcRanker.
  • FIG.3 presents Table 3: grouping of amino acids based on physiochemical properties.
  • FIG.4A-4B present purified Acr candidates and CRISPR/Cas effectors used.
  • FIG.5 present Table 4, which provides results for the leave-one-out cross-validation.
  • FIG.6 presents Table 5, which provides independent testing set validation results.
  • FIG.7 presents Table 6, which provides independent testing set validation results.
  • FIG.8A-8D presents Table 7, which provides a list of expected lethal self-targeting Streptococcus genomes obtained with Self-Target Spacer Searcher (STSS).
  • STSS Self-Target Spacer Searcher
  • Self-Targeting Spacer Sequences from top to bottom SEQ ID NOs:49-73, 73, 74-77, 68, 78-81, 76, 82-84, 77, 60, 62, 85, 86, 86, 87, 88-90, 90, 91-92, 92, 93-95, 93.
  • FIG.9A-9E presents Table 8, which provides top Acr gene candidates within each genome ranked by AcRanker.
  • FIG.10 depicts the genomic context of the Acr candidates selected for biochemical testing.
  • FIG.11A-11D depict inhibition of SpyCas9 and SauCas9 by Acr candidates.
  • FIG.12A-12D depict the results of an in vitro cleavage assay with SpyCas9.
  • FIG.13A-13B depict the results of an in vitro cleavage assay with SauCas9.
  • FIG.14A-14C depict the results of control experiments for the in vitro cleavage assays.
  • FIG.15A-15B depict inhibition of SinCas9 by ML1 and ML8.
  • FIG.16A-16B depict the sgRNA used for inhibition of SinCas9 (from top to bottom SEQ ID NOs:96-98).
  • FIG.17A-17D depict the results of an in vitro cleavage assay with SinCas9.
  • FIG.18A-18E depict competition between ML1 and AcrIIA2 for the same binding site.
  • FIG.19A-19C present Table 9, which provides primers (from top to bottom SEQ ID NOs:99- 127) used for amplification of DNA targets, cloning, and in vitro transcription (IVT) of sgRNAs.
  • FIG.20A-20B present Table 10, which provides amino acid sequences and accession numbers of Acr candidates.
  • FIG.21A-21D present Table 11, which provides amino acid sequences (from top to bottom SEQ ID NOs: 128-131) of CRISPR/Cas effector polypeptides used in this study.
  • FIG.22 presents Table 12, which provides nucleotide sequences (from top to bottom SEQ ID NOs:132-135) of sgRNAs used for in vitro cleavage assays.
  • FIG.23A-23B present Table 13, which provides nucleotide sequences of DNA targets (SEQ ID NO:136) used for in vitro cleavage assays.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • polynucleotide and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • oligonucleotide refers to a polynucleotide of between 4 and 100 nucleotides of single- or double-stranded nucleic acid (e.g., DNA, RNA, or a modified nucleic acid). However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes, transcribed (in vitro and/or in vivo), or chemically synthesized.
  • nucleic acid and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • hybridizable or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e.
  • Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/ uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C).
  • G can also base pair with U.
  • G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base- pairing with codons in mRNA.
  • a G (e.g., of a protein- binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA) is considered complementary to both a U and to C.
  • a G/U base-pair can be made at a given nucleotide position of a protein- binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
  • Tm melting temperature
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like).
  • a polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 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 complementarity to a target region within the target nucleic acid sequence to which it will hybridize.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
  • Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol.
  • Binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions are generally characterized by a dissociation constant (K d ) of less than 10 6 M, less than 10 -7 M, less than 10 -8 M, less than 10 9 M, less than 10 10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M, or less than 10 -15 M.
  • K d dissociation constant
  • Affinity refers to the strength of binding, increased binding affinity being correlated with a lower Kd.
  • a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine.
  • Exemplary conservative amino acid substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine- glutamine.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways.
  • sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi(dot)nlm(dot)nili(dot)gov/BLAST, ebi(dot)ac(dot)uk/Tools/msa/tcoffee/, ebi(dot)ac(dot)uk/Tools/msa/muscle/, mafft.cbrc(dot)jp/alignment/software/, www(dot)sbg(dot)bio(dot)ic(dot)ac(dot)uk/ ⁇ phyre2/.
  • DNA regulatory sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and/or regulate translation of an encoded polypeptide.
  • control elements refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and/or regulate translation of an encoded polypeptide.
  • regulatory elements refers transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e
  • a “promoter” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.
  • Various promoters including inducible promoters, may be used to drive the various nucleic acids (e.g., vectors) of the present disclosure.
  • "Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", below).
  • RNA sequences encoding RNA may also be considered recombinant.
  • the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
  • This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant polynucleotide encodes a polypeptide
  • the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence.
  • wild type wild type
  • variant e.g., a mutant
  • the term ''recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur.
  • a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non- naturally occurring (e.g., a variant, a mutant, etc.).
  • a ''recombinant" polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence.
  • a "vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.
  • An “expression cassette” comprises a DNA coding sequence operably linked to a promoter.
  • "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • the terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • Any given component, or combination of components can be unlabeled, or can be detectably labeled with a label moiety. In some cases, when two or more components are labeled, they can be labeled with label moieties that are distinguishable from one another.
  • General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al.
  • the present disclosure provides polypeptides that inhibit activity of a CRISPR/Cas effector polypeptide, nucleic acids encoding the polypeptides, and systems comprising the polypeptides and/or nucleic acids encoding the polypeptides.
  • the present disclosure provides methods of inhibiting activity of a CRISPR/Cas effector polypeptide.
  • ANTI-CRISPR POLYPEPTIDES [0053] The present disclosure provides polypeptides that inhibit activity of one or more CRISPR/Cas effector polypeptides.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 30 amino acids to 64 amino acids (e.g., from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 30 amino acids to 64 amino acids.
  • the Acr polypeptide has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 64 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 40 amino acids to 77 amino acids (e.g., from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, or from about 75 aa to 77 aa) of the following amino acid sequence: [0058] MGKTYWYNEGTDTLLTEKEYKELMEREAKALYEE
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML2 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 40 amino acids to 77 amino acids.
  • the Acr polypeptide has a length of from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, or from about 75 aa to 77 aa.
  • aa amino acids
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to 89 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, or from about 85 aa to 89 aa) of the following amino acid sequence: [0061] MSKTMYKNDVIELIKNAKTNNEELLFTSVERNTREAATQ
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML3 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 50 amino acids to 89 amino acids.
  • the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, or from about 85 aa to 89 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to about 95 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, from about 85 aa to about 90 aa, or from about 90 aa to 95 aa) of the following amino acid sequence: [0064] MLRRVNHVKNVLA
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML4 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 50 amino acids to 95 amino acids.
  • the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, from about 85 aa to about 90 aa, or from about 90 aa to 95 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 140 to about 210 amino acids (e.g., from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, or from about 200 aa to 210 aa) of the following amino acid sequence: [0067] MNRLKELRKEKKLTQEELAGEIGVSKITILRWENGERQIKPDKAKELAKYFN
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML5 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 140 amino acids to 210 amino acids.
  • the Acr polypeptide has a length of from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, or from about 200 aa to 210 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 40 amino acids to about 65 amino acids (e.g., from about from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 65 aa) of the following amino acid sequence: [0070] MLYIDEFKEAIDKGYILGGTVAIVRKNGKIFDYVLPHEEVREEEVVTVERVEDVMRELE (SEQ ID NO:6).
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML6 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 40 amino acids to 65 amino acids.
  • the Acr polypeptide has a length of from about from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 65 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 140 amino acids to about 219 amino acids (e.g., from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, from about 200 aa to about 210 aa, or from about 210 aa to 219 aa) of the following amino acid sequence: [0073] MIKIYFGKDAALNQAIQSRLDS
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML7 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 140 amino acids to 219 amino acids.
  • the Acr polypeptide has a length of from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, from about 200 aa to about 210 aa, or from about 210 aa to 219 aa.
  • aa amino acids
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 60 amino acids to about 114 amino acids (e.g., from about 60 amino acids (aa) to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, from about 95 aa to about 100 aa, from about 100 aa to about 105 aa, from about 105 aa to about 110 aa, or faro
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 60 amino acids to 114 amino acids.
  • the Acr polypeptide has a length of from about 60 amino acids (aa) to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, from about 95 aa to about 100 aa, from about 100 aa to about 105 aa, from about 105 aa to about 110 aa, or from about 110 aa to 114 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to about 99 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, or from about 95 aa to 99 aa) of the following amino acid sequence: [
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML9 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 50 amino acids to 99 amino acids.
  • the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, or from about 95 aa to 99 aa.
  • An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 35 to about 49 amino acids (e.g., from about 35 amino acids (aa) to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to 49 aa) of the following amino acid sequence: [0082] MNIEDIERIISEYLIFRSDIDGCAVIDIEDFLKHIRFSYERLK (SEQ ID NO:10).
  • an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML10 amino acid sequence depicted in FIG.20A-20B (and set forth above).
  • the Acr polypeptide has a length of from about 35 amino acids to 49 amino acids.
  • the Acr polypeptide has a length of from about 35 amino acids (aa) to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to 49 aa.
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9 polypeptide in a Cas9/guide RNA complex is not contacted with the Acr polypeptide).
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9/guide RNA complex is not contacted with the Acr polypeptide).
  • an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide, when the molar ratio of Acr to Cas9 polypeptide is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1.
  • an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide, when the molar ratio of Acr to Cas9 polypeptide is from about 2:1 to about 5:1, from about 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1 to about 50:1, or from about 50:1 to about 100:1.
  • the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 10:1.
  • the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 50:1.
  • the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 100:1.
  • a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 10:1, a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is from 10:1 to 50:1 (e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1).
  • a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 50:1.
  • a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 100:1.
  • Binding activity of a Cas9/guide RNA complex refers to binding of the Cas9/guide RNA complex to a target nucleic acid, where the target nucleic acid comprises a nucleotide sequence that has complementarity to a target-binding nucleotide sequence in the guide RNA.
  • “Cleavage” activity of a Cas9/guide RNA complex refers to generation by the Cas9/guide RNA complex of a single-strand or double-strand break in a target nucleic acid.
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Streptococcus pyogenes Cas9 (Spy Cas9) amino acid sequence provided in FIG.21A-21D.
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Staphylococcus aureus Cas9 (“SauCas9”) amino acid sequence provided in FIG.21A-21D.
  • SauCas9 Staphylococcus aureus Cas9
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Streptococcus iniae Cas9 (SinCas9) amino acid sequence provided in FIG.21A-21D.
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; and b) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sin Cas9 amino acid sequence provided in FIG.21A-21D.
  • an Acr polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity the ML1 amino acid sequence depicted in FIG.20A-20B inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; and b) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, or 100%
  • an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; b) a Sau Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sau Cas9 amino acid sequence provided in FIG.21A-21D; and c) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%,
  • an Acr polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity the ML8 amino acid sequence depicted in FIG.20A-20B inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50% at least 60% at least 70% , at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; b) a Sau Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity
  • an Acr polypeptide of the present disclosure does not substantially inhibit binding and/or cleavage activity of a SauCas9 in a Cas9/guide RNA complex.
  • an Acr polypeptide of the present disclosure does not substantially inhibit cleavage activity of a Cas9/guide RNA complex, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A-21D.
  • an Acr polypeptide of the present disclosure inhibits cleavage activity of a SauCas9/guide RNA complex by no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 2%, or no more than 1%, compared to the cleavage activity of the SauCas9/guide RNA complex in the absence of the Acr polypeptide, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A- 21D.
  • an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9/guide RNA complex by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1%, compared to the cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A-21D.
  • Whether an Acr polypeptide inhibits binding activity of a given Cas9/guide RNA complex can be readily determined.
  • a double stranded “Cas9 beacon” DNA fragment comprising a target can be constructed in which the target strand comprises a fluorophore and the non-target strand comprises a fluorescence quencher.
  • the non-target strand Upon binding of the beacon by a Cas9/guide RNA complex, the non-target strand is displaced, allowing unquenched fluorescence from the target strand.
  • Measurement of increased fluorescence signal in the presence of a Cas9/guide RNA complex indicates DNA binding activity of the Cas9/guide RNA complex (Mekler et al. (2016) Nuc.
  • a target DNA is mixed in vitro with: a) a complex of Cas9 and a guide RNA, where the guide RNA comprises both a nucleotide sequence (tracrRNA) that activates the Cas9 polypeptide and a nucleotide sequence (crRNA) that binds to the target DNA; and b) an Acr polypeptide.
  • TrarRNA nucleotide sequence
  • crRNA nucleotide sequence
  • Production of cleavage products of action of the Cas9 on the target DNA can be detected by resolving the cleavage products on a 1% agarose cell and staining the resolved cleavage products.
  • an Acr polypeptide of the present disclosure on cleavage of a target DNA by a Cas9/guide RNA complex can be tested in a cell.
  • a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide and a nucleic acid comprising a nucleotide sequence encoding a reporter e.g., a fluorescent protein such as a green fluorescent protein
  • a reporter e.g., a fluorescent protein such as a green fluorescent protein
  • a mammalian cell e.g., 293 cells, or other mammalian cell line
  • a ribonucleoprotein (RNP) complex comprising a Cas9 polypeptide and a guide RNA targeting the nucleotide sequence encoding the reporter is introduced into the reporter cell.
  • Inhibition of gene editing of the reporter can be determined by detecting the reporter.
  • the reporter is a fluorescent protein
  • fluorescence activated cell sorting FACS
  • a mixture of a Cas9/guide RNA complex and Acr polypeptide can be introduced into a mammalian cell line, and the effect of the Acr polypeptide on the ability of the Cas9/guide RNA complex to carry out gene editing can be determined by analyzing production of a gene product encoded by a nucleotide sequence targeted by the guide RNA.
  • an Acr polypeptide of the present disclosure comprises a non-peptidic moiety covalently linked to the Acr polypeptide.
  • the covalently linked non-peptidic moiety can confer a desirable attribute (e.g., increased protease resistance, increased membrane permeability, increased cell type or tissue specific targeting, increased in vivo half-life, increased in vivo stability, increased bioavailability), without substantially altering the ability of the linked Acr polypeptide to inhibit Cas9 activity.
  • the non-peptidic moiety confers increased in vivo half-life on the linked Acr polypeptide, compared to the in vivo half-life of the Acr not comprising the non-peptidic moiety.
  • the in vivo half-life of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold, greater than the in vivo half-life of the Acr polypeptide not comprising the non-peptidic moiety.
  • the non-peptidic moiety confers an increase in half-life of the linked Acr polypeptide in circulation in an animal.
  • the non-peptidic moiety confers increased in vivo stability on the linked Acr polypeptide, compared to the in vivo stability of the Acr polypeptide not comprising the non-peptidic moiety.
  • the in vivo stability of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5- fold, or at least 10-fold, greater than the in vivo stability of the Acr polypeptide not comprising the non-peptidic moiety.
  • the non-peptidic moiety confers increased bioavailability on the linked Acr polypeptide, compared to the bioavailability of the Acr polypeptide not comprising the non-peptidic moiety.
  • the bioavailability of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold, greater than the bioavailability of the Acr polypeptide not comprising the non-peptidic moiety.
  • Suitable non-peptidic moieties include, but are not limited to, lipids and non-peptidic polymers.
  • non-peptidic moieties include, but are not limited to, poly(ethylene glycol), polysialic acid, hydroxyethyl starch (HES), a dendrimer, a nanoparticle, and a liposome.
  • a non-peptidic moiety covalently linked to an Acr polypeptide of the present disclosure is a polymer.
  • Polymers may contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as “PGA”; lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA”; caprolactone units, such as poly(caprolactone), collectively referred to herein as “PCL”; copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as “PLGA”; and polyacrylates, and derivatives thereof.
  • PGA glycolic acid units
  • lactic acid units such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,
  • Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers".
  • PEG polyethylene glycol
  • the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker.
  • An Acr polypeptide of the present disclosure may include one or more covalently linked hydrophilic polymers.
  • Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide); poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol); polyoxazoline; and copolymers thereof.
  • PPS poly-L-glutamic acid
  • PES poly-L-glutamic acid
  • PES poly-L-gluta
  • An Acr polypeptide of the present disclosure may include one or more covalently linked hydrophobic polymers.
  • suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co- caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes; poly(oxy)
  • An Acr polypeptide of the present disclosure may include one or more covalently linked biodegradable polymers.
  • Suitable biodegradable polymers can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose such as methyl cellulose and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyleth
  • biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof.
  • the particle contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid).
  • an Acr polypeptide of the present disclosure is a fusion Acr polypeptide.
  • the present disclosure provides a fusion polypeptide (an “Acr fusion polypeptide”) comprising: a) an Acr polypeptide of the present disclosure; and b) a heterologous fusion partner (i.e., one or more heterologous fusion partners).
  • the heterologous fusion partner can provide one or more desirable attributes (where such attributes include, e.g., as increased protease resistance, increased membrane permeability, or increased half-life, increased nuclear localization, increased cell or tissue specific targeting and the like) without substantially altering the ability of the linked Acr polypeptide to inhibit Cas9 activity.
  • a fusion polypeptide comprising: a) an Acr polypeptide of the present disclosure; and b) a heterologous fusion partner is also referred to herein as an “Acr fusion polypeptide.”
  • a fusion polypeptide of the present disclosure can comprise two or more heterologous fusion partners.
  • Suitable heterologous fusion partners include, but are not limited to, a nuclear localization signal; a chloroplast transit peptide; an endosomal escape peptide; an epitope tag; a polypeptide that provides for ease of purification; a detectable protein; a protein that provides for increased in vivo half-life; a protein that provides for increased cell type or tissue specificity (e.g. an antibody or fragment thereof) and the like.
  • Suitable heterologous fusion partners include a hydroxine-binding protein, transthyretin, ⁇ 1-acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, ⁇ -2- macroglobulin, a lipoprotein, and a fragment of any of the foregoing.
  • Suitable heterologous fusion partners include a fluorescent protein, e.g., a green fluorescent protein (GFP), a yellow fluorescent protein, a red fluorescent protein a cyan fluorescent protein, and the like.
  • Suitable heterologous fusion partners include, e.g., a poly(histidine) tag (e.g., a 6XHis tag); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like.
  • a fusion polypeptide of the present disclosure comprises an Acr polypeptide fused to a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs).
  • NLS nuclear localization signal
  • a fusion Acr polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs).
  • one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus of the Acr polypeptide. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus of the Acr polypeptide. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus of the Acr polypeptide.
  • one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus of the Acr polypeptide. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus of the Acr polypeptide.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:11); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:12)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:13) or RQRRNELKRSP (SEQ ID NO:14); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:15); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:16) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:17) and
  • An Acr fusion polypeptide of the present disclosure can include, as the fusion partner, a "Protein Transduction Domain” or PTD (also known as a CPP – cell penetrating peptide).
  • 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:27); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al.
  • Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO:27), RKKRRQRRR (SEQ ID NO:32); an arginine homopolymer of from 3 arginine residues to 50 arginine residues;
  • Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:27); RKKRRQRR (SEQ ID NO:33); YARAAARQARA (SEQ ID NO:34); THRLPRRRRRR (SEQ ID NO:35); and GGRRARRRRRR (SEQ ID NO:36).
  • an Acr fusion polypeptide of the present disclosure comprises a linker between the Acr polypeptide and the fusion partner.
  • Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide.
  • the use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide.
  • the creation of such sequences is routine to those of skill in the art.
  • a variety of different linkers are commercially available and are considered suitable for use. In some cases, the linker is proteolytically cleavable.
  • linker polypeptides include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n (SEQ ID NO:37), GGSGGS n (SEQ ID NO:38), and GGGGS n (SEQ ID NO:39), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers.
  • Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:40), GGSGG (SEQ ID NO:41), GSGSG (SEQ ID NO:42), GSGGG (SEQ ID NO:43), GGGSG (SEQ ID NO:44), GSSSG (SEQ ID NO:45), and the like.
  • An Acr fusion polypeptide of the present disclosure can also comprise a covalently linked non-peptidic moiety, where suitable non-peptidic moieties are discussed above.
  • NUCLEIC ACIDS AND RECOMBINANT EXPRESSION VECTORS [00115] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • an Acr polypeptide of the present disclosure is a fusion polypeptide.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure.
  • the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the nucleic acid is RNA.
  • the nucleic acid is DNA.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure.
  • the nucleic acid is RNA.
  • the nucleic acid is DNA.
  • a nucleotide sequence encoding an Acr polypeptide of the present disclosure is codon optimized.
  • This type of optimization can entail a mutation of an Acr- encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein.
  • the codons can be changed, but the encoded protein remains unchanged.
  • a human codon- optimized Acr-encoding nucleotide sequence could be used.
  • a mouse codon-optimized Acr-encoding nucleotide sequence could be generated.
  • a plant codon-optimized Acr-encoding nucleotide sequence could be generated.
  • nucleotide sequence encoding an Acr polypeptide of the present disclosure, or encoding an Acr fusion polypeptide of the present disclosure is operably linked to one or more of a promoter, an enhancer, an internal ribosomal entry site, and a transcription termination signal.
  • nucleotide sequence encoding an Acr polypeptide of the present disclosure, or encoding an Acr fusion polypeptide of the present disclosure is operably linked to a transcriptional control element.
  • the transcriptional control element is a transcriptional control element that is functional in a eukaryotic cell.
  • the transcriptional control element can be a promoter.
  • the promoter is a constitutively active promoter.
  • the promoter is a regulatable promoter.
  • the promoter is an inducible promoter.
  • the promoter is a tissue-specific promoter.
  • the promoter is a cell type-specific promoter.
  • the transcriptional control element e.g., the promoter
  • the transcriptional control element is functional in a targeted cell type or targeted cell population.
  • the transcriptional control element can be functional in a T cell, a B cell, a hematopoietic stem cell, a liver cell, a lung cell, a muscle cell (e.g., a cardiac muscle cell; a skeletal muscle cell), a retinal cell, or other targeted cell.
  • a suitable promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/”ON” state).
  • a suitable promoter may be an inducible promoter (i.e., a promoter whose state, active/”ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein).
  • a suitable promoter can be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).
  • Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms.
  • Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III).
  • Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res.2003 Sep 1;31(17)), a human H1 promoter (H1), and the like.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMVIE cytomegalovirus
  • RSV Rou
  • inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art.
  • inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.
  • the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells.
  • Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell).
  • the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art.
  • Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art.
  • Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc
  • a suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat.
  • elements that are responsive to transactivation e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system
  • small molecule control systems such as tetracycl
  • examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc.
  • the promoter is an insect-specific promoter.
  • the promoter is a plant-specific promoter.
  • the promoter is a protozoan-specific promoter.
  • the promoter is a plant-specific promoter.
  • plant promoters include, but are not limited to, a cauliflower mosaic virus (CaMV) promoter, a nopaline synthetase promoter, a ribose bisphosphate carboxylase promoter, a ubiquitin promoter, a UBQ3 promoter, a cestrum virus promoter, a rice actin 1 promoter, a CaMV 35S promoter, a CaMV 19S promoter, a sucrose synthase promoter, and a figwort mosaic virus promoter.
  • CaMV cauliflower mosaic virus
  • Chemical agent-inducible promoters are known in the art and include, but are not limited to, the maize In2- 2 promoter, which is activated by benzenesulfonamide herbicide safeners; the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre- emergent herbicides; and the tobacco PR-1a promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci.
  • Tissue-preferred and tissue-specific promoters can be used to control expression in a particular plant tissue.
  • tissue-preferred and tissue-specific promoters include leaf-preferred promoter, root-preferred promoters; root-specific promoters, seed-preferred promoters; seed- specific promoters; and the like.
  • the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure.
  • Suitable expression vectors include viral expression vectors (e.g.
  • viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloprol
  • a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector.
  • a recombinant expression vector of the present disclosure is a recombinant lentivirus vector.
  • a recombinant expression vector of the present disclosure is a recombinant retroviral vector. [00130] Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.
  • nucleic acid e.g., DNA or RNA
  • a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure
  • nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure
  • a nucleic acid encoding a Cas9 polypeptide
  • Cas9 guide RNA a nucleic acid comprising a nucleotide sequence encoding a Cas9 guide RNA
  • a recombinant expression vector comprising one or more of the aforementioned nucleic acids; and the like
  • Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.
  • Introducing a recombinant expression vector of the present disclosure into a cell or cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo. Introducing the recombinant expression vector into a target cell can be carried out in vitro.
  • an Acr polypeptide-encoding nucleic acid can be provided as RNA.
  • the RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the Acr polypeptide). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).
  • Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g.
  • Vectors may be provided directly to a target host cell.
  • the cells are contacted with a recombinant expression vector comprising a nucleic acid (e.g., a recombinant expression vector comprising a nucleic acid encoding an Acr polypeptide; a recombinant expression vector comprising: i) a nucleic acid encoding an Acr polypeptide; ii) a nucleotide sequence encoding a Cas9 polypeptide; and iii) a nucleotide sequence encoding a Cas9 guide RNA; etc.) such that the vectors are taken up by the cells.
  • a recombinant expression vector comprising a nucleic acid
  • a recombinant expression vector comprising a nucleic acid encoding an Acr polypeptide
  • a recombinant expression vector comprising: i) a nucleic acid encoding an Acr polypeptide; ii) a nucleotide sequence encoding a Cas9 polypeptid
  • Retroviruses for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells).
  • the appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles.
  • Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).
  • a recombinant expression vector used for providing a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide (and, optionally, encoding a Cas9 polypeptide and/or a Cas9 guide RNA) to a target host cell can include suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest.
  • suitable promoters for driving the expression that is, transcriptional activation, of the nucleic acid of interest.
  • the nucleic acid of interest will be operably linked to a promoter.
  • recombinant expression vector used for providing a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide (and, optionally, encoding a Cas9 polypeptide and/or a Cas9 guide RNA) to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the recombinant expression vector.
  • an expression vector of the present disclosure comprises: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above).
  • the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the Cas9 polypeptide are operably linked to the same promoter.
  • nucleotide sequence encoding the Acr polypeptide is operably linked to a first promoter; and the nucleotide sequence encoding the Cas9 polypeptide is operably linked to a second promoter, where the second promoter is different from the first promoter.
  • an expression vector of the present disclosure comprises: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); and c) a nucleotide sequence encoding a Cas9 guide RNA.
  • nucleotide sequence encoding the Acr polypeptide, the nucleotide sequence encoding the Cas9 polypeptide, and the nucleotide sequence encoding the Cas9 guide RNA are operably linked to the same promoter.
  • nucleotide sequence encoding the Acr polypeptide is operably linked to a first promoter; and the nucleotide sequence encoding the Cas9 polypeptide and the nucleotide sequence encoding the Cas9 guide RNA are operably linked to a second promoter, where the second promoter is different from the first promoter.
  • the present disclosure provides a modified host cell comprising an Acr polypeptide of the present disclosure and/or a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the modified host cell is one that does not normally comprise an Acr polypeptide of the present disclosure; i.e., the Acr polypeptide is heterologous to the host cell.
  • the present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is inhibited by an Acr polypeptide of the present disclosure); and c) a nucleotide sequence encoding a Cas9guide RNA.
  • a cell that serves as a recipient for an Acr polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; insect cells; arachnid cells; etc.
  • a cell that serves as a recipient for an Acr polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure is referred to as a “host cell” or a “target cell.”
  • a host cell or a target cell can be a recipient of an Acr system of the present disclosure.
  • a host cell or a target cell can be a recipient of a single component of an Acr system of the present disclosure.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a prokaryotic cell
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep, a horse, a camel); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.) and the like.
  • the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).
  • modified host cells can be cells within a tissue in a vertebrate animal (e.g. a liver cell, a muscle cell, a pulmonary cell, a pancreatic cell, a skin cell, a renal cell, a cell in the CNS etc).
  • the cell can be a specific cell type within a tissue (e.g. a neuron or an astrocyte in brain tissue or a hepatocyte or Kupfer cell in liver tissue).
  • a cell can be an in vitro cell (e.g., a cell in culture, e.g., an established cultured cell line).
  • a cell can be an ex vivo cell (cultured cell from an individual).
  • a cell can be an in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • a cell can be a cell in a cell culture (e.g., in vitro cell culture).
  • a cell can be one of a collection of cells.
  • a cell can be a prokaryotic cell or derived from a prokaryotic cell.
  • a cell can be a bacterial cell or can be derived from a bacterial cell.
  • a cell can be an archaeal cell or derived from an archaeal cell.
  • a cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a cell can be a plant cell or derived from a plant cell.
  • a cell can be an animal cell or derived from an animal cell.
  • a cell can be an invertebrate cell or derived from an invertebrate cell.
  • a cell can be a vertebrate cell or derived from a vertebrate cell.
  • a cell can be a mammalian cell or derived from a mammalian cell.
  • a cell can be a rodent cell or derived from a rodent cell.
  • a cell can be a human cell or derived from a human cell.
  • a cell can be a microbe cell or derived from a microbe cell.
  • a cell can be a fungi cell or derived from a fungi cell.
  • a cell can be an insect cell.
  • a cell can be an arthropod cell.
  • a cell can be a protozoan cell.
  • a cell can be a helminth cell.
  • Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g.
  • a fibroblast an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.
  • Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells,
  • the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.
  • the immune cell is a cytotoxic T cell.
  • the immune cell is a helper T cell.
  • the immune cell is a regulatory T cell (Treg).
  • the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.
  • Adult stem cells are resident in differentiated tissue, but retain the properties of self- renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found.
  • somatic stem cells include muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
  • Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc.
  • the stem cell is a human stem cell.
  • the stem cell is a rodent (e.g., a mouse; a rat) stem cell.
  • the stem cell is a non-human primate stem cell.
  • Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.
  • the stem cell is a hematopoietic stem cell (HSC).
  • HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-.
  • HSCs can repopulate the erythroid, neutrophil- macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo.
  • HSCs can be induced in vitro to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
  • the stem cell is a neural stem cell (NSC).
  • Neural stem cells are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes).
  • a neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.
  • the stem cell is a mesenchymal stem cell (MSC).
  • a cell is in some cases a plant cell.
  • a plant cell can be a cell of a monocotyledon.
  • a cell can be a cell of a dicotyledon.
  • the cell is a plant cell.
  • the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like.
  • a major agricultural plant e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans,
  • the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese artichoke (crosnes), chinese cabbage, chinese celery, chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive,
  • a cell is in some cases an arthropod cell.
  • the cell can be a cell of a sub- order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hem
  • a cell is in some cases an insect cell.
  • the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.
  • COMPOSITIONS [00159]
  • the present disclosure provides a composition comprising an Acr polypeptide of the present disclosure.
  • the present disclosure comprises a composition comprising a nucleic acid or a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a composition comprising an Acr fusion polypeptide of the present disclosure.
  • the present disclosure provides a composition comprising a host cell comprising an Acr polypeptide of the present disclosure.
  • a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 poly
  • any one of, or any combination of, (a)-(g) as set out above can be delivered associated with, or encapsulated in, a nanoparticle.
  • a lipid composition e.g., a lipid composition (such as a liposome) comprising a lipid or lipidoid and a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and/or wherein the lipid composition further comprises cholesterol (e.g., particle from formulation 1
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DMPC 1,2-ditetradecanoy
  • a lipid composition can be formed using a multistep process in which an Acr polypeptide, a Cas9 polypeptide, and a Cas9 guideRNA are mixed together, e.g., at a 1:1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile, nuclease free 1 x phosphate- buffered saline (PBS); and separately, DOTAP, DMPC, PEG, and cholesterol as applicable for the formulation are dissolved in alcohol, e.g., 100% ethanol; and, the two solutions are mixed together to form particles containing the complexes).
  • PBS nuclease free 1 x phosphate- buffered saline
  • a biodegradable core-shell structured nanoparticle with a poly ( ⁇ -amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used.
  • particles/nanoparticles based on self assembling bioadhesive polymers are used; such particles/nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain.
  • Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated.
  • a molecular envelope technology which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used.
  • Lipidoid compounds are also useful in the administration of polynucleotides, and can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited
  • the aminoalcohol lipidoid compounds are combined with an agent (one or more of (a)-(f), above), to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles.
  • the aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.
  • a poly(beta-amino alcohol) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Ac
  • US Patent Publication No.20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization.
  • PBAAs poly(beta-amino alcohols)
  • Sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and
  • lipid nanoparticles are used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibit
  • Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge.
  • pH values e.g., pH 4
  • the LNPs exhibit a low surface charge compatible with longer circulation times.
  • ionizable cationic lipids Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium- propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).
  • DLinDAP 1,2-dilineoyl-3-dimethylammonium- propane
  • DLinDMA 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane
  • DLinKDMA 1,2- dilinoleyloxy-keto-N,N-dimethyl-3-amin
  • a nucleic acid (e.g., a Cas9 guide RNA; a nucleic acid of the present disclosure; etc.) may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C- DOMG at 40:10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated.
  • Spherical Nucleic Acid (SNATM) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the
  • Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).
  • PEI polyethyleneimine
  • RGD Arg-Gly-Asp
  • nanoparticle refers to any particle having a diameter of less than 1000 nm.
  • nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a nucleotide sequence encoding a Cas9 guide
  • nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where
  • nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where
  • nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9
  • Nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9
  • Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure.
  • Semi-solid and soft nanoparticles are also suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptid
  • an exosome is used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide
  • Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.
  • a liposome is used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties.
  • a liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3- phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.
  • DSPC 1,2-distearoryl-sn-glycero-3- phosphatidyl choline
  • sphingomyelin egg phosphatidylcholines and monosialoganglioside.
  • a stable nucleic-acid-lipid particle can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 guide RNA
  • the SNALP formulation may contain the lipids 3-N- [(methoxypoly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C- DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio.
  • PEG-C- DMA 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane
  • DSPC 1,2-distearoyl-sn- glycero-3-phosphocholine
  • cholesterol in a 2:40:10:48 molar percent ratio.
  • the SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA.
  • the resulting SNALP liposomes can be about 80-100 nm in size.
  • a SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2- dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane.
  • a SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N- dimethyl)aminopropane (DLinDMA).
  • DSPC 1,2-distearoyl-sn-glycero-3- phosphocholine
  • PEG-cDMA 1,2-dilinoleyloxy-3-(N;N- dimethyl)aminopropane
  • cationic lipids such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-KC2-DMA) can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibite
  • a preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3- bis(octadecyloxy) propyl-1-(methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w).
  • the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA.
  • Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity.
  • Lipids may be formulated with a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptid
  • Suitable lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a system, or component thereof, of the present disclosure, using a spontaneous vesicle formation procedure.
  • the component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12- 200/disteroylphosphatidyl choline/cholesterol/PEG-DMG).
  • a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibit
  • Supercharged proteins can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 poly
  • Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.
  • Cell Penetrating Peptides can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 poly
  • CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • An implantable device can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9
  • An implantable device suitable for use in delivering a polypeptide and/or a nucleic acid e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide
  • a suitable implantable device can comprise a polymeric substrate, such as a matrix for example, that is used as the device body, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging.
  • An implantable delivery device can be advantageous in providing release locally and over a prolonged period, where the polypeptide and/or nucleic acid to be delivered is released directly to a target site, e.g., the extracellular matrix (ECM), the vasculature surrounding a tumor, a diseased tissue, etc.
  • ECM extracellular matrix
  • Suitable implantable delivery devices include devices suitable for use in delivering to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix.
  • a suitable implantable drug delivery device comprises degradable polymers, wherein the main release mechanism is bulk erosion.
  • a suitable implantable drug delivery device comprises non degradable, or slowly degraded polymers, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months).
  • the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months).
  • Combinations of different polymers with different release mechanisms may also optionally be used.
  • the concentration gradient at the can be maintained effectively constant during a significant period of the total releasing period, and therefore the diffusion rate is effectively constant (termed "zero mode" diffusion).
  • the implantable delivery system is designed to shield a nucleic acid component from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject.
  • the site for implantation of the device, or target site can be selected for maximum therapeutic efficacy.
  • a delivery device can be implanted within or in the proximity of a tumor environment, or the blood supply associated with a tumor.
  • the target location can be, e.g.: 1) the brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2) the spine, as in the case of amyotrophic lateral sclerosis (ALS); 3) uterine cervix; 4) active and chronic inflammatory joints; 5) dermis as in the case of psoriasis; 7) sympathetic and sensoric nervous sites for analgesic effect; 7) a bone; 8) a site of acute or chronic infection; 9) Intra vaginal; 10) Inner ear--auditory system, labyrinth of the inner ear, vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary, epicardiac; 13) urinary tract or bladder; 14) biliary system; 15) parenchymal tissue including and not limited to the kidney
  • the present disclosure provides a system for controlling activity of a Cas9 polypeptide.
  • a system of the present disclosure can comprise two or more of: a) an Acr polypeptide of the present disclosure, an Acr fusion polypeptide of the present disclosure, or a modified Acr polypeptide of the present disclosure; b) an RNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; c) a DNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; d) a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; e) a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an
  • a system of the present disclosure comprises two or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a
  • a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide.
  • a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA.
  • a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide.
  • a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA.
  • a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA.
  • a system of the present disclosure comprises: a) a ribonucleoprotein comprising: i) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and ii) a Cas9 guide RNA; and b) an Acr polypeptide of the present disclosure.
  • a system of the present disclosure comprises: a) a ribonucleoprotein comprising: i) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and ii) a Cas9 guide RNA; and b) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • Acr polypeptides that are suitable for inclusion in a system of the present disclosure include: a) Acr polypeptides as described above (including truncated Acr polypeptides as described above); b) Acr fusion polypeptides as described above; and c) modified Acr polypeptides as described above.
  • a system of the present disclosure can comprise an Acr polypeptide of the present disclosure, or a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • a system of the present disclosure can comprise an Acr fusion polypeptide of the present disclosure, or a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure.
  • a system of the present disclosure can comprise a modified Acr polypeptide of the present disclosure.
  • Cas9 polypeptides [00189] As noted above, in some cases, a system of the present disclosure includes a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above.
  • a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide.
  • a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA.
  • a Cas9 polypeptide included in a system of the present disclosure comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence provided in FIG.5.
  • a Staphylococcus aureus Cas9 (SaCas9) suitable for inclusion in a system of the present disclosure can comprise one of the following sets of amino acid substitutions: N419A/R654A, Y211A/R654A, Y211A/Y212A, Y211A/N230A, Y211A/R245A, Y212A/Y230A, Y212A/R245A, Y230A/R245A, W229A/R654A, Y211A/Y212A/Y230A, Y211A/Y212A/R245A, Y211A/Y212A/Y651A, Y211A/Y230A/R245A, Y211A/Y230A/Y651A, Y211A/R245A/Y651A, Y211A/R245A/Y651A, Y211A/R245A/Y651A, Y211A/R245A
  • a Staphylococcus aureus Cas9 (SaCas9) suitable for inclusion in a system of the present disclosure comprises one of the following amino acid substitutions or sets of amino acid substitutions: E782K; K929R; N968K; R1015H; E782K/N968K/R1015H (KKH variant); E782K/K929R/R1015H (KRH variant); or E782K/K929R/N968K/R1015H (KRKH variant).
  • Cas9 guide RNA A nucleic acid molecule that binds to a Cas9 protein and targets the complex to a specific location within a target nucleic acid is referred to herein as a “Cas9 guide RNA.”
  • a Cas9 guide RNA can be said to include two segments, a first segment (referred to herein as a “targeting segment”); and a second segment (referred to herein as a “protein-binding segment”).
  • targeting segment referred to herein as a “targeting segment”
  • protein-binding segment referred to herein as a “protein-binding segment”.
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule.
  • a segment can also refer to a region/section of a complex such that a segment may comprise regions of more than one molecule.
  • the first segment (targeting segment) of a Cas9 guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.).
  • the protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a Cas9 polypeptide.
  • the protein-binding segment of a subject Cas9 guide RNA includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex).
  • Site-specific binding and/or cleavage of a target nucleic acid can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas9 guide RNA (the guide sequence of the Cas9 guide RNA) and the target nucleic acid.
  • a Cas9 guide RNA and a Cas9 protein form a complex (e.g., bind via non-covalent interactions).
  • the Cas9 guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid).
  • the Cas9 protein of the complex provides the site-specific activity (e.g., cleavage activity or an activity provided by the Cas9 protein when the Cas9 protein is a Cas9 fusion polypeptide, i.e., has a fusion partner).
  • the Cas9 protein is guided to a target nucleic acid sequence (e.g.
  • a target sequence in a chromosomal nucleic acid e.g., a chromosome
  • a target sequence in an extrachromosomal nucleic acid e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.
  • a target sequence in a mitochondrial nucleic acid e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.
  • a target sequence in a mitochondrial nucleic acid a target sequence in a chloroplast nucleic acid
  • a target sequence in a plasmid a target sequence in a viral nucleic acid; etc.
  • the “guide sequence” also referred to as the “targeting sequence” of a Cas9 guide RNA can be modified so that the Cas9 guide RNA can target a Cas9 protein to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence is taken into account.
  • PAM protospacer adjacent motif
  • a Cas9 guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.
  • a eukaryotic cell e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.
  • a Cas9 guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual Cas9 guide RNA”, a “double- molecule Cas9 guide RNA”, or a “two-molecule Cas9 guide RNA” a “dual guide RNA”, or a “dgRNA.”
  • the activator and targeter are covalently linked to one another (e.g., via intervening nucleotides) and the guide RNA is referred to as a “single guide RNA”, a “Cas9 single guide RNA”, a “single-molecule Cas9 guide RNA,” or a “one-molecule Cas9 guide RNA”, or simply “sgRNA.”
  • a Cas9 guide RNA comprises a crRNA-like (“CRISPR RNA” / “targeter” / “crRNA” / “crRNA repeat”) molecule and
  • a crRNA-like molecule comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch (“duplex-forming segment”) of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA.
  • a corresponding tracrRNA-like molecule comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide nucleic acid.
  • a stretch of nucleotides of a crRNA-like molecule are complementary to and hybridize with a stretch of nucleotides of a tracrRNA-like molecule to form the dsRNA duplex of the protein- binding domain of the Cas9 guide RNA.
  • each targeter molecule can be said to have a corresponding activator molecule (which has a region that hybridizes with the targeter).
  • the targeter molecule additionally provides the targeting segment.
  • a targeter and an activator molecule hybridize to form a Cas9 guide RNA.
  • the exact sequence of a given crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found.
  • a subject dual Cas9 guide RNA can include any corresponding activator and targeter pair.
  • activator or “activator RNA” is used herein to mean a tracrRNA-like molecule (tracrRNA: “trans-acting CRISPR RNA”) of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the “activator” and the “targeter” are linked together by, e.g., intervening nucleotides).
  • a Cas9 guide RNA (dgRNA or sgRNA) comprises an activator sequence (e.g., a tracrRNA sequence).
  • a tracr molecule is a naturally existing molecule that hybridizes with a CRISPR RNA molecule (a crRNA) to form a Cas9 dual guide RNA.
  • the term “activator” is used herein to encompass naturally existing tracrRNAs, but also to encompass tracrRNAs with modifications (e.g., truncations, sequence variations, base modifications, backbone modifications, linkage modifications, etc.) where the activator retains at least one function of a tracrRNA (e.g., contributes to the dsRNA duplex to which Cas9 protein binds).
  • the activator provides one or more stem loops that can interact with Cas9 protein.
  • an activator can be referred to as having a tracr sequence (tracrRNA sequence) and in some cases is a tracrRNA, but the term “activator” is not limited to naturally existing tracrRNAs.
  • the term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: “CRISPR RNA”) of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the “activator” and the “targeter” are linked together, e.g., by intervening nucleotides).
  • a Cas9 guide RNA comprises a targeting segment (which includes nucleotides that hybridize with (are complementary to) a target nucleic acid, and a duplex-forming segment (e.g., a duplex forming segment of a crRNA, which can also be referred to as a crRNA repeat).
  • a targeting segment the segment that hybridizes with a target sequence of a target nucleic acid
  • a crRNA repeat the sequence of a targeter will often be a non- naturally occurring sequence.
  • the duplex-forming segment of a targeter (described in more detail below), which hybridizes with the duplex-forming segment of an activator, can include a naturally existing sequence (e.g., can include the sequence of a duplex-forming segment of a naturally existing crRNA, which can also be referred to as a crRNA repeat).
  • targeter is used herein to distinguish from naturally occurring crRNAs, despite the fact that part of a targeter (e.g., the duplex-forming segment) often includes a naturally occurring sequence from a crRNA.
  • the term “targeter” encompasses naturally occurring crRNAs.
  • a Cas9 guide RNA can also be said to include 3 parts: (i) a targeting sequence (a nucleotide sequence that hybridizes with a sequence of the target nucleic acid); (ii) an activator sequence (as described above)(in some cases, referred to as a tracr sequence); and (iii) a sequence that hybridizes to at least a portion of the activator sequence to form a double stranded duplex.
  • a targeter has (i) and (iii); while an activator has (ii).
  • a Cas9 guide RNA e.g. a dual guide RNA or a single guide RNA
  • a Cas9 guide RNA can be comprised of any corresponding activator and targeter pair.
  • the duplex forming segments can be swapped between the activator and the targeter.
  • the targeter includes a sequence of nucleotides from a duplex forming segment of a tracrRNA (which sequence would normally be part of an activator) while the activator includes a sequence of nucleotides from a duplex forming segment of a crRNA (which sequence would normally be part of a targeter).
  • a targeter comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch (“duplex-forming segment”) of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA.
  • a corresponding tracrRNA-like molecule comprises a stretch of nucleotides (a duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA.
  • a stretch of nucleotides of the targeter is complementary to and hybridizes with a stretch of nucleotides of the activator to form the dsRNA duplex of the protein- binding segment of a Cas9 guide RNA.
  • each targeter can be said to have a corresponding activator (which has a region that hybridizes with the targeter).
  • the targeter molecule additionally provides the targeting segment.
  • a targeter and an activator hybridize to form a Cas9 guide RNA.
  • the particular sequence of a given naturally existing crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found. Examples of suitable activator and targeter are well known in the art.
  • Targeting segment of a Cas9 guide RNA [00205]
  • the first segment of a subject guide nucleic acid includes a guide sequence (i.e., a targeting sequence)(a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid).
  • the targeting segment of a subject guide nucleic acid can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA)) in a sequence-specific manner via hybridization (i.e., base pairing).
  • a target nucleic acid e.g., double stranded DNA (dsDNA)
  • the nucleotide sequence of the targeting segment may vary (depending on the target) and can determine the location within the target nucleic acid that the Cas9 guide RNA and the target nucleic acid will interact.
  • the targeting segment of a Cas9 guide RNA can be modified (e.g., by genetic engineering)/designed to hybridize to any desired sequence (target site) within a target nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA).
  • the targeting segment can have a length of 7 or more nucleotides (nt) (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more nucleotides).
  • nt nucleotides
  • the targeting segment can have a length of from 7 to 100 nucleotides (nt) (e.g., from 7 to 80 nt, from 7 to 60 nt, from 7 to 40 nt, from 7 to 30 nt, from 7 to 25 nt, from 7 to 22 nt, from 7 to 20 nt, from 7 to 18 nt, from 8 to 80 nt, from 8 to 60 nt, from 8 to 40 nt, from 8 to 30 nt, from 8 to 25 nt, from 8 to 22 nt, from 8 to 20 nt, from 8 to 18 nt, from 10 to 100 nt, from 10 to 80 nt, from 10 to 60 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 10 to 18 nt, from 12 to 100 nt, from 12 to 80 nt, from 12 to 60 nt
  • the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid can have a length of 10 nt or more.
  • the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid can have a length of 12 nt or more, 15 nt or more, 18 nt or more, 19 nt or more, or 20 nt or more.
  • the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 12 nt or more.
  • the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 18 nt or more.
  • the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid can have a length of from 10 to 100 nucleotides (nt) (e.g., from 10 to 90 nt, from 10 to 75 nt, from 10 to 60 nt, from 10 to 50 nt, from 10 to 35 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 12 to 100 nt, from 12 to 90 nt, from 12 to 75 nt, from 12 to 60 nt, from 12 to 50 nt, from 12 to 35 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12
  • the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 30 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 25 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 30 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 25 nt.
  • the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 22 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 20 nucleotides in length. In some cases, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 19 nucleotides in length.
  • the percent complementarity between the targeting sequence (guide sequence) of the targeting segment and the target site of the target nucleic acid can be 60% or more (e.g., 65% or more, 70% or more, 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%). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5’-most nucleotides of the target site of the target nucleic acid.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more over about 20 contiguous nucleotides. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the fourteen contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 20 nucleotides in length. [00210] In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5’- most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA).
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA).
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’- most nucleotides of the targeting sequence of the Cas9 guide RNA).
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more (e.g., e.g., 65% or more, 70% or more, 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%) over about 20 contiguous nucleotides.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 7 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 8 nucleotides in length.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 9 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 10 nucleotides in length.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 11 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 11 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 12 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 12 nucleotides in length.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 13 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 13 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 14 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length.
  • the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 17 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 18 nucleotides in length.
  • Examples of various Cas9 proteins and Cas9 guide RNAs can be found in the art, for example, see Jinek et al., Science.2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol.2013 May;10(5):726-37; Ma et al., Biomed Res Int.2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15644- 9; Jinek et al., Elife.2013;2:e00471; Pattanayak et al., Nat Biotechnol.2013 Sep;31(9):839-43; Qi et al, Cell.2013 Feb 28;152(5):1173-83; Wang et al., Cell.2013 May 9;153(4):9
  • a Cas9 guide RNA comprises has one or more modifications, e.g., a base modification, a backbone modification, etc.
  • Suitable nucleic acid modifications include, but are not limited to: 2’O-methyl modified nucleotides, 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7-methylguanylate cap (m7G)).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5'
  • Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
  • a Cas9 guide RNA comprises a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C 10 alkyl or C 2 to C10 alkenyl and alkynyl.
  • a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH 3 , OCN, C1, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a suitable modification includes 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 .
  • a subject nucleic acid may also include nucleobase (often referred to in the art simply as "base”) modifications or substitutions.
  • unmodified or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H- pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).
  • tricyclic pyrimidines such as
  • the present disclosure provides methods of inhibiting activity of a Cas9 polypeptide.
  • the methods generally involve contacting the Cas9 polypeptide with: a) an Acr polypeptide of the present disclosure; b) an Acr fusion polypeptide of the present disclosure; or b) a modified Acr polypeptide of the present disclosure.
  • the contacting occurs in a living cell in vitro.
  • the contacting occurs in a living cell in vivo.
  • the contacting occurs outside of a cell in vivo (e.g., the contacting occurs in an extracellular fluid in vivo).
  • an “Acr polypeptide of the present disclosure” includes an unmodified Acr polypeptide, a variant Acr polypeptide (as described above), a truncated Acr polypeptide (as described above), an Acr fusion polypeptide of the present disclosure, and a modified Acr polypeptide of the present disclosure.
  • a method of the present disclosure can inhibit binding and/or cleavage activity of a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9/guide RNA complex is not contacted with the Acr polypeptide).
  • a method of the present disclosure comprises introducing into a cell (a “target cell”) a nucleic acid (e.g., a recombinant expression vector; an mRNA; and the like) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, where the cell comprises, at the time the Acr-encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide.
  • a nucleic acid e.g., a recombinant expression vector; an mRNA; and the like
  • the cell comprises, at the time the Acr-encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nu
  • a method of the present disclosure comprises introducing into a cell (a “target cell”) a nucleic acid (e.g., a recombinant expression vector; an mRNA; and the like) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, where the cell does not comprise, at the time the Acr -encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide.
  • a target cell a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, where the cell does not comprise, at the time the Acr -encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Ac
  • a method of the present disclosure comprises introducing into a cell (a “target cell”) an Acr polypeptide of the present disclosure, where the cell comprises, at the time the Acr polypeptide is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide.
  • a method of the present disclosure comprises introducing into a cell (a “target cell”) an Acr polypeptide of the present disclosure, where the cell does not comprise, at the time the Acr polypeptide is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide.
  • a method of the present disclosure comprises introducing into a cell a nucleic acid (e.g., a DNA; a recombinant expression vector; an RNA) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure
  • the Acr-encoding nucleotide sequence is operably linked to one or more transcriptional control elements.
  • the one or more transcriptional control elements comprises a promoter, e.g., a promoter that is functional in a eukaryotic cell.
  • the promoter is a constitutive promoter.
  • the promoter is a regulated promoter, e.g., an inducible promoter.
  • the inducible promoter is a drug-inducible promoter
  • the method comprises contacting the cell with a drug that induces the drug-inducible promoter.
  • a method of the present disclosure provides for controlling gene drive. For example, where the gene drive limits viability of a target organism (or target population of an organism), a method of the present disclosure can restore viability to the target organism (or target population of an organism).
  • target organisms or target populations of an organism
  • ticks include ticks of the families Ixodidae and Argasidae, e.g., Ixodes ricinus, I. rubicundus, I.
  • mosquitoes e.g., mosquitoes that carry human pathogens such as malaria parasites, Yellow Fever Virus, Dengue virus, Zika virus, Chikungunya virus, and the like
  • mosquitoes include mosquitoes of the genera Culex, Culistea, Aedes, or Anopheles, e.g., Aedes aegypti, Aedes albopictus, and Anopheles gamiae
  • protozoans such as Plasmodium species (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium berghei, Plasmodium gallinaceum, and Plasmodium knowlesi), nematode species, Trypanosoma species, Trichomonadidae species, Leishmania species, and the like
  • insects e.g., mosquitoes that carry human pathogens such as malaria parasites, Yellow Fever
  • a method of the present disclosure provides for reducing off-target Cas9/guide RNA-mediated gene editing.
  • a method of the present disclosure reduces off-target Cas9/guide RNA-mediated gene editing by at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, or more than 95%, compared to the extent of off-target Cas9/guide RNA-mediated gene editing when the Cas9 is not contacted with an Acr polypeptide of the present disclosure.
  • a method of the present disclosure provides protection against deleterious effects of a “hostile” Cas9/guide RNA.
  • an individual can comprise immune cells genetically modified to include an Acr-encoding nucleic acid; if such an individual comes into contact with a hostile Cas9/guide RNA complex that targets immune cells in a deleterious manner, such an individual can be protected from deleterious effects of such a hostile Cas9/guide RNA.
  • an Acr polypeptide of the present disclosure is used to deliver a Cas9 polypeptide to a cell, e.g., a eukaryotic cell.
  • a complex of an Acr polypeptide and a Cas9 polypeptide is delivered to a cell.
  • the complex may further include a Cas9 guide RNA and/or a donor template.
  • Target nucleic acids and cells [00228]
  • An Acr polypeptide of the present disclosure inhibits a Cas9 polypeptide (when the Cas9 polypeptide is complexed with a Cas9 guide RNA) from binding and/or cleaving a target nucleic acid.
  • target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the Cas9 guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).
  • a chromosome genomic DNA
  • derived from a chromosome derived from a chromosome
  • chromosomal DNA plasmid
  • viral extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.
  • the Cas9 guide RNA comprises a nucleotide sequence that hybridize
  • a target nucleic acid (a target of a Cas9/guide RNA complex) can be DNA or RNA.
  • a target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA).
  • a target nucleic acid is single stranded.
  • a target nucleic acid is a single stranded RNA (ssRNA).
  • a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and microRNA (miRNA).
  • a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded.
  • a target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo.
  • Suitable target cells include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a fungal cell e.g., a yeast cell
  • an animal cell e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.
  • a cell of an insect e.g., a mosquito; a bee; an agricultural pest; etc.
  • a cell of an arachnid e.g., a spider; a tick; etc.
  • a cell from a vertebrate animal e.g., a fish, an amphibian, a reptile, a bird, a mammal
  • a cell from a mammal e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g.,
  • a stem cell e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • a germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.
  • a somatic cell e.g. a fibroblast,
  • Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
  • the primary cell lines are maintained for fewer than 10 passages in vitro.
  • Target cells can be unicellular organisms and/or can be grown in culture.
  • the cells may be harvest from an individual by any convenient method.
  • leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy.
  • Plant cells include cells of a monocotyledon, and cells of a dicotyledon.
  • the cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.
  • Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella), an algal cell, (e.g., Botryococcus braunii, Chla
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).
  • a cell can be an in vitro cell (e.g., established cultured cell line).
  • a cell can be an ex vivo cell (cultured cell from an individual).
  • a cell can be and in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • a cell can be a cell in a cell culture (e.g., in vitro cell culture).
  • a cell can be one of a collection of cells.
  • a cell can be a prokaryotic cell or derived from a prokaryotic cell.
  • a cell can be a bacterial cell or can be derived from a bacterial cell.
  • a cell can be an archaeal cell or derived from an archaeal cell.
  • a cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a cell can be a plant cell or derived from a plant cell.
  • a cell can be an animal cell or derived from an animal cell.
  • a cell can be an invertebrate cell or derived from an invertebrate cell.
  • a cell can be a vertebrate cell or derived from a vertebrate cell.
  • a cell can be a mammalian cell or derived from a mammalian cell.
  • a cell can be a rodent cell or derived from a rodent cell.
  • a cell can be a human cell or derived from a human cell.
  • a cell can be a microbe cell or derived from a microbe cell.
  • a cell can be a fungi cell or derived from a fungi cell.
  • a cell can be an insect cell.
  • a cell can be an arthropod cell.
  • a cell can be a protozoan cell.
  • a cell can be a helminth cell.
  • Suitable cells include a stem cell (e.g.
  • an embryonic stem (ES) cell an induced pluripotent stem (iPS) cell
  • a germ cell e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.
  • a somatic cell e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.
  • Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, adult cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-
  • the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.
  • the immune cell is a cytotoxic T cell.
  • the immune cell is a helper T cell.
  • the immune cell is a regulatory T cell (Treg).
  • the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.
  • Adult stem cells are resident in differentiated tissue, but retain the properties of self- renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found.
  • somatic stem cells include muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.
  • Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc.
  • the stem cell is a human stem cell.
  • the stem cell is a rodent (e.g., a mouse; a rat) stem cell.
  • the stem cell is a non-human primate stem cell.
  • Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.
  • the stem cell is a hematopoietic stem cell (HSC).
  • HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-.
  • HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo.
  • HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo.
  • HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.
  • the stem cell is a neural stem cell (NSC).
  • Neural stem cells are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes).
  • a neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.
  • the stem cell is a mesenchymal stem cell (MSC).
  • a cell is in some cases a plant cell.
  • a plant cell can be a cell of a monocotyledon.
  • a cell can be a cell of a dicotyledon.
  • the cell is a plant cell.
  • the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like.
  • a major agricultural plant e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans
  • the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese artichoke (crosnes), chinese cabbage, chinese celery, chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive,
  • a cell is in some cases an arthropod cell.
  • the cell can be a cell of a sub- order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, He
  • a cell is in some cases an insect cell.
  • the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.
  • KITS [00249]
  • the present disclosure provides a kit comprising an Acr polypeptide of the present disclosure or a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure.
  • the present disclosure provides a kit comprising an Acr system of the present disclosure or a component of an Acr system of the present disclosure.
  • a kit of the present can comprise: a) any combination of an Acr system, as described above; b) and one or more additional components and/or reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a positive and/or negative control target DNA; v) a positive and/or negative control Cas9 guide RNA; and the like.
  • additional components and/or reagents e.g., i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a positive and/or negative control target DNA; v) a positive and/or negative control Cas9 guide RNA; and the like.
  • a kit of the present disclosure comprises: a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and b) an Acr polypeptide that is an inhibitor of an activity of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% (at least 70%, at least 85%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, or a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide, wherein the enzymatic activity is nucleic acid cleavage.
  • a kit of the present disclosure comprises: [00253] a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to any one of the amino acid sequences depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and [00254] b) i) an Acr polypeptide that is an inhibitor of an activity of the Cas9 polypeptide; or ii) an Acr fusion polypeptide of the present disclosure; or iii) a modified Acr polypeptide of the present disclosure, wherein the Acr polypeptide (or the Acr polypeptide present in the fusion polypeptide, or the modified Acr polypeptide)) comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; or iv) a nucleic acid comprising a nucleotide
  • a kit of the present disclosure can also include a positive control and/or a negative control.
  • a suitable control for a protein that inhibits SpyCas9-mediated, but not SauCas9-mediated, cleavage of a target nucleic acid is AcrIIA4.
  • a suitable control for a protein that inhibits both SpyCas9-mediated and SauCas9-mediated cleavage of a target nucleic acid is AcrIIA5.
  • the present disclosure provides a method of training a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, the method comprising: a) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases; b) representing each of the one or more Acr proteins as a feature vector based on amino acid composition of the Acr protein; c) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity from one or more databases; d) representing each non-Acr protein as a feature vector based on amino acid composition of the protein; and e) training the machine learning model using the feature vectors representing one or more anti-CRISPR proteins and the feature vectors representing one or more non-Acr proteins.
  • Acr anti-CRISPR
  • the present disclosure provides method of identifying one or more proteins based on their potential to inhibit CRISPR-Cas activity, the method comprising: a) using sequence information for one or more known anti-CRISPR (Acr) proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity; b) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins; and c) identifying one or more potential Acr proteins based on each such protein’s position in the ordered list.
  • Acr anti-CRISPR
  • aspects of the methods include: (1) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases, including, for example, from the Anti-CRISPRdb data base.
  • Acr anti-CRISPR
  • aspects of the methods include: (1) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases, including, for example, from the Anti-CRISPRdb data base.
  • Acr anti-CRISPR
  • the collection of one or more known Acr proteins may comprise a positive class training set for the machine learning model; (2) representing each of the one or more Acr proteins as a feature vector based on the amino acid composition of the Acr protein.
  • the Acr proteins may be represented based on sequence features such as amino acid composition and grouped dimer or trimer frequency counts.
  • Amino acids may be grouped into, for example, seven or more classes based on their physicochemical properties, and the frequency counts of all possible groups labeled as dimers and trimers in a given protein sequence may be used in conjunction with amino acid composition to represent the protein.
  • a feature vector representation may be generated for each protein sequence; (3) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity (each such protein, a “non-anti-CRISPR” or “non-Acr”) from one or more databases.
  • non-anti-CRISPR proteins may be collected from the proteomes of the source species to which each of the known Acr proteins belongs.
  • each non-anti-CRISPR protein selected includes no more than a threshold level of sequence similarity with each of the Acr proteins, for example a threshold of between 20-60% redundancy, such as a 40% threshold or a 20% threshold or a 60% threshold of redundancy.
  • the collection of one or more known non-anti-CRISPR proteins may comprise a negative class training set for the machine learning model; (4) representing each non-anti- CRISPR protein as a feature vector based on amino acid composition of the protein, as described above; and (5) training the machine learning model using the feature vectors representing one or more Acr proteins and the feature vectors representing one or more non-anti-CRISPR proteins.
  • Training the machine learning model may entail, for example, employing a boosting technique to combine multiple weak classifiers to produce a strong classifier.
  • the machine learning model may comprise a tree-based method of developing predictions, and further may apply pairwise ranking to potential anti-CRISPR proteins based on each protein’s expected anti-CRISPR behavior.
  • aspects of the methods include: (1) using sequence information for one or more known Acr proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, including, for example, the method of training a machine learning model discussed above; (2) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins.
  • the plurality of proteins belongs to the same species.
  • each protein of the plurality of proteins is found within one or more prophage regions.
  • the prophage regions may be identified using a database or a software tool, for example, PHASTER (PHAge Search Tool Enhanced Release); and (3) identifying one or more potential anti-CRISPR proteins based on each such protein’s position in the ordered list, for example identifying the potential Acr proteins ranked above a threshold position on the ranked list of anti-CRISPR proteins, such as those proteins within the top 10th percentile or within the top 5th percentile.
  • identifying one or more potential Acr proteins further comprises inspecting whether each potential anti-CRISPR protein exhibits specific features, including being a strong promoter, having a strong ribosome binding site or having an intrinsic terminator, for example by visually inspecting sequence information.
  • a fusion polypeptide comprising: a) anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a heterologous fusion partner.
  • Aspect 2 The fusion polypeptide of aspect 1, wherein the heterologous fusion partner is a nuclear localization sequence.
  • Aspect 3 The fusion polypeptide of aspect 1, wherein the heterologous fusion partner is an epitope tag.
  • Aspect 5 The fusion polypeptide of any one of aspects 1-3, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence set forth in FIG.20A-20B; and wherein the Acr polypeptide has a length of from 50 amino acids to 114 amino acids.
  • a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of aspects 1-5.
  • Aspect 7. The nucleic acid of aspect 6, wherein the nucleotide sequence is operably linked to a promoter.
  • Aspect 8. A recombinant expression vector comprising the nucleic acid of aspect 6 or aspect 7.
  • Aspect 9. A cell comprising the nucleic acid of aspect 6 or aspect 7, or the recombinant expression vector of aspect 8.
  • Aspect 10 The cell of aspect 9, wherein the cell is a eukaryotic cell.
  • Aspect 11 The cell of aspect 9 or aspect 10, wherein the cell is in vitro.
  • a modified anti-CRISPR (Acr) polypeptide comprising: a) an Acr polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a non-peptidic moiety covalently linked to the Acr polypeptide.
  • the modified Acr polypeptide of aspect 13, wherein the non-peptidic moiety provides for one or more of an increase in in vivo half-life, in vivo stability, and bioavailability of the Acr polypeptide, compared to the unmodified Acr polypeptide.
  • Aspect 15 The modified Acr polypeptide of aspect 13 or aspect 14, wherein the non- peptidic moiety comprises poly(ethylene glycol).
  • Aspect 16 The modified Acr polypeptide of aspect 13 or aspect 14, wherein the non- peptidic moiety comprises poly(ethylene glycol).
  • a recombinant expression vector comprising a nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1- 10.
  • Aspect 17 The recombinant expression vector of aspect 16, wherein the Acr polypeptide-encoding nucleotide sequence is operably linked to a promoter.
  • Aspect 18 The recombinant expression vector of aspect 17, wherein the promoter is functional in a eukaryotic cell. [00281] Aspect 19.
  • Aspect 20 The recombinant expression vector of aspect 19, wherein the regulated promoter is an inducible promoter.
  • Aspect 21 The recombinant expression vector of aspect 20, wherein the inducible promoter is a heat-inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone-inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter.
  • Aspect 22 Aspect 22.
  • Aspect 23 The recombinant expression vector of any one of aspects 16-22, further comprising a nucleotide sequence encoding a guide RNA that binds to and activates a Cas9 polypeptide.
  • Aspect 24 The recombinant expression vector of any one of aspects 15-22, wherein the recombinant expression vector is a recombinant viral vector.
  • Aspect 25 A cell comprising the recombinant expression vector of any one of aspects 16-24.
  • Aspect 26 Aspect 26.
  • Aspect 27 The cell of aspect 25, wherein the cell is in vitro.
  • Aspect 28 The cell of any one of aspects 24-27, wherein the cell is a eukaryotic cell.
  • Aspect 29 The cell of aspect 28, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
  • Aspect 30 Aspect 30.
  • a nucleic acid comprising: [00293] a) a first nucleotide sequence encoding the constant region of a guide RNA; [00294] b) a second nucleotide sequence encoding a Cas9 polypeptide; and [00295] c) a third nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10. [00296] Aspect 31.
  • the nucleic acid of aspect 30 comprising an insertion site for inserting a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the insertion site is 5’ of and immediately adjacent to first nucleotide sequence.
  • Aspect 32 The nucleic acid of aspect 30, comprising a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the guide sequence-encoding nucleotide sequence is 5’ of and immediately adjacent to first nucleotide sequence.
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to any one of the amino acid sequences depicted in FIG.21A-21D, optionally wherein: [00299] a) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B;
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B;
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; [00302] d) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or [00303] e) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B. [00304] Aspect 34. The nucleic acid of any one of aspects 30-33, wherein the third nucleotide sequence is operably linked to a promoter.
  • Aspect 35 The nucleic acid of aspect 34, wherein the promoter is functional in a eukaryotic cell.
  • Aspect 36 The nucleic acid of aspect 33 or aspect 34, wherein the promoter is an inducible promoter.
  • Aspect 37 The nucleic acid of aspect 36, wherein the inducible promoter is a heat- inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone- inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter.
  • Aspect 38 Aspect 38.
  • Aspect 39 A recombinant expression vector comprising the nucleic acid of any one of aspects 30-38.
  • Aspect 40 A cell comprising the nucleic acid of any one of aspects 30-38 or the recombinant expression vector of aspect 39.
  • Aspect 41 The cell of aspect 40, wherein the cell is in vitro.
  • Aspect 42 The cell of aspect 40, wherein the cell is in vivo.
  • Aspect 43 The cell of any one of aspects 40-42, wherein the cell is a eukaryotic cell.
  • Aspect 44 The cell of aspect 43, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
  • Aspect 45 The cell of aspect 43, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
  • a nucleic acid comprising a nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, and wherein the nucleotide sequence is optimized for expression in a mammalian cell.
  • Aspect 46 The nucleic acid of aspect 45, wherein the nucleotide sequence is operably linked to a promoter.
  • Aspect 47 A recombinant expression vector comprising the nucleic acid of aspect 45 or aspect 46.
  • a kit comprising: [00319] a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to a Cas9 amino acid sequence depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and [00320] b) an anti-CRISPR (Acr) polypeptide that is an inhibitor of an activity of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1- 10, or a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide, wherein the enzymatic activity is nucleic acid cleavage, optionally wherein: [00321] i) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphyloc
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B;
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B;
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B;
  • the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.
  • the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or [00325] v) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B. [00326] Aspect 49. The kit of aspect 48, wherein component (a) and component (b) are in separate containers. [00327] Aspect 50.
  • a method for inhibiting an activity of a Cas9 polypeptide comprising contacting the Cas9 polypeptide with: a) an anti-CRISPR (Acr) polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; or b) the Acr fusion polypeptide of any one of aspects 1-5.
  • Aspect 51 The method of aspect 50, wherein the Cas9 polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence depicted in FIG.21A-21D.
  • Aspect 52 The method of aspect 50 or 51, wherein said contacting occurs in a living cell in vitro.
  • Aspect 53 The method of aspect 50 or 51, wherein said contacting occurs in a living cell in vivo.
  • Aspect 54 The method of aspect 52 or aspect 53, comprising introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide.
  • Aspect 55 The method of any one of aspects 52-54, wherein the nucleotide sequence encoding the Acr polypeptide is operably linked to an inducible promoter.
  • Aspect 56 The method of any one of aspects 52-55, wherein the cell is a eukaryotic cell.
  • Aspect 57 The method of any one of aspects 52-55, wherein the cell is a eukaryotic cell.
  • Aspect 56 wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
  • Aspect 58 The method of aspect 55, wherein the inducible promoter is a drug-inducible promoter, and wherein the method comprises contacting the cell with a drug that induces the drug-inducible promoter.
  • Aspect 59 The method of any one of aspects 52-55, wherein the cell is a prokaryotic cell.
  • Aspect 60 The method of any one of aspects 52-55, wherein the cell is a prokaryotic cell.
  • a method of training a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity comprising: a) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases; b) representing each of the one or more Acr proteins as a feature vector based on amino acid composition of the Acr protein; c) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity from one or more databases; d) representing each non-Acr protein as a feature vector based on amino acid composition of the protein; and e) training the machine learning model using the feature vectors representing one or more anti-CRISPR proteins and the feature vectors representing one or more non-Acr proteins.
  • Acr anti-CRISPR
  • Aspect 61 The method of aspect 60, wherein collecting sequence information for one or more Acr proteins comprises collecting one or more non-redundant Acr proteins.
  • Aspect 62 The method of aspect 61, wherein collecting sequence information for one or more non-Acr proteins comprises selecting one or more non-Acr proteins that are not redundant with the one or more Acr proteins.
  • Aspect 63 The method of aspect 62, wherein collecting sequence information for one or more non-Acr proteins further comprises selecting one or more non-Acr proteins that belong to the same species as one or more of the Acr proteins.
  • Aspect 64 The method of aspect 60, wherein the machine learning model is a tree-based model.
  • Aspect 65 The method of aspect 64, wherein the machine learning model utilizes pairwise ranking.
  • Aspect 66 A method of identifying one or more proteins based on their potential to inhibit CRISPR-Cas activity, the method comprising: a) using sequence information for one or more known anti-CRISPR (Acr) proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity; b) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins; and c) identifying one or more potential Acr proteins based on each such protein’s position in the ordered list.
  • Acr anti-CRISPR
  • Aspect 67 The method of aspect 66, wherein the plurality of proteins belongs to the same species.
  • Aspect 68 The method of aspect 67, wherein each of the plurality of proteins is found within one or more prophage regions.
  • Aspect 69 The method of aspect 68, wherein identifying one or more potential anti- CRISPR proteins further comprises inspecting whether each potential anti-CRISPR protein exhibits specific features.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Example 1 MATERIALS AND METHODS Data collection and preprocessing
  • a dataset consisting of examples from both positive (anti-CRISPR) and negative (non-anti- CRISPR) classes was needed.
  • Anti-CRISPR information for proteins from Anti-CRISPRdb was collected (Dong et al.2018).
  • the database contained information for 432 anti-CRISPR proteins.
  • CD-HIT was used to identify a non-redundant set (at 40% sequence similarity threshold) of 2F0 experimentally verified Acrs (FIG.1; providing Table 1) (Huang et al.2010).
  • FIG.1 contains a list of Acrs used for training and cross-validation of the Acranker model. These proteins belong to different Acr classes: 12 of the proteins are active against class I-F CRISPR Cas systems, 4 against I-E and 4 against II-A (Bondy-Denomy et al.2013; Pawluk et al.2016; Hynes et al. 2017; Pawluk et al.2014; Rauch et al.2017). This set constitutes positive class of the dataset. Complete proteomes of source species to which each of these proteins belong were downloaded. Proteins in these proteomes with ⁇ 40% sequence similarity with the set of known anti-CRISPR proteins were used to construct the negative dataset.
  • FIG.2 contains a list of Acrs used for independent testing of AcRanker.
  • the proteins used for development of the machine learning model in AcRanker belonged to classes I-F, I-E, and II-A (Dong et al.2018), so only the Acrs that belonged to either of these classes were chosen.
  • Source proteomes for all these proteins were downloaded.
  • Feature Extraction [00349] In line with existing machine learning based protein function prediction techniques, sequence features were used (Saidi et al.2010) based on amino acid composition and grouped dimer and trimer frequency counts (Shen et al.2007).
  • amino acids were first grouped into seven classes based on their physicochemical properties (Shen et al.2007) (FIG.3; providing Table 3) and the frequency counts of all possible groups labeled as dimers and trimers in a given protein sequence were used in conjunction with amino acid composition. All three types of features (amino acid composition, di- and tri- meric frequency counts) were normalized to unit norm resulting in a -dimensional feature vector representation for a given protein sequence (Leslie et al.2001; Ben-Hur and Weston 2010). [00350] FIG.3 (Table 3). Grouping of amino acids based on physiochemical properties. Groups of amino acids with similar side chains are grouped together to reduce the number of features to test in the machine learning model.
  • Machine learning model [00351] The underlying machine learning model for AcRanker has been built using EXtreme Gradient Boosting (XGBoost) (Chen and Guestrin 2016).
  • XGBoost EXtreme Gradient Boosting
  • boosting is a technique in which multiple weak classifiers are combined to produce a strong classifier (Chen and Guestrin 2016).
  • XGBoost is a tree based method (Chen and Guestrin 2016) that uses boosting in an end-to-end fashion, i.e., every next tree tries to minimize the error produced by its predecessor.
  • XGBoost has been shown to be a fast and scalable learning algorithm and has been widely used in many machine learning applications.
  • XGBoost has been used as a pairwise ranking model to rank constituent proteins in a given proteome in descending order of their expected Acr behavior.
  • the XGBoost model is trained in a species-specific manner to produce higher scores for anti-CRISPR proteins as compared to non-anti-CRISPR proteins in a given proteome.
  • the pairwise ranking model performed better in terms of correctly identifying known anti-CRISPR proteins in test proteomes in cross-validation (comparison not shown for brevity).
  • the objective is to obtain an XGBoost predictor f (x; ⁇ ) with learnable parameters ⁇ that generates a prediction score for a given protein sequence represented in terms of its feature vector .
  • the model it is required that the model to learn optimal parameters ⁇ uch that the score f ( ⁇ ; ⁇ ) for a positive example ⁇ (known Anti-CRISPR protein) should be higher than f (n; ⁇ ) all negative examples n (non-Anti-CRISPR proteins) within the same species.
  • the hyperparameters of the learning model are selected through cross validation and optimal results are obtained with: number of estimators set at 120, learning rate of 0.1, subsampling of 0.6 and maximum tree depth of 3.
  • Performance Evaluation [00353] To evaluate the performance of the machine learning model, leave-one-proteome-out cross-validation, as well as validation over an independent test set, was performed. In a single fold of leave-one-proteome-out cross-validation, the source proteome of a given anti-CRISPR protein was set aside for testing and train on all other proteomes. To ensure an unbiased evaluation, all sequences in the training set with a sequence identity of 40% or higher with any test protein or among themselves were removed from the training set.
  • AcRanker Webserver [00357] A webserver implementation of AcRanker is publicly available at http://acranker(dot)pythonanywhere(dot)com/. The webserver accepts a proteome file in FASTA format and returns a ranked list of proteins. The Python code for the webserver implementation is available at https://github(dot)com/amina01/AcRanker.
  • Prophage regions were predicted using PHASTER (Arndt et al.2016). Proteins within the prophage regions were ranked with AcRanker. [00359] To select individual gene candidates for synthesis and biochemical validation, the six highest ranked proteins from each genome were examined by visual inspection for a strong promoter, a strong ribosome binding site, and an intrinsic terminator. Promoters were searched manually by looking for sequences closely matching the strong consensus promoter sequence TTGACA-17(+/-1)N-TATAAT upstream of the Acr candidate gene, or any genes immediately preceding it. The presence of a strong ribosome binding site (resembling AGGAGG) near the start codon was similarly searched for, and was required to be upstream of a gene candidate for selection.
  • E. coli Rosetta2 (DE3) containing Acr or Cas9 expression plasmids were grown in Terrific Broth (100 ⁇ g/mL ampicillin) to an OD600 of 0.6-0.8, cooled on ice, induced with 0.5 mM isopropyl-b-D-thiogalactoside and incubated at 16°C for 16 h.
  • Cells were harvested by centrifugation, resuspended in wash buffer (20 mM Tris-C1 (pH 7.5), 500 mM NaC1, 1 mM TCEP (tris(2-carboxyethyl)phosphine), 5% (v/v) glycerol) supplemented with 0.5 mM PMSF (phenylmethanesulfonyl fluoride) and cOmplete protease inhibitor (Roche), lysed by sonication, clarified by centrifugation and purified over Ni-NTA Superflow resin (Qiagen) in wash buffer supplemented with 10 mM (wash) or 300 mM imidazole (elution).
  • wash buffer 20 mM Tris-C1 (pH 7.5), 500 mM NaC1, 1 mM TCEP (tris(2-carboxyethyl)phosphine), 5% (v/v) glycerol
  • PMSF phenyl
  • Elution fractions were pooled and digested overnight with recombinantly expressed TEV protease while dialysed against dialysis buffer (20 mM Tris-C1 (pH 7.5), 125 mM NaC1, 1 mM TCEP, 5% (v/v) glycerol) at 4°C.
  • the cleaved proteins were loaded onto an MBP-Trap (GE Healthcare) upstream of a Heparin Hi- Trap (GE Healthcare) in the case of SpyCas9, SauCas9 and SinCas9.
  • digested Acrs were loaded onto a Q (ML1, ML2, ML3, ML6, ML8 and ML10), heparin (ML4, ML5), or SP (ML7 and ML9) Hi-Trap column. Proteins were eluted over a salt gradient (20 mM Tris-Cl (pH 7.5), 1 mM TCEP, 5% (v/v) glycerol, 125 mM – 1 M KCl).
  • the eluted proteins were concentrated and loaded onto a Superdex S200 Increase 10/300 (GE Healthcare) for SpyCas9, SauCas9, SinCas9 and Superdex S75 Increase 10/300 (GE Healthcare) for all the Acr candidates.
  • Columns were pre-equilibrated with gel filtration buffer (20 mM HEPES-K (pH 7.5), 200 mM KCl, 1 mM TCEP and 5% (v/v) glycerol).
  • Purified proteins were concentrated to approximately 50 ⁇ M for Cas9 effectors and 100 ⁇ M for Acr candidates.
  • the absorbance at 280 nm was measured by Nanodrop and the concentration was determined using an extinction coefficient estimated based on the primary amino acid sequence of each protein.
  • FIG.4A-4B Purified Acr candidates and Cas effectors used in this study.
  • A 4-20% gradient SDS-PAGE of purified machine learning Acr candidates and Cas effectors used in this study.
  • B Mass spectra of all purified Acr candidates used. (* 23,510 Da, unknown protein).
  • RNA preparation [00362] All RNAs were transcribed in vitro using recombinant T7 RNA polymerase and purified by gel extraction as described previously (East-Seletsky et al.2016). Briefly, 100 ⁇ g/mL T7 polymerase, 1 ⁇ g/mL pyrophosphatase (Roche), 800 units RNase inhibitor, 5 mM ATP, 5 mM CTP, 5 mM GTP, 5 mM UTP, 10 mM dithiothreitol (DTT), were incubated with DNA target in transcription buffer (30 mM Tris-C1 pH 8.1, 25 mM MgCl 2 , 0.01% Triton X-100, 2 mM spermidine) and incubated overnight at 37°C.
  • T7 polymerase 100 ⁇ g/mL T7 polymerase, 1 ⁇ g/mL pyrophosphatase (Roche), 800 units RNase inhibitor, 5 mM ATP,
  • the sgRNA was first incubated at 95°C for 5 min and cooled down to room temperature.
  • the Cas effectors (SpyCas9, SauCas9, AsCas12a at 100 nM and SinCas9 at 200 nM respectively) were incubated with each candidate Acr protein at 37°C for 10 min before the addition of sgRNA (SpyCas9, SauCas9, AsCas12a sgRNA at 160 nM and SinCas9 sgRNA at 320 nM) to form the RNP at 37°C for 10 min.
  • the DNA cleavage reaction was then initiated with the addition of DNA target and reactions incubated for 30 min at 37°C before quenching in 1X quench buffer (5% glycerol, 0.2% SDS, 50 mM EDTA). Samples were then directly loaded to a 1% (w/v) agarose gel stained with SYBRGold (ThermoFisher) and imaged with a BioRad ChemiDoc. Competition binding experiment [00364] The reconstitution of the SinCas9-sgRNA-AcrIIA16 and SinCas9-sgRNA-AcrIIA2 complex was carried out as previously described (Jiang et al.2019).
  • the reconstituted ternary complex was further purified by analytical size-exclusion chromatography (Superdex S200 Increase 10//300 GL column, GE Healthcare) pre-equilibrated with the gel filtration buffer (20 mM HEPES-K (pH 7.5), 200 mM KCl, 1 mM TCEP and 5% (v/v) glycerol) containing 1 mM MgCl 2 .
  • FIG.19A-19C present Table 9, which provides primers used for amplification of DNA targets, cloning, and in vitro transcription (IVT) of sgRNAs.
  • FIG.20A-20B present Table 10, which provides amino acid sequences and accession numbers of Acr candidates.
  • FIG.21A-21D present Table 11, which provides amino acid sequences of CRISPR/Cas effector polypeptides used in this study.
  • FIG.22 presents Table 12, which provides nucleotide sequences of sgRNAs used for in vitro cleavage assays.
  • FIG.23A-23B present Table 13, which provides nucleotide sequences of DNA targets used for in vitro cleavage assays.
  • Target sequences are shown in italics (GATGGTAAGCCCTCCCGTAT; SEQ ID NO:46)) (Streptococcus pyogenes, Streptococcus iniae); bold and italics (ACGTTTTCCAATGATGAGCACTTT; SEQ ID NO:47) (Acidaminococcus sp.) or bold (TATCGTAGTTATCTACACG; SEQ ID NO:48) (Staphylococcus aureus).
  • Each each row of the table indicates which Acr was excluded from the training dataset and used as a test dataset, and each number displayed is the ranking of the known Acr received from the indicated test proteome using either the blastp search against all other known Acrs (BLAST) or AcRanker.
  • the Acrs from bacterial proteomes - AcrIF6, AcrIF9, AcrIF10, AcrIIA1, AcrIIA2, and AcrIIA4 - were also ranked using only the subset of proteins predicted to reside within prophages as predicted by PHASTER(Arndt et al.2016). Two Acrs from bacterial proteomes did not occur in the predicted prophages and are indicated by dash placeholders.
  • Prophage proteome subset fields have been left empty for Acrs from phage proteomes.
  • machine learning rankings for Acrs contained in phage proteomes are much better than those contained in bacterial proteomes, likely due to their smaller size.
  • the relative rankings of the known Acrs found within bacterial proteomes would improve in the context of only prophage-derived proteins, which proteins in the bacterial proteomes were found within prophages were identified using PHASTER (Arndt et al.2016) and used only that subset to test both models. With the prophage subsets, a higher ranking for the known Acrs was observed due to the removal of higher-ranking proteins not found in the predicted prophages.
  • proteomes containing non-redundant ( ⁇ 40% sequence identity) Acrs from bacterial and phage sources were ranked using AcRanker.
  • Bacterial proteomes that had Acrs within PHASTER-predicted prophages were also tested with a subset of the proteome containing only the prophage proteins. However, of the five proteins found within a phage/prophage, AcRanker ranked three within the top five, including one with the highest rank (FIG.7; providing Table 6).
  • FIG.6 (Table 5). Independent testing set validation results. Ten proteomes containing non-redundant ( ⁇ 40% sequence identity) Acrs from bacterial and phage sources were ranked using AcRanker.
  • FIG.7 (Table 6). Independent testing set validation results. Five proteomes containing non-redundant ( ⁇ 40% sequence identity) Acrs from bacterial proteomes that had Acrs within PHASTER-predicted prophages, as well as their AcRanker results. Anti-CRISPR candidate selection [00378] AcRanker was applied to predict novel anti-CRISPRs from self-targeting genomes.
  • SpyCas9 Streptococcus pyogenes Cas9
  • a list of Streptococcus genomes was generated containing at least one self-targeting type II-A CRISPR system using Self-Target Spacer Searcher, which has been previously described (Watters et al.2018).385 instances of self-targeting were found from type II-A CRISPR arrays occurring within 241 Streptococcus genome assemblies, six of which contained known Acrs. Of these 241 self-targeting arrays, instances where the target sequence was flanked by the 3′ NRG protospacer adjacent motif (PAM) characteristic of SpyCas9 were sought and it was observed that it was present in 20 genomes.
  • PAM NRG protospacer adjacent motif
  • PHASTER (Arndt et al.2016) was first used to predict all of the prophages residing within the 20 self-targeting Streptococcus genomes as well as an additional Listeria monocytogenes genome (strain R2-502) containing a type II-A self- targeting CRISPR system (with six self-targets) and three well-known AcrIIA genes (Rauch et al.2017).
  • the Listeria strain was included to determine if the known Acrs within it were returned as the top ranked genes, and if not, test the higher ranking genes as potential additional Acrs within a known Acr-harboring strain.
  • FIG.9A-9E Table 8
  • Top Acr gene candidates within each genome ranked by AcRanker.
  • the proteins found within the prophages of 20 Streptococcus genomes were ranked using AcRanker; up to the top 10 highest ranking genes are listed in ascending order.
  • Known Acr genes and the 10 genes synthesized for biochemical testing are indicated in the rightmost column. Genomes with fewer than 10 listed have very few annotated proteins found within predicted prophages.
  • FIG.10 Acr candidates selected for biochemical testing. Ten Acr candidates were selected from manual inspection for further biochemical testing (blue). Each candidate is shown in its genomic context with its assigned rank from AcRanker noted in red. Homologous proteins share the same color border (green, blue). Homologs of AcrIIA3 (orange border) and AcrIIA1 (red border) are indicated.
  • FIG.11A-11D Inhibition of SpyCas9 and SauCas9 by newly discovered Acr candidates.
  • Presence of uncleaved DNA target indicates the ability of the Acr candidate to inhibit SpyCas9 at 50-fold excess.
  • AcrllA4 is used as a positive control.
  • B In vitro cleavage assay with an increasing concentration of AcrIIA4, ML1 and ML8 (Acr:RNP 0.1-, 1-, 2- ,10-, 50- and 100-fold excess from left to right) showing the ability of ML1 and ML8 to inhibit SpyCas9 at high concentrations.
  • C In vitro cleavage assay used to test the inhibition of SauCas9 by Acr candidates.
  • FIG.12A-12D In vitro cleavage assay with SpyCas9.
  • AcrllA4 positive control: The same controls and dilution series are used in all of the cleavage assays unless otherwise indicated.
  • FIG.13A-13B In vitro cleavage assay with SauCas9.
  • A In vitro cleavage assay with a dilution series of AcrllA5 (positive control) and BSA (negative control).
  • FIG.14A-14C Control experiments for the in vitro cleavage assay.
  • A Cleavage assay with the restriction enzyme AlwN1 and AcrllA4, ML1, ML3 and ML8 showing that none of the Acrs is able to inhibit AlwN1.
  • B Different dilution series with ML8 showing that even 10,000- fold excess of Acr does not lead to AlwN1 inhibition.
  • C Cleavage assay with AsCas12a and AcrVA1 (positive control), ML1, ML3 and ML8.
  • ML1 a potent inhibitor of SinCas9
  • Sin Streptococcus iniae
  • Previous studies have reported anti-CRISPRs can exhibit either selective or broad-spectrum inhibition of divergent Cas effectors (Knott et al.2019; Harrington et al.2017). Given that SinCas9 is 70.10% identical to SpyCas9 and only 25.58% identical to SauCas9 it was investigated if ML1 might be a more potent inhibitor of SinCas9. To explore this, SinCas9 protein was cloned, expressed, and purified for use in in vitro DNA targeting assays.
  • SinCas9 was capable of cleaving dsDNA targets proximal to an NGG PAM using a sgRNA derived from a fusion of the tracrRNA and crRNA (FIG.15A, FIG.16A-16B).
  • both ML1 and ML8 inhibited DNA cleavage by SinCas9.
  • the potency of SinCas9 inhibition was assayed again (FIG.15B).
  • ML1 was able to potently inhibit DNA cleavage by SinCas9 (FIG.15B).
  • SinCas9 was susceptible to inhibition by AcrIIA4 (FIG.18A) and AcrIIA2 (FIG.17D), both PAM mimics that inhibit PAM recognition by SpyCas9 (Shin et al.2017; Jiang et al.2019).
  • ML1 is a small protein with a predicted negatively charged surface potential (isoelectric point of 4.3), suggesting that it too might compete with negatively charged DNA.
  • a competition binding experiment was developed to assay if the association of ML1 with SinCas9 might prevent the binding of AcrIIA2 (FIG.18A).
  • FIG.16A-16B SinCas9 works with a similar sgRNA as SpyCas9.
  • A Schematic of the pre-crRNA:tracrRNA complex.
  • B Schematic of the single-guide RNA.20-bp spacer sequence is shown in blue, tracrRNA is shown in red and the direct repeat sequence in grey.
  • FIG.17A-17D In vitro cleavage assay with SinCas9.
  • A In vitro cleavage assay with a dilution series of ML1.
  • FIG.18A-18E ML1 competes with AcrIIA2 for the same binding site.
  • A Flowchart for the competition binding experiment between ML1 and AcrIIA2. Binding of the Acr to the SinCas9-sgRNA RNP was reconstituted using size-exclusion chromatography (SEC).
  • Last lane (III) shows the competition of ML1 with AcrIIA2 for the same binding site.

Abstract

The present disclosure provides polypeptides that inhibit activity of a CRISPR/Cas effector polypeptide, nucleic acids encoding the polypeptides, and systems comprising the polypeptides and/or nucleic acids encoding the polypeptides. The present disclosure provides methods of inhibiting activity of a CRISPR/Cas effector polypeptide.

Description

MODULATORS OF CAS9 POLYPEPTIDE ACTIVITY AND METHODS OF USE THEREOF CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/941,332, filed November 27, 2019, which application is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under HR0011-17-2-0043 awarded by the Defense Advanced Research Projects Agency and 1244557 awarded by the National Science Foundation. The government has certain rights in the invention. INTRODUCTION [0003] Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 nucleases, when complexed with a guide RNA, effect genome editing in a sequence-specific manner. RNA- guided Cas9 has proven to be a versatile tool for genome engineering in multiple cell types and organisms. [0004] There is a need in the art for compositions and methods for controlling genome editing activity of CRISPR/Cas9. SUMMARY [0005] The present disclosure provides polypeptides that inhibit activity of a CRISPR/Cas effector polypeptide, nucleic acids encoding the polypeptides, and systems comprising the polypeptides and/or nucleic acids encoding the polypeptides. The present disclosure provides methods of inhibiting activity of a CRISPR/Cas effector polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS [0006] FIG.1 presents Table 1, which provides a list of Acrs used for training and cross-validation of the AcRanker model. [0007] FIG.2 presents Table 2, which provides a list of Acrs used for independent testing of AcRanker. [0008] FIG.3 presents Table 3: grouping of amino acids based on physiochemical properties. [0009] FIG.4A-4B present purified Acr candidates and CRISPR/Cas effectors used. [0010] FIG.5 present Table 4, which provides results for the leave-one-out cross-validation. [0011] FIG.6 presents Table 5, which provides independent testing set validation results. [0012] FIG.7 presents Table 6, which provides independent testing set validation results. [0013] FIG.8A-8D presents Table 7, which provides a list of expected lethal self-targeting Streptococcus genomes obtained with Self-Target Spacer Searcher (STSS). Self-Targeting Spacer Sequences: from top to bottom SEQ ID NOs:49-73, 73, 74-77, 68, 78-81, 76, 82-84, 77, 60, 62, 85, 86, 86, 87, 88-90, 90, 91-92, 92, 93-95, 93. [0014] FIG.9A-9E presents Table 8, which provides top Acr gene candidates within each genome ranked by AcRanker. [0015] FIG.10 depicts the genomic context of the Acr candidates selected for biochemical testing. [0016] FIG.11A-11D depict inhibition of SpyCas9 and SauCas9 by Acr candidates. [0017] FIG.12A-12D depict the results of an in vitro cleavage assay with SpyCas9. [0018] FIG.13A-13B depict the results of an in vitro cleavage assay with SauCas9. [0019] FIG.14A-14C depict the results of control experiments for the in vitro cleavage assays. [0020] FIG.15A-15B depict inhibition of SinCas9 by ML1 and ML8. [0021] FIG.16A-16B depict the sgRNA used for inhibition of SinCas9 (from top to bottom SEQ ID NOs:96-98). [0022] FIG.17A-17D depict the results of an in vitro cleavage assay with SinCas9. [0023] FIG.18A-18E depict competition between ML1 and AcrIIA2 for the same binding site. [0024] FIG.19A-19C present Table 9, which provides primers (from top to bottom SEQ ID NOs:99- 127) used for amplification of DNA targets, cloning, and in vitro transcription (IVT) of sgRNAs. [0025] FIG.20A-20B present Table 10, which provides amino acid sequences and accession numbers of Acr candidates. [0026] FIG.21A-21D present Table 11, which provides amino acid sequences (from top to bottom SEQ ID NOs: 128-131) of CRISPR/Cas effector polypeptides used in this study. [0027] FIG.22 presents Table 12, which provides nucleotide sequences (from top to bottom SEQ ID NOs:132-135) of sgRNAs used for in vitro cleavage assays. [0028] FIG.23A-23B present Table 13, which provides nucleotide sequences of DNA targets (SEQ ID NO:136) used for in vitro cleavage assays. DEFINITIONS [0029] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. [0030] The term “oligonucleotide” refers to a polynucleotide of between 4 and 100 nucleotides of single- or double-stranded nucleic acid (e.g., DNA, RNA, or a modified nucleic acid). However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes, transcribed (in vitro and/or in vivo), or chemically synthesized. The terms “polynucleotide" and "nucleic acid" should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. [0031] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/ uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base- pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein- binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA) is considered complementary to both a U and to C. For example, when a G/U base-pair can be made at a given nucleotide position of a protein- binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. [0032] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). [0033] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 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 complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). [0034] The terms ''peptide," ''polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. [0035] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 106 M, less than 10-7 M, less than 10-8 M, less than 109 M, less than 1010 M, less than 10-11 M, less than 10-12 M, less than 10-13 M, less than 10-14 M, or less than 10-15 M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd. [0036] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine- glutamine. [0037] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi(dot)nlm(dot)nili(dot)gov/BLAST, ebi(dot)ac(dot)uk/Tools/msa/tcoffee/, ebi(dot)ac(dot)uk/Tools/msa/muscle/, mafft.cbrc(dot)jp/alignment/software/, www(dot)sbg(dot)bio(dot)ic(dot)ac(dot)uk/~phyre2/. See, e.g., Altschul et al. (1990), J. Mol. Bioi.215:403-10. [0038] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and/or regulate translation of an encoded polypeptide. [0039] As used herein, a "promoter" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. Various promoters, including inducible promoters, may be used to drive the various nucleic acids (e.g., vectors) of the present disclosure. [0040] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", below). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term ''recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non- naturally occurring (e.g., a variant, a mutant, etc.). Thus, a ''recombinant" polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence. [0041] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell. [0042] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. [0043] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences. [0044] Any given component, or combination of components can be unlabeled, or can be detectably labeled with a label moiety. In some cases, when two or more components are labeled, they can be labeled with label moieties that are distinguishable from one another. [0045] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. [0046] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0047] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0049] It must 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 “an Acr polypeptide” includes a plurality of such polypeptides and reference to “the CRISPR/Cas effector polypeptide” includes reference to one or more CRISPR/Cas effector polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0050] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. [0051] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION [0052] The present disclosure provides polypeptides that inhibit activity of a CRISPR/Cas effector polypeptide, nucleic acids encoding the polypeptides, and systems comprising the polypeptides and/or nucleic acids encoding the polypeptides. The present disclosure provides methods of inhibiting activity of a CRISPR/Cas effector polypeptide. ANTI-CRISPR POLYPEPTIDES [0053] The present disclosure provides polypeptides that inhibit activity of one or more CRISPR/Cas effector polypeptides. A polypeptide of the present disclosure that inhibits activity of a CRISPR/Cas effector polypeptide is also referred to herein as an “anti-CRISPR polypeptide” or an “Acr polypeptide.” ML1 [0054] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 30 amino acids to 64 amino acids (e.g., from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 64 aa) of the following amino acid sequence: [0055] MKNYEVTNEVKNLNTQVETIGQAVDLYKEYGSNTIVWSIDKNEDLIDEVTELVAEYAE KGTVIK (SEQ ID NO:1). This Acr is referred to herein as “ML1” or “AcrIIA16.” [0056] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 30 amino acids to 64 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 30 amino acids (aa) to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 64 aa. ML2 [0057] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 40 amino acids to 77 amino acids (e.g., from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, or from about 75 aa to 77 aa) of the following amino acid sequence: [0058] MGKTYWYNEGTDTLLTEKEYKELMEREAKALYEEVQEEEKDFESSEKTSFEEFLKTCY ENESDFVLSDNEGNKLEEW (SEQ ID NO:2). This Acr is referred to herein as “ML2.” [0059] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML2 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 40 amino acids to 77 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, or from about 75 aa to 77 aa. ML3 [0060] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to 89 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, or from about 85 aa to 89 aa) of the following amino acid sequence: [0061] MSKTMYKNDVIELIKNAKTNNEELLFTSVERNTREAATQYFRCPEKHVSDAGVYYGED FEFDGFEIFEDDLIYTRSYDKEELN (SEQ ID NO:3). This Acr is referred to herein as “ML3.” [0062] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML3 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 50 amino acids to 89 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, or from about 85 aa to 89 aa. ML4 [0063] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to about 95 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, from about 85 aa to about 90 aa, or from about 90 aa to 95 aa) of the following amino acid sequence: [0064] MLRRVNHVKNVLAHGEFAEWIENKIGIHYREANRMMTVAKQIPNVSTLKYLGATAKH VNGVAKRKQNFLSQISLIPTNPQLPHQTIINTYLYWQP (SEQ ID NO:4). This Acr is referred to herein as “ML4.” [0065] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML4 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 50 amino acids to 95 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aaa to about 85 aa, from about 85 aa to about 90 aa, or from about 90 aa to 95 aa. ML5 [0066] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 140 to about 210 amino acids (e.g., from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, or from about 200 aa to 210 aa) of the following amino acid sequence: [0067] MNRLKELRKEKKLTQEELAGEIGVSKITILRWENGERQIKPDKAKELAKYFNVSVGYLL GYAPNKKIDFQLNLDGTTLHLTKEQFLALENTSKSIKKIKNTINESVKQEEYIKNASKYY DFEKVSRRLTDRLFEIHTDLIELLMMLDHFPSGELSKSQQEAIFKFYKQLDYFVTDTPASF DYFKKNLESYGYKIYTEGDKIDFD (SEQ ID NO:5). This Acr is referred to herein as “ML5.” [0068] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML5 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 140 amino acids to 210 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, or from about 200 aa to 210 aa. ML6 [0069] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 40 amino acids to about 65 amino acids (e.g., from about from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 65 aa) of the following amino acid sequence: [0070] MLYIDEFKEAIDKGYILGGTVAIVRKNGKIFDYVLPHEEVREEEVVTVERVEDVMRELE (SEQ ID NO:6). This Acr is referred to herein as “ML6.” [0071] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML6 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 40 amino acids to 65 amino acids. For example, in some cases, the Acr polypeptide has a length of from about from about 40 amino acids (aa) to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, or from about 60 aa to 65 aa. ML7 [0072] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 140 amino acids to about 219 amino acids (e.g., from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, from about 200 aa to about 210 aa, or from about 210 aa to 219 aa) of the following amino acid sequence: [0073] MIKIYFGKDAALNQAIQSRLDSYQIDYQAFSSKDIDAKTLMEWLFKSTDIFELLSTKMLK YKLNTQITLSQFVRKILKDVNSTLKLPIVVTDEVIYSNMSPDYVTVLLPKEYRKIKRIQLM RKMEQLDEGRLFWKNFELFRKQSELRWFELNELLFADVSDDLGEIKKAKDRFFSYKKN NQVPPNEIIERILKIFLVDREDFFKKSPSDLQNF (SEQ ID NO:7). This Acr is referred to herein as “ML7.” [0074] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML7 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 140 amino acids to 219 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 140 amino acids (aa) to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, from about 190 aa to about 200 aa, from about 200 aa to about 210 aa, or from about 210 aa to 219 aa. ML8 [0075] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 60 amino acids to about 114 amino acids (e.g., from about 60 amino acids (aa) to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, from about 95 aa to about 100 aa, from about 100 aa to about 105 aa, from about 105 aa to about 110 aa, or farom about 110 aa to 114 aa) of the following amino acid sequence: [0076] MDYDNENYLIPKILLQDDFYSSLSAKDILVYAVLKDRQIEALEKGWIDTDGSIYLNFKLIE LAKMFSCSRTTMIDVMQRLEEVNLIERERVDVFYGYSLPYKTYINEV (SEQ ID NO:8). This Acr is referred to herein as “ML8” or “AcrIIA17.” [0077] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 60 amino acids to 114 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 60 amino acids (aa) to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, from about 95 aa to about 100 aa, from about 100 aa to about 105 aa, from about 105 aa to about 110 aa, or from about 110 aa to 114 aa. ML9 [0078] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 to about 99 amino acids (e.g., from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, or from about 95 aa to 99 aa) of the following amino acid sequence: [0079] MTEGFTIQLPKVTEKKLLARYDDMLQKAIEKALEDKELYKPIVRMAGLCRWLDVSTTT VVKWQKQGGMPHMVIDGVTLYDKHKVAQWLQQFER (SEQ ID NO:9). This Acr is referred to herein as “ML9.” [0080] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML9 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 50 amino acids to 99 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 50 amino acids (aa) to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, from about 65 aa to about 70 aa, from about 70 aa to about 75 aa, from about 75 aa to about 80 aa, from about 80 aa to about 85 aa, from about 85 aa to about 90 aa, from about 90 aa to about 95 aa, or from about 95 aa to 99 aa. ML10 [0081] An Acr polypeptide of the present disclosure can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 35 to about 49 amino acids (e.g., from about 35 amino acids (aa) to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to 49 aa) of the following amino acid sequence: [0082] MNIEDIERIISEYLIFRSDIDGCAVIDIEDFLKHIRFSYERLK (SEQ ID NO:10). This Acr is referred to herein as “ML10.” [0083] In some cases, an Acr polypeptide of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ML10 amino acid sequence depicted in FIG.20A-20B (and set forth above). In some cases, the Acr polypeptide has a length of from about 35 amino acids to 49 amino acids. For example, in some cases, the Acr polypeptide has a length of from about 35 amino acids (aa) to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to 49 aa. Activity [0084] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9 polypeptide in a Cas9/guide RNA complex is not contacted with the Acr polypeptide). [0085] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9/guide RNA complex is not contacted with the Acr polypeptide). [0086] In some cases, an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide, when the molar ratio of Acr to Cas9 polypeptide is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1. In some cases, an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the cleavage activity of the Cas9 polypeptide in a Cas9/guide RNA complex in the absence of the Acr polypeptide, when the molar ratio of Acr to Cas9 polypeptide is from about 2:1 to about 5:1, from about 5:1 to about 10:1, from about 10:1 to about 20:1, from about 20:1 to about 50:1, or from about 50:1 to about 100:1. [0087] In some cases, the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 10:1. In some cases, the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 50:1. In some cases, the cleavage activity of a Cas9 polypeptide present in a Cas9/guide RNA complex is inhibited by an Acr polypeptide of the present disclosure to 50% of the maximal cleavage activity, when the molar ratio of Acr to Cas9 polypeptide is 100:1. In other words, in some cases, a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 10:1, a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is from 10:1 to 50:1 (e.g., 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1). In some cases, a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 50:1. a Cas9 polypeptide present in a Cas9/guide RNA complex exhibits half maximal activity when contacted with an Acr polypeptide of the present disclosure, when the molar ratio of Acr to Cas9 polypeptide is 100:1. [0088] “Binding” activity of a Cas9/guide RNA complex refers to binding of the Cas9/guide RNA complex to a target nucleic acid, where the target nucleic acid comprises a nucleotide sequence that has complementarity to a target-binding nucleotide sequence in the guide RNA. [0089] “Cleavage” activity of a Cas9/guide RNA complex refers to generation by the Cas9/guide RNA complex of a single-strand or double-strand break in a target nucleic acid. [0090] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Streptococcus pyogenes Cas9 (Spy Cas9) amino acid sequence provided in FIG.21A-21D. [0091] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Staphylococcus aureus Cas9 (“SauCas9”) amino acid sequence provided in FIG.21A-21D. [0092] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of a Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Streptococcus iniae Cas9 (SinCas9) amino acid sequence provided in FIG.21A-21D. [0093] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; and b) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sin Cas9 amino acid sequence provided in FIG.21A-21D. For example, an Acr polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity the ML1 amino acid sequence depicted in FIG.20A-20B inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; and b) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sin Cas9 amino acid sequence provided in FIG.21A-21D. [0094] In some cases, an Acr polypeptide of the present disclosure inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; b) a Sau Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sau Cas9 amino acid sequence provided in FIG.21A-21D; and c) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sin Cas9 amino acid sequence provided in FIG.21A-21D. For example, an Acr polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity the ML8 amino acid sequence depicted in FIG.20A-20B inhibits binding and/or cleavage activity of: a) a Spy Cas9 polypeptide in a Cas9/guide RNA complex having at least 50% at least 60% at least 70% , at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Spy Cas9 amino acid sequence provided in FIG.21A-21D; b) a Sau Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sau Cas9 amino acid sequence provided in FIG.21A-21D; and c) a Sin Cas9 polypeptide in a Cas9/guide RNA complex having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Sin Cas9 amino acid sequence provided in FIG.21A-21D. [0095] In some cases, an Acr polypeptide of the present disclosure does not substantially inhibit binding and/or cleavage activity of a SauCas9 in a Cas9/guide RNA complex. For example, in some cases, an Acr polypeptide of the present disclosure does not substantially inhibit cleavage activity of a Cas9/guide RNA complex, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A-21D. In some cases, an Acr polypeptide of the present disclosure inhibits cleavage activity of a SauCas9/guide RNA complex by no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 2%, or no more than 1%, compared to the cleavage activity of the SauCas9/guide RNA complex in the absence of the Acr polypeptide, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A- 21D. In some cases, an Acr polypeptide of the present disclosure inhibits cleavage activity of a Cas9/guide RNA complex by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1%, compared to the cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide, where the Cas9 present in the Cas9/guide RNA complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the SauCas9 amino acid sequence provided in FIG.21A-21D. [0096] Whether an Acr polypeptide inhibits binding activity of a given Cas9/guide RNA complex can be readily determined. For example, a double stranded “Cas9 beacon” DNA fragment comprising a target can be constructed in which the target strand comprises a fluorophore and the non-target strand comprises a fluorescence quencher. Upon binding of the beacon by a Cas9/guide RNA complex, the non-target strand is displaced, allowing unquenched fluorescence from the target strand. Measurement of increased fluorescence signal in the presence of a Cas9/guide RNA complex indicates DNA binding activity of the Cas9/guide RNA complex (Mekler et al. (2016) Nuc. Acids Res.44(6):2837-2845). [0097] Whether an Acr polypeptide inhibits cleavage activity of a given Cas9 polypeptide present in a Cas9/guide RNA complex can be readily determined. For example, the effect of an Acr polypeptide of the present disclosure on cleavage of a target DNA by a Cas9/guide RNA complex can be tested in a cell-free system in vitro, as described in the Examples section. For example, a target DNA is mixed in vitro with: a) a complex of Cas9 and a guide RNA, where the guide RNA comprises both a nucleotide sequence (tracrRNA) that activates the Cas9 polypeptide and a nucleotide sequence (crRNA) that binds to the target DNA; and b) an Acr polypeptide. Production of cleavage products of action of the Cas9 on the target DNA can be detected by resolving the cleavage products on a 1% agarose cell and staining the resolved cleavage products. [0098] As another example, the effect of an Acr polypeptide of the present disclosure on cleavage of a target DNA by a Cas9/guide RNA complex can be tested in a cell. For example, a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide and a nucleic acid comprising a nucleotide sequence encoding a reporter (e.g., a fluorescent protein such as a green fluorescent protein) can be integrated into the genome of a mammalian cell (e.g., 293 cells, or other mammalian cell line), generating a reporter cell; and a ribonucleoprotein (RNP) complex comprising a Cas9 polypeptide and a guide RNA targeting the nucleotide sequence encoding the reporter is introduced into the reporter cell. Inhibition of gene editing of the reporter can be determined by detecting the reporter. For example, where the reporter is a fluorescent protein, fluorescence activated cell sorting (FACS) can be used to determine whether gene editing has been inhibited. As another example, a mixture of a Cas9/guide RNA complex and Acr polypeptide can be introduced into a mammalian cell line, and the effect of the Acr polypeptide on the ability of the Cas9/guide RNA complex to carry out gene editing can be determined by analyzing production of a gene product encoded by a nucleotide sequence targeted by the guide RNA. Covalently linked non-peptidic moiety [0099] In some cases, an Acr polypeptide of the present disclosure comprises a non-peptidic moiety covalently linked to the Acr polypeptide. The covalently linked non-peptidic moiety can confer a desirable attribute (e.g., increased protease resistance, increased membrane permeability, increased cell type or tissue specific targeting, increased in vivo half-life, increased in vivo stability, increased bioavailability), without substantially altering the ability of the linked Acr polypeptide to inhibit Cas9 activity. [00100] In some cases, the non-peptidic moiety confers increased in vivo half-life on the linked Acr polypeptide, compared to the in vivo half-life of the Acr not comprising the non-peptidic moiety. For example, in some cases, the in vivo half-life of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold, greater than the in vivo half-life of the Acr polypeptide not comprising the non-peptidic moiety. For example, in some cases, the non-peptidic moiety confers an increase in half-life of the linked Acr polypeptide in circulation in an animal. In some cases, the non-peptidic moiety confers increased in vivo stability on the linked Acr polypeptide, compared to the in vivo stability of the Acr polypeptide not comprising the non-peptidic moiety. For example, in some cases, the in vivo stability of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5- fold, or at least 10-fold, greater than the in vivo stability of the Acr polypeptide not comprising the non-peptidic moiety. In some cases, the non-peptidic moiety confers increased bioavailability on the linked Acr polypeptide, compared to the bioavailability of the Acr polypeptide not comprising the non-peptidic moiety. For example, in some cases, the bioavailability of an Acr polypeptide comprising a covalently linked non-peptidic moiety is at least 10%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold, greater than the bioavailability of the Acr polypeptide not comprising the non-peptidic moiety. [00101] Suitable non-peptidic moieties include, but are not limited to, lipids and non-peptidic polymers. Suitable non-peptidic moieties include, but are not limited to, poly(ethylene glycol), polysialic acid, hydroxyethyl starch (HES), a dendrimer, a nanoparticle, and a liposome. [00102] In some cases, a non-peptidic moiety covalently linked to an Acr polypeptide of the present disclosure is a polymer. Polymers may contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as “PGA”; lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA”; caprolactone units, such as poly(caprolactone), collectively referred to herein as “PCL”; copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as “PLGA”; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers". In certain cases, the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker. [00103] An Acr polypeptide of the present disclosure may include one or more covalently linked hydrophilic polymers. Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide); poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol); polyoxazoline; and copolymers thereof. [00104] An Acr polypeptide of the present disclosure may include one or more covalently linked hydrophobic polymers. Examples of suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co- caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof. [00105] An Acr polypeptide of the present disclosure may include one or more covalently linked biodegradable polymers. Suitable biodegradable polymers can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose such as methyl cellulose and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, polymers of acrylic and methacrylic esters such as poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene and polyvinylpryrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. Exemplary biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. In some embodiments the particle contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). Fusion polypeptides [00106] In some cases, an Acr polypeptide of the present disclosure is a fusion Acr polypeptide. The present disclosure provides a fusion polypeptide (an “Acr fusion polypeptide”) comprising: a) an Acr polypeptide of the present disclosure; and b) a heterologous fusion partner (i.e., one or more heterologous fusion partners). The heterologous fusion partner can provide one or more desirable attributes (where such attributes include, e.g., as increased protease resistance, increased membrane permeability, or increased half-life, increased nuclear localization, increased cell or tissue specific targeting and the like) without substantially altering the ability of the linked Acr polypeptide to inhibit Cas9 activity. A fusion polypeptide comprising: a) an Acr polypeptide of the present disclosure; and b) a heterologous fusion partner is also referred to herein as an “Acr fusion polypeptide.” A fusion polypeptide of the present disclosure can comprise two or more heterologous fusion partners. [00107] Suitable heterologous fusion partners include, but are not limited to, a nuclear localization signal; a chloroplast transit peptide; an endosomal escape peptide; an epitope tag; a polypeptide that provides for ease of purification; a detectable protein; a protein that provides for increased in vivo half-life; a protein that provides for increased cell type or tissue specificity (e.g. an antibody or fragment thereof) and the like. [00108] Suitable heterologous fusion partners include a hydroxine-binding protein, transthyretin, α1-acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, α-2- macroglobulin, a lipoprotein, and a fragment of any of the foregoing. Suitable heterologous fusion partners include a fluorescent protein, e.g., a green fluorescent protein (GFP), a yellow fluorescent protein, a red fluorescent protein a cyan fluorescent protein, and the like. Suitable heterologous fusion partners include, e.g., a poly(histidine) tag (e.g., a 6XHis tag); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like. [00109] In some cases, a fusion polypeptide of the present disclosure comprises an Acr polypeptide fused to a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a fusion Acr polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus of the Acr polypeptide. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus of the Acr polypeptide. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus of the Acr polypeptide. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus of the Acr polypeptide. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus of the Acr polypeptide. [00110] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:11); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:12)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:13) or RQRRNELKRSP (SEQ ID NO:14); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:15); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:16) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:17) and PPKKARED (SEQ ID NO:18) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:19) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:20) of mouse c- abl IV; the sequences DRLRR (SEQ ID NO:21) and PKQKKRK (SEQ ID NO:22) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:23) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:24) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:25) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:26) of the steroid hormone receptors (human) glucocorticoid. [00111] An Acr fusion polypeptide of the present disclosure can include, as the fusion partner, a "Protein Transduction Domain" or PTD (also known as a CPP – cell penetrating peptide). 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:27); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther.9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:28); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:29); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:30); and RQIKIWFQNRRMKWKK (SEQ ID NO:31). Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO:27), RKKRRQRRR (SEQ ID NO:32); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:27); RKKRRQRR (SEQ ID NO:33); YARAAARQARA (SEQ ID NO:34); THRLPRRRRRR (SEQ ID NO:35); and GGRRARRRRRR (SEQ ID NO:36). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). [00112] In some cases, an Acr fusion polypeptide of the present disclosure comprises a linker between the Acr polypeptide and the fusion partner. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. In some cases, the linker is proteolytically cleavable. [00113] Examples of linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO:37), GGSGGSn (SEQ ID NO:38), and GGGGSn (SEQ ID NO:39), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:40), GGSGG (SEQ ID NO:41), GSGSG (SEQ ID NO:42), GSGGG (SEQ ID NO:43), GGGSG (SEQ ID NO:44), GSSSG (SEQ ID NO:45), and the like. [00114] An Acr fusion polypeptide of the present disclosure can also comprise a covalently linked non-peptidic moiety, where suitable non-peptidic moieties are discussed above. NUCLEIC ACIDS AND RECOMBINANT EXPRESSION VECTORS [00115] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. As noted above, in some cases, an Acr polypeptide of the present disclosure is a fusion polypeptide. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure. The present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure. [00116] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. In some cases, the nucleic acid is RNA. In some cases, the nucleic acid is DNA. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure. In some cases, the nucleic acid is RNA. In some cases, the nucleic acid is DNA. [00117] In some cases, a nucleotide sequence encoding an Acr polypeptide of the present disclosure is codon optimized. This type of optimization can entail a mutation of an Acr- encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon- optimized Acr-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized Acr-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized Acr-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were an insect cell, then an insect codon-optimized Acr-encoding nucleotide sequence could be generated. [00118] In some cases, the nucleotide sequence encoding an Acr polypeptide of the present disclosure, or encoding an Acr fusion polypeptide of the present disclosure, is operably linked to one or more of a promoter, an enhancer, an internal ribosomal entry site, and a transcription termination signal. [00119] In some cases, the nucleotide sequence encoding an Acr polypeptide of the present disclosure, or encoding an Acr fusion polypeptide of the present disclosure, is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a transcriptional control element that is functional in a eukaryotic cell. [00120] The transcriptional control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in a T cell, a B cell, a hematopoietic stem cell, a liver cell, a lung cell, a muscle cell (e.g., a cardiac muscle cell; a skeletal muscle cell), a retinal cell, or other targeted cell. [00121] A suitable promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/”ON” state). A suitable promoter may be an inducible promoter (i.e., a promoter whose state, active/”ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). A suitable promoter can be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process). [00122] Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res.2003 Sep 1;31(17)), a human H1 promoter (H1), and the like. [00123] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells). [00124] In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell). [00125] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis- related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. A suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761). [00126] For illustration purposes, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. In some cases, the promoter is an insect-specific promoter. In some cases, the promoter is a plant-specific promoter. In some cases, the promoter is a protozoan-specific promoter. [00127] In some cases, the promoter is a plant-specific promoter. Examples of plant promoters include, but are not limited to, a cauliflower mosaic virus (CaMV) promoter, a nopaline synthetase promoter, a ribose bisphosphate carboxylase promoter, a ubiquitin promoter, a UBQ3 promoter, a cestrum virus promoter, a rice actin 1 promoter, a CaMV 35S promoter, a CaMV 19S promoter, a sucrose synthase promoter, and a figwort mosaic virus promoter. Chemical agent-inducible promoters are known in the art and include, but are not limited to, the maize In2- 2 promoter, which is activated by benzenesulfonamide herbicide safeners; the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre- emergent herbicides; and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J.14(2):247-257); copper-inducible system promoters; salicylate-inducible system promoters (e.g., the PR1a system); glucocorticoid- inducible promoters (Aoyama et al. (1997) Plant J.11:605-612); and ecdysone-inducible system promoters. Tissue-preferred and tissue-specific promoters can be used to control expression in a particular plant tissue. Such tissue-preferred and tissue-specific promoters include leaf-preferred promoter, root-preferred promoters; root-specific promoters, seed-preferred promoters; seed- specific promoters; and the like. [00128] As noted above, the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. As noted above, the present disclosure provides a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure. [00129] Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:38223828; Mendelson et al., Virol. (1988) 166:154165; and Flotte et al., PNAS (1993) 90:1061310617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant retroviral vector. [00130] Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector. [00131] Methods of introducing a nucleic acid (e.g., DNA or RNA) (e.g., a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; a nucleic acid comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure; a nucleic acid encoding a Cas9 polypeptide; Cas9 guide RNA; a nucleic acid comprising a nucleotide sequence encoding a Cas9 guide RNA; a recombinant expression vector comprising one or more of the aforementioned nucleic acids; and the like) into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. [00132] Introducing a recombinant expression vector of the present disclosure into a cell or cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo. Introducing the recombinant expression vector into a target cell can be carried out in vitro. [00133] In some embodiments, an Acr polypeptide-encoding nucleic acid can be provided as RNA. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the Acr polypeptide). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.). [00134] Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS One 5(7): e11756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer et al. (2008) Proc. Natl. Acad. Sci. USA 105(50):19821-19826. [00135] Vectors may be provided directly to a target host cell. In other words, the cells are contacted with a recombinant expression vector comprising a nucleic acid (e.g., a recombinant expression vector comprising a nucleic acid encoding an Acr polypeptide; a recombinant expression vector comprising: i) a nucleic acid encoding an Acr polypeptide; ii) a nucleotide sequence encoding a Cas9 polypeptide; and iii) a nucleotide sequence encoding a Cas9 guide RNA; etc.) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors. [00136] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA). [00137] A recombinant expression vector used for providing a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide (and, optionally, encoding a Cas9 polypeptide and/or a Cas9 guide RNA) to a target host cell can include suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest. In other words, in some cases, the nucleic acid of interest will be operably linked to a promoter. In addition, recombinant expression vector used for providing a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide (and, optionally, encoding a Cas9 polypeptide and/or a Cas9 guide RNA) to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the recombinant expression vector. [00138] In some cases, an expression vector of the present disclosure comprises: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above). In some cases, the nucleotide sequence encoding the Acr polypeptide and the nucleotide sequence encoding the Cas9 polypeptide are operably linked to the same promoter. In some cases, the nucleotide sequence encoding the Acr polypeptide is operably linked to a first promoter; and the nucleotide sequence encoding the Cas9 polypeptide is operably linked to a second promoter, where the second promoter is different from the first promoter. [00139] In some cases, an expression vector of the present disclosure comprises: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); and c) a nucleotide sequence encoding a Cas9 guide RNA. In some cases, the nucleotide sequence encoding the Acr polypeptide, the nucleotide sequence encoding the Cas9 polypeptide, and the nucleotide sequence encoding the Cas9 guide RNA are operably linked to the same promoter. In some cases, the nucleotide sequence encoding the Acr polypeptide is operably linked to a first promoter; and the nucleotide sequence encoding the Cas9 polypeptide and the nucleotide sequence encoding the Cas9 guide RNA are operably linked to a second promoter, where the second promoter is different from the first promoter. MODIFIED HOST CELLS [00140] The present disclosure provides a modified host cell comprising an Acr polypeptide of the present disclosure and/or a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. In some cases, the modified host cell is one that does not normally comprise an Acr polypeptide of the present disclosure; i.e., the Acr polypeptide is heterologous to the host cell. [00141] The present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is inhibited by an Acr polypeptide of the present disclosure); and c) a nucleotide sequence encoding a Cas9guide RNA. [00142] A cell that serves as a recipient for an Acr polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; insect cells; arachnid cells; etc. A cell that serves as a recipient for an Acr polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure is referred to as a “host cell” or a “target cell.” A host cell or a target cell can be a recipient of an Acr system of the present disclosure. A host cell or a target cell can be a recipient of a single component of an Acr system of the present disclosure. [00143] Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep, a horse, a camel); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.) and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell). In some instances, modified host cells can be cells within a tissue in a vertebrate animal (e.g. a liver cell, a muscle cell, a pulmonary cell, a pancreatic cell, a skin cell, a renal cell, a cell in the CNS etc). In some instances, the cell can be a specific cell type within a tissue (e.g. a neuron or an astrocyte in brain tissue or a hepatocyte or Kupfer cell in liver tissue). [00144] A cell can be an in vitro cell (e.g., a cell in culture, e.g., an established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell. [00145] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc. [00146] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. [00147] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). [00148] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells. [00149] Adult stem cells are resident in differentiated tissue, but retain the properties of self- renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like. [00150] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell. [00151] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A. [00152] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate the erythroid, neutrophil- macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. HSCs can be induced in vitro to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells. [00153] In other instances, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art. [00154] In other cases, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC. [00155] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon. [00156] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese artichoke (crosnes), chinese cabbage, chinese celery, chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like. [00157] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a sub- order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera. [00158] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle. COMPOSITIONS [00159] The present disclosure provides a composition comprising an Acr polypeptide of the present disclosure. The present disclosure comprises a composition comprising a nucleic acid or a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a composition comprising an Acr fusion polypeptide of the present disclosure. The present disclosure provides a composition comprising a host cell comprising an Acr polypeptide of the present disclosure. [00160] For example, one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, can be delivered (non-contemporaneously or simultaneously) using particles or lipids (e.g., liposomes). For example, any one of, or any combination of, (a)-(g) as set out above can be delivered associated with, or encapsulated in, a nanoparticle. For example, any one of, or any combination of, (a)-(g) as set out above can be delivered associated with, or encapsulated in, a lipid composition, e.g., a lipid composition (such as a liposome) comprising a lipid or lipidoid and a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and/or wherein the lipid composition further comprises cholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). For example, a lipid composition can be formed using a multistep process in which an Acr polypeptide, a Cas9 polypeptide, and a Cas9 guideRNA are mixed together, e.g., at a 1:1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile, nuclease free 1 x phosphate- buffered saline (PBS); and separately, DOTAP, DMPC, PEG, and cholesterol as applicable for the formulation are dissolved in alcohol, e.g., 100% ethanol; and, the two solutions are mixed together to form particles containing the complexes). [00161] In some cases, one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, are delivered using particles or lipid envelopes. For example, a biodegradable core-shell structured nanoparticle with a poly (β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles/nanoparticles based on self assembling bioadhesive polymers are used; such particles/nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used. Doses of about 5 mg/kg can be used, with single or multiple doses, depending on various factors, e.g., the target tissue. [00162] Lipidoid compounds (e.g., as described in US patent application 20110293703) are also useful in the administration of polynucleotides, and can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure. In one aspect, the aminoalcohol lipidoid compounds are combined with an agent (one or more of (a)-(f), above), to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition. [00163] A poly(beta-amino alcohol) (PBAA) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, to a target cell. US Patent Publication No.20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization. [00164] Sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, to a target cell. [00165] In some cases, lipid nanoparticles (LNPs) are used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium- propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Preparation of LNPs and is described in, e.g., Rosin et al. (2011) Molecular Therapy 19:1286-2200). The cationic lipids 1,2-dilineoyl- 3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2''- (methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R- 3-[(omega-methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. A nucleic acid (e.g., a Cas9 guide RNA; a nucleic acid of the present disclosure; etc.) may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C- DOMG at 40:10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated. [00166] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure, to a target cell.. See, e.g., Cutler et al., J. Am. Chem. Soc.2011133:9254-9257, Hao et al., Small.20117:3158-3162, Zhang et al., ACS Nano. 20115:6962-6970, Cutler et al., J. Am. Chem. Soc.2012134:1376-1391, Young et al., Nano Lett.201212:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA.2012109:11975-80, Mirkin, Nanomedicine 20127:635-638 Zhang et al., J. Am. Chem. Soc.2012134:16488-1691, Weintraub, Nature 2013495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA.2013110(19): 7625-7630, Jensen et al., Sci. Transl. Med.5, 209ra152 (2013) and Mirkin, et al., Small, 10:186- 192. [00167] Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG). [00168] In general, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. [00169] In some cases, nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell have a diameter of from 25 nm to 200 nm. [00170] In some cases, nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell have a diameter of 100 nm or less. In some cases, nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell have a diameter of from 35 nm to 60 nm. [00171] Nanoparticles suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure. [00172] Semi-solid and soft nanoparticles are also suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. A prototype nanoparticle of semi-solid nature is the liposome. [00173] In some cases, an exosome is used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs. [00174] In some cases, a liposome is used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3- phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside. [00175] A stable nucleic-acid-lipid particle (SNALP) can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. The SNALP formulation may contain the lipids 3-N- [(methoxypoly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C- DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulting SNALP liposomes can be about 80-100 nm in size. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2- dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N- dimethyl)aminopropane (DLinDMA). [00176] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-KC2-DMA) can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3- bis(octadecyloxy) propyl-1-(methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04 (n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity. [00177] Lipids may be formulated with a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a system, or component thereof, of the present disclosure, using a spontaneous vesicle formation procedure. The component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12- 200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). [00178] A polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) may be delivered encapsulated in PLGA microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279. [00179] Supercharged proteins can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. [00180] Cell Penetrating Peptides (CPPs) can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. [00181] An implantable device can be used to deliver a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.). An implantable device suitable for use in delivering a polypeptide and/or a nucleic acid (e.g., one or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); e) a Cas9 guide RNA; f) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and g) an Acr polypeptide or an Acr fusion polypeptide of the present disclosure) to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.) can include a container (e.g., a reservoir, a matrix, etc.) that comprises the polypeptide or nucleic acid (or combinations). [00182] A suitable implantable device can comprise a polymeric substrate, such as a matrix for example, that is used as the device body, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging. An implantable delivery device can be advantageous in providing release locally and over a prolonged period, where the polypeptide and/or nucleic acid to be delivered is released directly to a target site, e.g., the extracellular matrix (ECM), the vasculature surrounding a tumor, a diseased tissue, etc. Suitable implantable delivery devices include devices suitable for use in delivering to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix. In some cases, a suitable implantable drug delivery device comprises degradable polymers, wherein the main release mechanism is bulk erosion. In some cases, a suitable implantable drug delivery device comprises non degradable, or slowly degraded polymers, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of different polymers with different release mechanisms may also optionally be used. The concentration gradient at the can be maintained effectively constant during a significant period of the total releasing period, and therefore the diffusion rate is effectively constant (termed "zero mode" diffusion). By the term "constant" it is meant a diffusion rate that is maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or may fluctuate, for example increasing and decreasing to a certain degree. The diffusion rate can be so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period. [00183] In some cases, the implantable delivery system is designed to shield a nucleic acid component from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject. [00184] The site for implantation of the device, or target site, can be selected for maximum therapeutic efficacy. For example, a delivery device can be implanted within or in the proximity of a tumor environment, or the blood supply associated with a tumor. The target location can be, e.g.: 1) the brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2) the spine, as in the case of amyotrophic lateral sclerosis (ALS); 3) uterine cervix; 4) active and chronic inflammatory joints; 5) dermis as in the case of psoriasis; 7) sympathetic and sensoric nervous sites for analgesic effect; 7) a bone; 8) a site of acute or chronic infection; 9) Intra vaginal; 10) Inner ear--auditory system, labyrinth of the inner ear, vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary, epicardiac; 13) urinary tract or bladder; 14) biliary system; 15) parenchymal tissue including and not limited to the kidney, liver, spleen; 16) lymph nodes; 17) salivary glands; 18) dental gums; 19) Intra-articular (into joints); 20) Intra-ocular; 21) Brain tissue; 22) Brain ventricles; 23) Cavities, including abdominal cavity (for example but without limitation, for ovary cancer); 24) Intra esophageal; and 25) Intra rectal; and 26) into the vasculature. SYSTEMS [00185] The present disclosure provides a system for controlling activity of a Cas9 polypeptide. A system of the present disclosure can comprise two or more of: a) an Acr polypeptide of the present disclosure, an Acr fusion polypeptide of the present disclosure, or a modified Acr polypeptide of the present disclosure; b) an RNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; c) a DNA comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; d) a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure or an Acr fusion polypeptide of the present disclosure; e) a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); f) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); g) a DNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); h) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); i) a Cas9 guide RNA; j) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 guide RNA; l) a recombinant expression vector comprising a nucleotide sequence encoding a constant region (tracRNA region) of a Cas9 guide RNA and an insertion site for inserting a nucleotide sequence encoding a crRNA portion of the Cas9 guide RNA; m) a recombinant expression vector comprising: i) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); n) a recombinant expression vector comprising: i) a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and ii) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above); and iii) a nucleotide sequence encoding a Cas9 guide RNA; o) a host cell of the present disclosure. [00186] In some cases, a system of the present disclosure comprises two or more of: a) recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure (where the recombinant expression vector may optionally include one or both of: i) a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); and ii) a nucleotide sequence encoding a Cas9 guide RNA); b) a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); c) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) an RNA comprising a nucleotide sequence encoding a Cas9 polypeptide (where the Cas9 polypeptide is one whose activity can be inhibited by the Acr polypeptide); d) a Cas9 guide RNA; e) a DNA comprising a nucleotide sequence encoding a Cas9 guide RNA; and f) an Acr polypeptide. [00187] In some cases, a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide. In some cases, a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA. In some cases, a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide. In some cases, a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA. In some cases, a system of the present disclosure comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure; b) a recombinant expression vector comprising a nucleotide sequence encoding a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA. In some cases, a system of the present disclosure comprises: a) a ribonucleoprotein comprising: i) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and ii) a Cas9 guide RNA; and b) an Acr polypeptide of the present disclosure. In some cases, a system of the present disclosure comprises: a) a ribonucleoprotein comprising: i) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and ii) a Cas9 guide RNA; and b) a recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. Acr polypeptides [00188] Acr polypeptides that are suitable for inclusion in a system of the present disclosure include: a) Acr polypeptides as described above (including truncated Acr polypeptides as described above); b) Acr fusion polypeptides as described above; and c) modified Acr polypeptides as described above. A system of the present disclosure can comprise an Acr polypeptide of the present disclosure, or a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. A system of the present disclosure can comprise an Acr fusion polypeptide of the present disclosure, or a nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding an Acr fusion polypeptide of the present disclosure. A system of the present disclosure can comprise a modified Acr polypeptide of the present disclosure. Cas9 polypeptides [00189] As noted above, in some cases, a system of the present disclosure includes a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by an Acr polypeptide of the present disclosure, where such Cas9 polypeptides are described above. In some cases, a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; and b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide. In some cases, a system of the present disclosure comprises: a) an Acr polypeptide of the present disclosure; b) a Cas9 polypeptide, where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide; and c) a Cas9 guide RNA. [00190] In some cases, a Cas9 polypeptide included in a system of the present disclosure comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence provided in FIG.5. [00191] A Staphylococcus aureus Cas9 (SaCas9) suitable for inclusion in a system of the present disclosure can comprise one of the following sets of amino acid substitutions: N419A/R654A, Y211A/R654A, Y211A/Y212A, Y211A/N230A, Y211A/R245A, Y212A/Y230A, Y212A/R245A, Y230A/R245A, W229A/R654A, Y211A/Y212A/Y230A, Y211A/Y212A/R245A, Y211A/Y212A/Y651A, Y211A/Y230A/R245A, Y211A/Y230A/Y651A, Y211A/R245A/Y651A, Y211A/R245A/R654A, Y211A/R245A/N419A, Y211A/N419A/R654A, Y212A/Y230A/R245A, Y212A/Y230A/Y651A, Y212A/R245A/Y651A, Y230A/R245A/Y651A, R245A/N419A/R654A, T392A/N419A/R654A, R245A/T392AN419A/R654A, Y211A/R245A/N419A/R654A, W229A/R245A/N419A/R654A, Y211A/R245A/T392A/N419A/R654A, and Y211A1W229A/R245A/N419A/R654A. [00192] A Staphylococcus aureus Cas9 (SaCas9) suitable for inclusion in a system of the present disclosure comprises one of the following amino acid substitutions or sets of amino acid substitutions: E782K; K929R; N968K; R1015H; E782K/N968K/R1015H (KKH variant); E782K/K929R/R1015H (KRH variant); or E782K/K929R/N968K/R1015H (KRKH variant). Cas9 guide RNA [00193] A nucleic acid molecule that binds to a Cas9 protein and targets the complex to a specific location within a target nucleic acid is referred to herein as a “Cas9 guide RNA.” [00194] A Cas9 guide RNA (can be said to include two segments, a first segment (referred to herein as a “targeting segment”); and a second segment (referred to herein as a “protein-binding segment”). By “segment” it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule. A segment can also refer to a region/section of a complex such that a segment may comprise regions of more than one molecule. [00195] The first segment (targeting segment) of a Cas9 guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.). The protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a Cas9 polypeptide. The protein-binding segment of a subject Cas9 guide RNA includes two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex). Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas9 guide RNA (the guide sequence of the Cas9 guide RNA) and the target nucleic acid. [00196] A Cas9 guide RNA and a Cas9 protein form a complex (e.g., bind via non-covalent interactions). The Cas9 guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The Cas9 protein of the complex provides the site-specific activity (e.g., cleavage activity or an activity provided by the Cas9 protein when the Cas9 protein is a Cas9 fusion polypeptide, i.e., has a fusion partner). In other words, the Cas9 protein is guided to a target nucleic acid sequence (e.g. a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle, an ssRNA, an ssDNA, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid; etc.) by virtue of its association with the Cas9 guide RNA. [00197] The “guide sequence” also referred to as the “targeting sequence” of a Cas9 guide RNA can be modified so that the Cas9 guide RNA can target a Cas9 protein to any desired sequence of any desired target nucleic acid, with the exception that the protospacer adjacent motif (PAM) sequence is taken into account. Thus, for example, a Cas9 guide RNA can have a targeting segment with a sequence (a guide sequence) that has complementarity with (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like. [00198] In some embodiments, a Cas9 guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual Cas9 guide RNA”, a “double- molecule Cas9 guide RNA”, or a “two-molecule Cas9 guide RNA” a “dual guide RNA”, or a “dgRNA.” In some embodiments, the activator and targeter are covalently linked to one another (e.g., via intervening nucleotides) and the guide RNA is referred to as a “single guide RNA”, a “Cas9 single guide RNA”, a “single-molecule Cas9 guide RNA,” or a “one-molecule Cas9 guide RNA”, or simply “sgRNA.” [00199] A Cas9 guide RNA comprises a crRNA-like (“CRISPR RNA” / “targeter” / “crRNA” / “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-acting CRISPR RNA” / “activator” / “tracrRNA”) molecule. A crRNA-like molecule (targeter) comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch (“duplex-forming segment”) of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. A corresponding tracrRNA-like molecule (activator / tracrRNA) comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide nucleic acid. In other words, a stretch of nucleotides of a crRNA-like molecule are complementary to and hybridize with a stretch of nucleotides of a tracrRNA-like molecule to form the dsRNA duplex of the protein- binding domain of the Cas9 guide RNA. As such, each targeter molecule can be said to have a corresponding activator molecule (which has a region that hybridizes with the targeter). The targeter molecule additionally provides the targeting segment. Thus, a targeter and an activator molecule (as a corresponding pair) hybridize to form a Cas9 guide RNA. The exact sequence of a given crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found. A subject dual Cas9 guide RNA can include any corresponding activator and targeter pair. [00200] The term “activator” or “activator RNA” is used herein to mean a tracrRNA-like molecule (tracrRNA: “trans-acting CRISPR RNA”) of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the “activator” and the “targeter” are linked together by, e.g., intervening nucleotides). Thus, for example, a Cas9 guide RNA (dgRNA or sgRNA) comprises an activator sequence (e.g., a tracrRNA sequence). A tracr molecule (a tracrRNA) is a naturally existing molecule that hybridizes with a CRISPR RNA molecule (a crRNA) to form a Cas9 dual guide RNA. The term “activator” is used herein to encompass naturally existing tracrRNAs, but also to encompass tracrRNAs with modifications (e.g., truncations, sequence variations, base modifications, backbone modifications, linkage modifications, etc.) where the activator retains at least one function of a tracrRNA (e.g., contributes to the dsRNA duplex to which Cas9 protein binds). In some cases, the activator provides one or more stem loops that can interact with Cas9 protein. An activator can be referred to as having a tracr sequence (tracrRNA sequence) and in some cases is a tracrRNA, but the term “activator” is not limited to naturally existing tracrRNAs. [00201] The term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: “CRISPR RNA”) of a Cas9 dual guide RNA (and therefore of a Cas9 single guide RNA when the “activator” and the “targeter” are linked together, e.g., by intervening nucleotides). Thus, for example, a Cas9 guide RNA (dgRNA or sgRNA) comprises a targeting segment (which includes nucleotides that hybridize with (are complementary to) a target nucleic acid, and a duplex-forming segment (e.g., a duplex forming segment of a crRNA, which can also be referred to as a crRNA repeat). Because the sequence of a targeting segment (the segment that hybridizes with a target sequence of a target nucleic acid) of a targeter is modified by a user to hybridize with a desired target nucleic acid, the sequence of a targeter will often be a non- naturally occurring sequence. However, the duplex-forming segment of a targeter (described in more detail below), which hybridizes with the duplex-forming segment of an activator, can include a naturally existing sequence (e.g., can include the sequence of a duplex-forming segment of a naturally existing crRNA, which can also be referred to as a crRNA repeat). Thus, the term targeter is used herein to distinguish from naturally occurring crRNAs, despite the fact that part of a targeter (e.g., the duplex-forming segment) often includes a naturally occurring sequence from a crRNA. However, the term “targeter” encompasses naturally occurring crRNAs. [00202] A Cas9 guide RNA can also be said to include 3 parts: (i) a targeting sequence (a nucleotide sequence that hybridizes with a sequence of the target nucleic acid); (ii) an activator sequence (as described above)(in some cases, referred to as a tracr sequence); and (iii) a sequence that hybridizes to at least a portion of the activator sequence to form a double stranded duplex. A targeter has (i) and (iii); while an activator has (ii). [00203] A Cas9 guide RNA (e.g. a dual guide RNA or a single guide RNA) can be comprised of any corresponding activator and targeter pair. In some cases, the duplex forming segments can be swapped between the activator and the targeter. In other words, in some cases, the targeter includes a sequence of nucleotides from a duplex forming segment of a tracrRNA (which sequence would normally be part of an activator) while the activator includes a sequence of nucleotides from a duplex forming segment of a crRNA (which sequence would normally be part of a targeter). [00204] As noted above, a targeter comprises both the targeting segment (single stranded) of the Cas9 guide RNA and a stretch (“duplex-forming segment”) of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. A corresponding tracrRNA-like molecule (activator) comprises a stretch of nucleotides (a duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the Cas9 guide RNA. In other words, a stretch of nucleotides of the targeter is complementary to and hybridizes with a stretch of nucleotides of the activator to form the dsRNA duplex of the protein- binding segment of a Cas9 guide RNA. As such, each targeter can be said to have a corresponding activator (which has a region that hybridizes with the targeter). The targeter molecule additionally provides the targeting segment. Thus, a targeter and an activator (as a corresponding pair) hybridize to form a Cas9 guide RNA. The particular sequence of a given naturally existing crRNA or tracrRNA molecule is characteristic of the species in which the RNA molecules are found. Examples of suitable activator and targeter are well known in the art. Targeting segment of a Cas9 guide RNA [00205] The first segment of a subject guide nucleic acid includes a guide sequence (i.e., a targeting sequence)(a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid). In other words, the targeting segment of a subject guide nucleic acid can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA)) in a sequence-specific manner via hybridization (i.e., base pairing). As such, the nucleotide sequence of the targeting segment may vary (depending on the target) and can determine the location within the target nucleic acid that the Cas9 guide RNA and the target nucleic acid will interact. The targeting segment of a Cas9 guide RNA can be modified (e.g., by genetic engineering)/designed to hybridize to any desired sequence (target site) within a target nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA). [00206] The targeting segment can have a length of 7 or more nucleotides (nt) (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more nucleotides). In some cases, the targeting segment can have a length of from 7 to 100 nucleotides (nt) (e.g., from 7 to 80 nt, from 7 to 60 nt, from 7 to 40 nt, from 7 to 30 nt, from 7 to 25 nt, from 7 to 22 nt, from 7 to 20 nt, from 7 to 18 nt, from 8 to 80 nt, from 8 to 60 nt, from 8 to 40 nt, from 8 to 30 nt, from 8 to 25 nt, from 8 to 22 nt, from 8 to 20 nt, from 8 to 18 nt, from 10 to 100 nt, from 10 to 80 nt, from 10 to 60 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 10 to 18 nt, from 12 to 100 nt, from 12 to 80 nt, from 12 to 60 nt, from 12 to 40 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12 to 20 nt, from 12 to 18 nt, from 14 to 100 nt, from 14 to 80 nt, from 14 to 60 nt, from 14 to 40 nt, from 14 to 30 nt, from 14 to 25 nt, from 14 to 22 nt, from 14 to 20 nt, from 14 to 18 nt, from 16 to 100 nt, from 16 to 80 nt, from 16 to 60 nt, from 16 to 40 nt, from 16 to 30 nt, from 16 to 25 nt, from 16 to 22 nt, from 16 to 20 nt, from 16 to 18 nt, from 18 to 100 nt, from 18 to 80 nt, from 18 to 60 nt, from 18 to 40 nt, from 18 to 30 nt, from 18 to 25 nt, from 18 to 22 nt, or from 18 to 20 nt). [00207] The nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid can have a length of 10 nt or more. For example, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid can have a length of 12 nt or more, 15 nt or more, 18 nt or more, 19 nt or more, or 20 nt or more. In some cases, the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 12 nt or more. In some cases, the nucleotide sequence (the targeting sequence) of the targeting segment that is complementary to a nucleotide sequence (target site) of the target nucleic acid has a length of 18 nt or more. [00208] For example, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid can have a length of from 10 to 100 nucleotides (nt) (e.g., from 10 to 90 nt, from 10 to 75 nt, from 10 to 60 nt, from 10 to 50 nt, from 10 to 35 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 22 nt, from 10 to 20 nt, from 12 to 100 nt, from 12 to 90 nt, from 12 to 75 nt, from 12 to 60 nt, from 12 to 50 nt, from 12 to 35 nt, from 12 to 30 nt, from 12 to 25 nt, from 12 to 22 nt, from 12 to 20 nt, from 15 to 100 nt, from 15 to 90 nt, from 15 to 75 nt, from 15 to 60 nt, from 15 to 50 nt, from 15 to 35 nt, from 15 to 30 nt, from 15 to 25 nt, from 15 to 22 nt, from 15 to 20 nt, from 17 to 100 nt, from 17 to 90 nt, from 17 to 75 nt, from 17 to 60 nt, from 17 to 50 nt, from 17 to 35 nt, from 17 to 30 nt, from 17 to 25 nt, from 17 to 22 nt, from 17 to 20 nt, from 18 to 100 nt, from 18 to 90 nt, from 18 to 75 nt, from 18 to 60 nt, from 18 to 50 nt, from 18 to 35 nt, from 18 to 30 nt, from 18 to 25 nt, from 18 to 22 nt, or from 18 to 20 nt). In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 30 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 15 nt to 25 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 30 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 25 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target sequence of the target nucleic acid has a length of from 18 nt to 22 nt. In some cases, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 20 nucleotides in length. In some cases, the targeting sequence of the targeting segment that is complementary to a target site of the target nucleic acid is 19 nucleotides in length. [00209] The percent complementarity between the targeting sequence (guide sequence) of the targeting segment and the target site of the target nucleic acid can be 60% or more (e.g., 65% or more, 70% or more, 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%). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5’-most nucleotides of the target site of the target nucleic acid. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more over about 20 contiguous nucleotides. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the fourteen contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the seven contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 20 nucleotides in length. [00210] In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5’- most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’- most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5’-most nucleotides of the target site of the target nucleic acid (which can be complementary to the 3’-most nucleotides of the targeting sequence of the Cas9 guide RNA). In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or more (e.g., e.g., 65% or more, 70% or more, 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%) over about 20 contiguous nucleotides. [00211] In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 7 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 7 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 8 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 8 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 9 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 9 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 10 contiguous 5’-most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 10 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 11 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 11 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 12 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 12 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 13 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 13 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 14 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 14 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 17 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 17 nucleotides in length. In some cases, the percent complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over the 18 contiguous 5’- most nucleotides of the target site of the target nucleic acid and as low as 0% or more over the remainder. In such a case, the targeting sequence can be considered to be 18 nucleotides in length. [00212] Examples of various Cas9 proteins and Cas9 guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art, for example, see Jinek et al., Science.2012 Aug 17;337(6096):816-21; Chylinski et al., RNA Biol.2013 May;10(5):726-37; Ma et al., Biomed Res Int.2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15644- 9; Jinek et al., Elife.2013;2:e00471; Pattanayak et al., Nat Biotechnol.2013 Sep;31(9):839-43; Qi et al, Cell.2013 Feb 28;152(5):1173-83; Wang et al., Cell.2013 May 9;153(4):910-8; Auer et al., Genome Res.2013 Oct 31; Chen et al., Nucleic Acids Res.2013 Nov 1;41(20):e19; Cheng et al., Cell Res.2013 Oct;23(10):1163-71; Cho et al., Genetics.2013 Nov;195(3):1177-80; DiCarlo et al., Nucleic Acids Res.2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods.2013 Oct;10(10):1028-34; Ebina et al., Sci Rep.2013;3:2510; Fujii et al., Nucleic Acids Res.2013 Nov 1;41(20):e187; Hu et al., Cell Res.2013 Nov;23(11):1322-5; Jiang et al., Nucleic Acids Res.2013 Nov 1;41(20):e188; Larson et al., Nat Protoc.2013 Nov;8(11):2180-96; Mali et al., Nat Methods.2013 Oct;10(10):957-63; Nakayama et al., Genesis.2013 Dec;51(12):835-43; Ran et al., Nat Protoc.2013 Nov;8(11):2281-308; Ran et al., Cell.2013 Sep 12;154(6):1380-9; Upadhyay et al., G3 (Bethesda).2013 Dec 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U S A.2013 Sep 24;110(39):15514-5; Xie et al., Mol Plant.2013 Oct 9; Yang et al., Cell.2013 Sep 12;154(6):1370-9; Briner et al., Mol Cell.2014 Oct 23;56(2):333-9; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; all of which are hereby incorporated by reference in their entirety. [00213] In some cases, a Cas9 guide RNA comprises has one or more modifications, e.g., a base modification, a backbone modification, etc. Suitable nucleic acid modifications include, but are not limited to: 2’O-methyl modified nucleotides, 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7-methylguanylate cap (m7G)). [00214] Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included. [00215] In some cases, a Cas9 guide RNA comprises a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO) mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable modifications include a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, C1, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2'-methoxyethoxy (2'-O-CH2 CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group. A further suitable modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2. [00216] A subject nucleic acid may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H- pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one). METHODS [00217] The present disclosure provides methods of inhibiting activity of a Cas9 polypeptide. The methods generally involve contacting the Cas9 polypeptide with: a) an Acr polypeptide of the present disclosure; b) an Acr fusion polypeptide of the present disclosure; or b) a modified Acr polypeptide of the present disclosure. In some cases, the contacting occurs in a living cell in vitro. In some cases, the contacting occurs in a living cell in vivo. In some cases, the contacting occurs outside of a cell in vivo (e.g., the contacting occurs in an extracellular fluid in vivo). For simplicity, unless stated otherwise, an “Acr polypeptide of the present disclosure” includes an unmodified Acr polypeptide, a variant Acr polypeptide (as described above), a truncated Acr polypeptide (as described above), an Acr fusion polypeptide of the present disclosure, and a modified Acr polypeptide of the present disclosure. [00218] A method of the present disclosure can inhibit binding and/or cleavage activity of a Cas9/guide RNA complex by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, compared to the binding and/or cleavage activity of the Cas9/guide RNA complex in the absence of the Acr polypeptide (i.e., where the Cas9/guide RNA complex is not contacted with the Acr polypeptide). [00219] In some cases, a method of the present disclosure comprises introducing into a cell (a “target cell”) a nucleic acid (e.g., a recombinant expression vector; an mRNA; and the like) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, where the cell comprises, at the time the Acr-encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide. In some cases, the Acr-encoding nucleotide sequence is integrated into the genome of the cell. In some cases, the Acr-encoding nucleotide sequence is extrachromosomal. [00220] In some cases, a method of the present disclosure comprises introducing into a cell (a “target cell”) a nucleic acid (e.g., a recombinant expression vector; an mRNA; and the like) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, where the cell does not comprise, at the time the Acr -encoding nucleic acid is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide. In some cases, the Acr-encoding nucleotide sequence is integrated into the genome of the cell. In some cases, the Acr-encoding nucleotide sequence is extrachromosomal. [00221] In some cases, a method of the present disclosure comprises introducing into a cell (a “target cell”) an Acr polypeptide of the present disclosure, where the cell comprises, at the time the Acr polypeptide is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide. [00222] In some cases, a method of the present disclosure comprises introducing into a cell (a “target cell”) an Acr polypeptide of the present disclosure, where the cell does not comprise, at the time the Acr polypeptide is introduced into the cell, a Cas9 polypeptide (where the Cas9 polypeptide is one that can be inhibited by the Acr polypeptide) or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide. [00223] Where a method of the present disclosure comprises introducing into a cell a nucleic acid (e.g., a DNA; a recombinant expression vector; an RNA) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure, in some cases, the Acr-encoding nucleotide sequence is operably linked to one or more transcriptional control elements. In some cases, the one or more transcriptional control elements comprises a promoter, e.g., a promoter that is functional in a eukaryotic cell. In some cases, the promoter is a constitutive promoter. In some cases, the promoter is a regulated promoter, e.g., an inducible promoter. In some case, the inducible promoter is a drug-inducible promoter, and the method comprises contacting the cell with a drug that induces the drug-inducible promoter. [00224] In some cases, a method of the present disclosure provides for controlling gene drive. For example, where the gene drive limits viability of a target organism (or target population of an organism), a method of the present disclosure can restore viability to the target organism (or target population of an organism). Examples of target organisms (or target populations of an organism) include ticks (e.g., ticks that carry human pathogens), where ticks include ticks of the families Ixodidae and Argasidae, e.g., Ixodes ricinus, I. rubicundus, I. scapularis, I. holocyclus, and I. pacificus mites; mosquitoes (e.g., mosquitoes that carry human pathogens such as malaria parasites, Yellow Fever Virus, Dengue virus, Zika virus, Chikungunya virus, and the like), where examples of such mosquitoes include mosquitoes of the genera Culex, Culistea, Aedes, or Anopheles, e.g., Aedes aegypti, Aedes albopictus, and Anopheles gamiae; protozoans such as Plasmodium species (e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium berghei, Plasmodium gallinaceum, and Plasmodium knowlesi), nematode species, Trypanosoma species, Trichomonadidae species, Leishmania species, and the like; insects that are harmful to plants; arthropods that are harmful to plants; and the like. [00225] In some cases, a method of the present disclosure provides for reducing off-target Cas9/guide RNA-mediated gene editing. In some cases, a method of the present disclosure reduces off-target Cas9/guide RNA-mediated gene editing by at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, or more than 95%, compared to the extent of off-target Cas9/guide RNA-mediated gene editing when the Cas9 is not contacted with an Acr polypeptide of the present disclosure. [00226] In some cases, a method of the present disclosure provides protection against deleterious effects of a “hostile” Cas9/guide RNA. For example, an individual can comprise immune cells genetically modified to include an Acr-encoding nucleic acid; if such an individual comes into contact with a hostile Cas9/guide RNA complex that targets immune cells in a deleterious manner, such an individual can be protected from deleterious effects of such a hostile Cas9/guide RNA. [00227] In some cases, an Acr polypeptide of the present disclosure is used to deliver a Cas9 polypeptide to a cell, e.g., a eukaryotic cell. For example, in some cases, a complex of an Acr polypeptide and a Cas9 polypeptide is delivered to a cell. The complex may further include a Cas9 guide RNA and/or a donor template. Target nucleic acids and cells [00228] An Acr polypeptide of the present disclosure inhibits a Cas9 polypeptide (when the Cas9 polypeptide is complexed with a Cas9 guide RNA) from binding and/or cleaving a target nucleic acid. target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the Cas9 guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted). [00229] A target nucleic acid (a target of a Cas9/guide RNA complex) can be DNA or RNA. A target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded. [00230] A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo. Suitable target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuña, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). [00231] Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in vitro. Target cells can be unicellular organisms and/or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy. [00232] Plant cells include cells of a monocotyledon, and cells of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc. [00233] Additional examples of target cells are listed above in the section titled “Modified cells.” Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell). [00234] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be and in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell. [00235] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc. [00236] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, adult cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells. [00237] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). [00238] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells. [00239] Adult stem cells are resident in differentiated tissue, but retain the properties of self- renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like. [00240] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell. [00241] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A. [00242] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells. [00243] In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art. [00244] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No.5,736,396, which describes isolation of human MSC. [00245] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon. [00246] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese artichoke (crosnes), chinese cabbage, chinese celery, chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like. [00247] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a sub- order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera. [00248] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle. KITS [00249] The present disclosure provides a kit comprising an Acr polypeptide of the present disclosure or a nucleic acid (e.g., a recombinant expression vector) comprising a nucleotide sequence encoding an Acr polypeptide of the present disclosure. The present disclosure provides a kit comprising an Acr system of the present disclosure or a component of an Acr system of the present disclosure. [00250] A kit of the present can comprise: a) any combination of an Acr system, as described above; b) and one or more additional components and/or reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a positive and/or negative control target DNA; v) a positive and/or negative control Cas9 guide RNA; and the like. [00251] In some cases, a kit of the present disclosure comprises: a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and b) an Acr polypeptide that is an inhibitor of an activity of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% (at least 70%, at least 85%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, or a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide, wherein the enzymatic activity is nucleic acid cleavage. [00252] In some cases, a kit of the present disclosure comprises: [00253] a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to any one of the amino acid sequences depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and [00254] b) i) an Acr polypeptide that is an inhibitor of an activity of the Cas9 polypeptide; or ii) an Acr fusion polypeptide of the present disclosure; or iii) a modified Acr polypeptide of the present disclosure, wherein the Acr polypeptide (or the Acr polypeptide present in the fusion polypeptide, or the modified Acr polypeptide)) comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; or iv) a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide; or v) a nucleic acid comprising a nucleotide sequence encoding the Acr fusion polypeptide, wherein the enzymatic activity is nucleic acid cleavage. [00255] A kit of the present disclosure can also include a positive control and/or a negative control. For example, a suitable control for a protein that inhibits SpyCas9-mediated, but not SauCas9-mediated, cleavage of a target nucleic acid is AcrIIA4. A suitable control for a protein that inhibits both SpyCas9-mediated and SauCas9-mediated cleavage of a target nucleic acid is AcrIIA5. ACRANKER [00256] The present disclosure provides a method of training a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, the method comprising: a) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases; b) representing each of the one or more Acr proteins as a feature vector based on amino acid composition of the Acr protein; c) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity from one or more databases; d) representing each non-Acr protein as a feature vector based on amino acid composition of the protein; and e) training the machine learning model using the feature vectors representing one or more anti-CRISPR proteins and the feature vectors representing one or more non-Acr proteins. [00257] The present disclosure provides method of identifying one or more proteins based on their potential to inhibit CRISPR-Cas activity, the method comprising: a) using sequence information for one or more known anti-CRISPR (Acr) proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity; b) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins; and c) identifying one or more potential Acr proteins based on each such protein’s position in the ordered list. [00258] In practicing methods of the invention to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, aspects of the methods include: (1) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases, including, for example, from the Anti-CRISPRdb data base. In some instances, only non-redundant Acr proteins are used, meaning each Acr protein selected includes no more than a threshold level of sequence similarity with the other Acr proteins selected, for example a threshold between 20- 60% redundancy, such as a 40% threshold or a 20% threshold or a 60% threshold of redundancy. [00259] The collection of one or more known Acr proteins may comprise a positive class training set for the machine learning model; (2) representing each of the one or more Acr proteins as a feature vector based on the amino acid composition of the Acr protein. For example, the Acr proteins may be represented based on sequence features such as amino acid composition and grouped dimer or trimer frequency counts. Amino acids may be grouped into, for example, seven or more classes based on their physicochemical properties, and the frequency counts of all possible groups labeled as dimers and trimers in a given protein sequence may be used in conjunction with amino acid composition to represent the protein. Based on such grouping, a feature vector representation may be generated for each protein sequence; (3) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity (each such protein, a “non-anti-CRISPR” or “non-Acr”) from one or more databases. [00260] In some instances, non-anti-CRISPR proteins may be collected from the proteomes of the source species to which each of the known Acr proteins belongs. In some instances, only non-anti-CRISPR proteins are used that are not redundant with the one or more known Acr proteins, meaning each non-anti-CRISPR protein selected includes no more than a threshold level of sequence similarity with each of the Acr proteins, for example a threshold of between 20-60% redundancy, such as a 40% threshold or a 20% threshold or a 60% threshold of redundancy. The collection of one or more known non-anti-CRISPR proteins may comprise a negative class training set for the machine learning model; (4) representing each non-anti- CRISPR protein as a feature vector based on amino acid composition of the protein, as described above; and (5) training the machine learning model using the feature vectors representing one or more Acr proteins and the feature vectors representing one or more non-anti-CRISPR proteins. Training the machine learning model may entail, for example, employing a boosting technique to combine multiple weak classifiers to produce a strong classifier. The machine learning model may comprise a tree-based method of developing predictions, and further may apply pairwise ranking to potential anti-CRISPR proteins based on each protein’s expected anti-CRISPR behavior. [00261] In practicing methods of the invention to identify one or more proteins based on their potential to inhibit CRISPR-Cas activity aspects of the methods include: (1) using sequence information for one or more known Acr proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, including, for example, the method of training a machine learning model discussed above; (2) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins. In some instances, the plurality of proteins belongs to the same species. In some instances, each protein of the plurality of proteins is found within one or more prophage regions. The prophage regions may be identified using a database or a software tool, for example, PHASTER (PHAge Search Tool Enhanced Release); and (3) identifying one or more potential anti-CRISPR proteins based on each such protein’s position in the ordered list, for example identifying the potential Acr proteins ranked above a threshold position on the ranked list of anti-CRISPR proteins, such as those proteins within the top 10th percentile or within the top 5th percentile. In some instances, identifying one or more potential Acr proteins further comprises inspecting whether each potential anti-CRISPR protein exhibits specific features, including being a strong promoter, having a strong ribosome binding site or having an intrinsic terminator, for example by visually inspecting sequence information. Examples of Non-Limiting Aspects of the Disclosure [00262] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-69 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: [00263] Aspect 1. A fusion polypeptide comprising: a) anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a heterologous fusion partner. [00264] Aspect 2. The fusion polypeptide of aspect 1, wherein the heterologous fusion partner is a nuclear localization sequence. [00265] Aspect 3. The fusion polypeptide of aspect 1, wherein the heterologous fusion partner is an epitope tag. [00266] Aspect 4. The fusion polypeptide of any one of aspects 1-3, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence set forth in FIG.20A-20B; and wherein the Acr polypeptide has a length of from 30 amino acids to 64 amino acids. [00267] Aspect 5. The fusion polypeptide of any one of aspects 1-3, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence set forth in FIG.20A-20B; and wherein the Acr polypeptide has a length of from 50 amino acids to 114 amino acids. [00268] Aspect 6. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of aspects 1-5. [00269] Aspect 7. The nucleic acid of aspect 6, wherein the nucleotide sequence is operably linked to a promoter. [00270] Aspect 8. A recombinant expression vector comprising the nucleic acid of aspect 6 or aspect 7. [00271] Aspect 9. A cell comprising the nucleic acid of aspect 6 or aspect 7, or the recombinant expression vector of aspect 8. [00272] Aspect 10. The cell of aspect 9, wherein the cell is a eukaryotic cell. [00273] Aspect 11. The cell of aspect 9 or aspect 10, wherein the cell is in vitro. [00274] Aspect 12. The cell of aspect 9 or aspect 10, wherein the cell is in vivo. [00275] Aspect 13. A modified anti-CRISPR (Acr) polypeptide comprising: a) an Acr polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a non-peptidic moiety covalently linked to the Acr polypeptide. [00276] Aspect 14. The modified Acr polypeptide of aspect 13, wherein the non-peptidic moiety provides for one or more of an increase in in vivo half-life, in vivo stability, and bioavailability of the Acr polypeptide, compared to the unmodified Acr polypeptide. [00277] Aspect 15. The modified Acr polypeptide of aspect 13 or aspect 14, wherein the non- peptidic moiety comprises poly(ethylene glycol). [00278] Aspect 16. A recombinant expression vector comprising a nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1- 10. [00279] Aspect 17. The recombinant expression vector of aspect 16, wherein the Acr polypeptide-encoding nucleotide sequence is operably linked to a promoter. [00280] Aspect 18. The recombinant expression vector of aspect 17, wherein the promoter is functional in a eukaryotic cell. [00281] Aspect 19. The recombinant expression vector of aspect 17 or aspect 18, wherein the promoter is a regulated promoter. [00282] Aspect 20. The recombinant expression vector of aspect 19, wherein the regulated promoter is an inducible promoter. [00283] Aspect 21. The recombinant expression vector of aspect 20, wherein the inducible promoter is a heat-inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone-inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter. [00284] Aspect 22. The recombinant expression vector of aspect 17, wherein the promoter is a tissue-specific promoter or a cell type-specific promoter. [00285] Aspect 23. The recombinant expression vector of any one of aspects 16-22, further comprising a nucleotide sequence encoding a guide RNA that binds to and activates a Cas9 polypeptide. [00286] Aspect 24. The recombinant expression vector of any one of aspects 15-22, wherein the recombinant expression vector is a recombinant viral vector. [00287] Aspect 25. A cell comprising the recombinant expression vector of any one of aspects 16-24. [00288] Aspect 26. The cell of aspect 25, wherein the cell is in vitro. [00289] Aspect 27. The cell of aspect 25, wherein the cell is in vivo. [00290] Aspect 28. The cell of any one of aspects 24-27, wherein the cell is a eukaryotic cell. [00291] Aspect 29. The cell of aspect 28, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell. [00292] Aspect 30. A nucleic acid comprising: [00293] a) a first nucleotide sequence encoding the constant region of a guide RNA; [00294] b) a second nucleotide sequence encoding a Cas9 polypeptide; and [00295] c) a third nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10. [00296] Aspect 31. The nucleic acid of aspect 30, comprising an insertion site for inserting a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the insertion site is 5’ of and immediately adjacent to first nucleotide sequence. [00297] Aspect 32. The nucleic acid of aspect 30, comprising a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the guide sequence-encoding nucleotide sequence is 5’ of and immediately adjacent to first nucleotide sequence. [00298] Aspect 33. The nucleic acid of any one of aspects 30-32, wherein the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to any one of the amino acid sequences depicted in FIG.21A-21D, optionally wherein: [00299] a) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; [00300] b) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; [00301] c) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; [00302] d) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or [00303] e) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B. [00304] Aspect 34. The nucleic acid of any one of aspects 30-33, wherein the third nucleotide sequence is operably linked to a promoter. [00305] Aspect 35. The nucleic acid of aspect 34, wherein the promoter is functional in a eukaryotic cell. [00306] Aspect 36. The nucleic acid of aspect 33 or aspect 34, wherein the promoter is an inducible promoter. [00307] Aspect 37. The nucleic acid of aspect 36, wherein the inducible promoter is a heat- inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone- inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter. [00308] Aspect 38. The nucleic acid of aspect 34 or 35, wherein the promoter is a tissue-specific promoter or a cell type-specific promoter. [00309] Aspect 39. A recombinant expression vector comprising the nucleic acid of any one of aspects 30-38. [00310] Aspect 40. A cell comprising the nucleic acid of any one of aspects 30-38 or the recombinant expression vector of aspect 39. [00311] Aspect 41. The cell of aspect 40, wherein the cell is in vitro. [00312] Aspect 42. The cell of aspect 40, wherein the cell is in vivo. [00313] Aspect 43. The cell of any one of aspects 40-42, wherein the cell is a eukaryotic cell. [00314] Aspect 44. The cell of aspect 43, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell. [00315] Aspect 45. A nucleic acid comprising a nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, and wherein the nucleotide sequence is optimized for expression in a mammalian cell. [00316] Aspect 46. The nucleic acid of aspect 45, wherein the nucleotide sequence is operably linked to a promoter. [00317] Aspect 47. A recombinant expression vector comprising the nucleic acid of aspect 45 or aspect 46. [00318] Aspect 48. A kit comprising: [00319] a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to a Cas9 amino acid sequence depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and [00320] b) an anti-CRISPR (Acr) polypeptide that is an inhibitor of an activity of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1- 10, or a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide, wherein the enzymatic activity is nucleic acid cleavage, optionally wherein: [00321] i) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; [00322] ii) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; [00323] iii) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; [00324] iv) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or [00325] v) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG. 21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B. [00326] Aspect 49. The kit of aspect 48, wherein component (a) and component (b) are in separate containers. [00327] Aspect 50. A method for inhibiting an activity of a Cas9 polypeptide, the method comprising contacting the Cas9 polypeptide with: a) an anti-CRISPR (Acr) polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; or b) the Acr fusion polypeptide of any one of aspects 1-5. [00328] Aspect 51. The method of aspect 50, wherein the Cas9 polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence depicted in FIG.21A-21D. [00329] Aspect 52. The method of aspect 50 or 51, wherein said contacting occurs in a living cell in vitro. [00330] Aspect 53. The method of aspect 50 or 51, wherein said contacting occurs in a living cell in vivo. [00331] Aspect 54. The method of aspect 52 or aspect 53, comprising introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide. [00332] Aspect 55. The method of any one of aspects 52-54, wherein the nucleotide sequence encoding the Acr polypeptide is operably linked to an inducible promoter. [00333] Aspect 56. The method of any one of aspects 52-55, wherein the cell is a eukaryotic cell. [00334] Aspect 57. The method of aspect 56, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell. [00335] Aspect 58. The method of aspect 55, wherein the inducible promoter is a drug-inducible promoter, and wherein the method comprises contacting the cell with a drug that induces the drug-inducible promoter. [00336] Aspect 59. The method of any one of aspects 52-55, wherein the cell is a prokaryotic cell. [00337] Aspect 60. A method of training a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, the method comprising: a) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases; b) representing each of the one or more Acr proteins as a feature vector based on amino acid composition of the Acr protein; c) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity from one or more databases; d) representing each non-Acr protein as a feature vector based on amino acid composition of the protein; and e) training the machine learning model using the feature vectors representing one or more anti-CRISPR proteins and the feature vectors representing one or more non-Acr proteins. [00338] Aspect 61. The method of aspect 60, wherein collecting sequence information for one or more Acr proteins comprises collecting one or more non-redundant Acr proteins. [00339] Aspect 62. The method of aspect 61, wherein collecting sequence information for one or more non-Acr proteins comprises selecting one or more non-Acr proteins that are not redundant with the one or more Acr proteins. [00340] Aspect 63. The method of aspect 62, wherein collecting sequence information for one or more non-Acr proteins further comprises selecting one or more non-Acr proteins that belong to the same species as one or more of the Acr proteins. [00341] Aspect 64. The method of aspect 60, wherein the machine learning model is a tree-based model. [00342] Aspect 65. The method of aspect 64, wherein the machine learning model utilizes pairwise ranking. [00343] Aspect 66. A method of identifying one or more proteins based on their potential to inhibit CRISPR-Cas activity, the method comprising: a) using sequence information for one or more known anti-CRISPR (Acr) proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity; b) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins; and c) identifying one or more potential Acr proteins based on each such protein’s position in the ordered list. [00344] Aspect 67. The method of aspect 66, wherein the plurality of proteins belongs to the same species. [00345] Aspect 68. The method of aspect 67, wherein each of the plurality of proteins is found within one or more prophage regions. [00346] Aspect 69. The method of aspect 68, wherein identifying one or more potential anti- CRISPR proteins further comprises inspecting whether each potential anti-CRISPR protein exhibits specific features. EXAMPLES [00347] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. Example 1 MATERIALS AND METHODS Data collection and preprocessing [00348] To model the task of anti-CRISPR protein identification as a machine learning problem, a dataset consisting of examples from both positive (anti-CRISPR) and negative (non-anti- CRISPR) classes was needed. Anti-CRISPR information for proteins from Anti-CRISPRdb was collected (Dong et al.2018). The database contained information for 432 anti-CRISPR proteins. CD-HIT was used to identify a non-redundant set (at 40% sequence similarity threshold) of 2F0 experimentally verified Acrs (FIG.1; providing Table 1) (Huang et al.2010). FIG.1 contains a list of Acrs used for training and cross-validation of the Acranker model. These proteins belong to different Acr classes: 12 of the proteins are active against class I-F CRISPR Cas systems, 4 against I-E and 4 against II-A (Bondy-Denomy et al.2013; Pawluk et al.2016; Hynes et al. 2017; Pawluk et al.2014; Rauch et al.2017). This set constitutes positive class of the dataset. Complete proteomes of source species to which each of these proteins belong were downloaded. Proteins in these proteomes with <40% sequence similarity with the set of known anti-CRISPR proteins were used to construct the negative dataset. For independent testing of the method, a dataset comprising recently found Acrs (Marino et al.2018) was used (FIG.2; providing Table 2). FIG.2 contains a list of Acrs used for independent testing of AcRanker. The proteins used for development of the machine learning model in AcRanker belonged to classes I-F, I-E, and II-A (Dong et al.2018), so only the Acrs that belonged to either of these classes were chosen. Source proteomes for all these proteins were downloaded. Feature Extraction [00349] In line with existing machine learning based protein function prediction techniques, sequence features were used (Saidi et al.2010) based on amino acid composition and grouped dimer and trimer frequency counts (Shen et al.2007). For this purpose, amino acids were first grouped into seven classes based on their physicochemical properties (Shen et al.2007) (FIG.3; providing Table 3) and the frequency counts of all possible groups labeled as dimers and trimers in a given protein sequence were used in conjunction with amino acid composition. All three types of features (amino acid composition, di- and tri- meric frequency counts) were normalized to unit norm resulting in a -dimensional feature vector representation for a given protein sequence (Leslie et al.2001; Ben-Hur and Weston 2010). [00350] FIG.3 (Table 3). Grouping of amino acids based on physiochemical properties. Groups of amino acids with similar side chains are grouped together to reduce the number of features to test in the machine learning model. Machine learning model [00351] The underlying machine learning model for AcRanker has been built using EXtreme Gradient Boosting (XGBoost) (Chen and Guestrin 2016). In machine learning, boosting is a technique in which multiple weak classifiers are combined to produce a strong classifier (Chen and Guestrin 2016). XGBoost is a tree based method (Chen and Guestrin 2016) that uses boosting in an end-to-end fashion, i.e., every next tree tries to minimize the error produced by its predecessor. XGBoost has been shown to be a fast and scalable learning algorithm and has been widely used in many machine learning applications. [00352] In this work, XGBoost has been used as a pairwise ranking model to rank constituent proteins in a given proteome in descending order of their expected Acr behavior. The XGBoost model is trained in a species-specific manner to produce higher scores for anti-CRISPR proteins as compared to non-anti-CRISPR proteins in a given proteome. In comparison to conventional XGBoost classification, the pairwise ranking model performed better in terms of correctly identifying known anti-CRISPR proteins in test proteomes in cross-validation (comparison not shown for brevity). Specifically, given a set of training proteomes each with one or more known anti-CRISPR proteins, the objective is to obtain an XGBoost predictor f (x;θ) with learnable parameters θ that generates a prediction score for a given protein sequence represented in terms of its feature vector . In training, it is required that the model to learn optimal parameters θ uch that the score f (ρ;θ) for a positive example ρ (known Anti-CRISPR protein) should be higher than f (n;θ) all negative examples n (non-Anti-CRISPR proteins) within the same species. The hyperparameters of the learning model are selected through cross validation and optimal results are obtained with: number of estimators set at 120, learning rate of 0.1, subsampling of 0.6 and maximum tree depth of 3. Performance Evaluation [00353] To evaluate the performance of the machine learning model, leave-one-proteome-out cross-validation, as well as validation over an independent test set, was performed. In a single fold of leave-one-proteome-out cross-validation, the source proteome of a given anti-CRISPR protein was set aside for testing and train on all other proteomes. To ensure an unbiased evaluation, all sequences in the training set with a sequence identity of 40% or higher with any test protein or among themselves were removed from the training set. Furthermore, all proteins in the test set with more than 40% sequence identity with known anti-CRISPR proteins in the training set were also removed. This ensures that there is only one known anti-CRISPR protein in the test set in a single fold. The XGBoost ranking model was then trained and the prediction scores for all proteins in the test set was computed. Ideally, the known anti-CRISPR protein in the proteome should score the highest across all proteins in the given test proteome. This process was then repeated for all proteomes in the dataset. The rank of the known anti-CRISPR protein in its source proteome was used as a performance metric. [00354] In bacteria, Acrs are usually located within prophage regions (Rauch et al.2017; Koonin and Makarova 2018). Based on this premise, in another experiment for model evaluation, only the proteins found within prophage regions were passed to the model. To identify the prophage regions for a given bacterial proteome, PHASTER (PHAge Search Tool Enhanced Release) web server was used, (Arndt et al.2016) which accepts a bacterial genome and annotates prophage regions in it. The decision scores were computed for all phage proteins identified by PHASTER in the test proteome. [00355] As a baseline for comparison in leave-one-out cross-validation, BLAST (Basic Local Alignment Search Tool) (Baulcombe et al.1998) similarity was used. For each protein in a given test proteome, BLASTp scores were computed with the set of known Acrs and rank proteins in the increasing order of the respective e-values. [00356] For independent validation, the ranking based XGBoost model trained over sequence features for all 20 source proteomes (FIG.1, providing Table 1) has been tested for recently discovered Acrs (FIG.2, providing Table 2) by Marino et al. (Marino et al.2018) which are not part of the training set. The rank of known Acr in its corresponding proteome was computed. Here again, the model for both complete proteomes and respective MGE subset identified by PHASTER was evaluated. AcRanker Webserver [00357] A webserver implementation of AcRanker is publicly available at http://acranker(dot)pythonanywhere(dot)com/. The webserver accepts a proteome file in FASTA format and returns a ranked list of proteins. The Python code for the webserver implementation is available at https://github(dot)com/amina01/AcRanker. Acr candidate selection [00358] Self-targeting Spacer Searcher (STSS; https://github(dot)com/kew222/Self-Targeting- Spacer-Searcher) (Watters et al.2018) was run with default parameters using ‘Streptococcus’ as a search term for the NCBI genomes database, which returned a list of all self-targets found in those genomes. Whether known Acr genes were present in each of the self-targeting genomes was checked using a simple blastp search using default parameters with the Acr proteins stored within STSS. Twenty self-targeting genomes that contained at least one self-target with a 3′- NRG PAM were chosen for further analysis with AcRanker. Prophage regions were predicted using PHASTER (Arndt et al.2016). Proteins within the prophage regions were ranked with AcRanker. [00359] To select individual gene candidates for synthesis and biochemical validation, the six highest ranked proteins from each genome were examined by visual inspection for a strong promoter, a strong ribosome binding site, and an intrinsic terminator. Promoters were searched manually by looking for sequences closely matching the strong consensus promoter sequence TTGACA-17(+/-1)N-TATAAT upstream of the Acr candidate gene, or any genes immediately preceding it. The presence of a strong ribosome binding site (resembling AGGAGG) near the start codon was similarly searched for, and was required to be upstream of a gene candidate for selection. Last, given the nature of Acrs to be clustered together, genes neighboring the best candidates were also selected for further testing/validation. Protein expression and purification [00360] Each of the Acr candidates was cloned into a custom vector (pET-based expression vector) such that each protein was N-terminally tagged with a 10xHis sequence, superfolder GFP, and a tobacco etch virus protease cleavage site. Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9) and Streptococcus iniae Cas9 (SinCas9) were expressed as N-terminal MBP fusions. Proteins were produced and purified as previously described (Knott et al.2019). Briefly, E. coli Rosetta2 (DE3) containing Acr or Cas9 expression plasmids were grown in Terrific Broth (100 µg/mL ampicillin) to an OD600 of 0.6-0.8, cooled on ice, induced with 0.5 mM isopropyl-b-D-thiogalactoside and incubated at 16°C for 16 h. Cells were harvested by centrifugation, resuspended in wash buffer (20 mM Tris-C1 (pH 7.5), 500 mM NaC1, 1 mM TCEP (tris(2-carboxyethyl)phosphine), 5% (v/v) glycerol) supplemented with 0.5 mM PMSF (phenylmethanesulfonyl fluoride) and cOmplete protease inhibitor (Roche), lysed by sonication, clarified by centrifugation and purified over Ni-NTA Superflow resin (Qiagen) in wash buffer supplemented with 10 mM (wash) or 300 mM imidazole (elution). Elution fractions were pooled and digested overnight with recombinantly expressed TEV protease while dialysed against dialysis buffer (20 mM Tris-C1 (pH 7.5), 125 mM NaC1, 1 mM TCEP, 5% (v/v) glycerol) at 4°C. The cleaved proteins were loaded onto an MBP-Trap (GE Healthcare) upstream of a Heparin Hi- Trap (GE Healthcare) in the case of SpyCas9, SauCas9 and SinCas9. Depending on the pI, digested Acrs were loaded onto a Q (ML1, ML2, ML3, ML6, ML8 and ML10), heparin (ML4, ML5), or SP (ML7 and ML9) Hi-Trap column. Proteins were eluted over a salt gradient (20 mM Tris-Cl (pH 7.5), 1 mM TCEP, 5% (v/v) glycerol, 125 mM – 1 M KCl). The eluted proteins were concentrated and loaded onto a Superdex S200 Increase 10/300 (GE Healthcare) for SpyCas9, SauCas9, SinCas9 and Superdex S75 Increase 10/300 (GE Healthcare) for all the Acr candidates. Columns were pre-equilibrated with gel filtration buffer (20 mM HEPES-K (pH 7.5), 200 mM KCl, 1 mM TCEP and 5% (v/v) glycerol). Purified proteins were concentrated to approximately 50 µM for Cas9 effectors and 100 µM for Acr candidates. The absorbance at 280 nm was measured by Nanodrop and the concentration was determined using an extinction coefficient estimated based on the primary amino acid sequence of each protein. Proteins were then snap- frozen in liquid nitrogen for storage at -80 °C. Purity and integrity of proteins was assessed by 4- 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 4A; Coomassie blue staining) and LC-MS (FIG.4B). [00361] FIG.4A-4B. Purified Acr candidates and Cas effectors used in this study. (A) 4-20% gradient SDS-PAGE of purified machine learning Acr candidates and Cas effectors used in this study. (B) Mass spectra of all purified Acr candidates used. (* 23,510 Da, unknown protein). RNA preparation [00362] All RNAs were transcribed in vitro using recombinant T7 RNA polymerase and purified by gel extraction as described previously (East-Seletsky et al.2016). Briefly, 100 µg/mL T7 polymerase, 1 µg/mL pyrophosphatase (Roche), 800 units RNase inhibitor, 5 mM ATP, 5 mM CTP, 5 mM GTP, 5 mM UTP, 10 mM dithiothreitol (DTT), were incubated with DNA target in transcription buffer (30 mM Tris-C1 pH 8.1, 25 mM MgCl2, 0.01% Triton X-100, 2 mM spermidine) and incubated overnight at 37°C. The reaction was quenched by adding 5 units RNase-free DNase (Promega). Transcription reactions were purified by 12.5% (v/v) urea- denaturing PAGE (0.5x Tris-borate-EDTA (TBE)) and ethanol precipitation. In vitro cleavage assay [00363] In vitro cleavage assays were performed at 37°C in 1X cleavage buffer (20 mM Tris- HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol (v/v)) targeting a PCR amplified fragment of double-stranded DNA (FIG.23A-23B; Table 13). For all cleavage reactions, the sgRNA was first incubated at 95°C for 5 min and cooled down to room temperature. The Cas effectors (SpyCas9, SauCas9, AsCas12a at 100 nM and SinCas9 at 200 nM respectively) were incubated with each candidate Acr protein at 37°C for 10 min before the addition of sgRNA (SpyCas9, SauCas9, AsCas12a sgRNA at 160 nM and SinCas9 sgRNA at 320 nM) to form the RNP at 37°C for 10 min. The DNA cleavage reaction was then initiated with the addition of DNA target and reactions incubated for 30 min at 37°C before quenching in 1X quench buffer (5% glycerol, 0.2% SDS, 50 mM EDTA). Samples were then directly loaded to a 1% (w/v) agarose gel stained with SYBRGold (ThermoFisher) and imaged with a BioRad ChemiDoc. Competition binding experiment [00364] The reconstitution of the SinCas9-sgRNA-AcrIIA16 and SinCas9-sgRNA-AcrIIA2 complex was carried out as previously described (Jiang et al.2019). Briefly, purified SinCas9 and in vitro transcribed sgRNA were incubated in a 1:1.6 molar ratio at 37°C for 10 min.10 molar excess of AcrIIA16 respectively AcrIIA2 were added and incubated with the preformed RNP complex at 37°C for 10 min. For the competition binding experiment, 10 molar excess of AcrIIA16 was incubated with the RNP complex at 37°C before incubation with 10 molar excess of AcrIIA2 at 37°C for 10 min. The reconstituted ternary complex was further purified by analytical size-exclusion chromatography (Superdex S200 Increase 10//300 GL column, GE Healthcare) pre-equilibrated with the gel filtration buffer (20 mM HEPES-K (pH 7.5), 200 mM KCl, 1 mM TCEP and 5% (v/v) glycerol) containing 1 mM MgCl2. The relevant fractions containing the ternary complex as well as the Acrs and SinCas9 were further concentrated by a spin concentrator (3-kDa cutoff).2X SDS-Loading dye (4% w/v SDS, 0.2% w/v bromophenol blue and 20% v/v glycerol) was added and the samples were boiled down to 20 µl before loading onto a 4-20% gradient SDS-PAGE. Mass spectrometry [00365] Protein samples were analyzed using a Synapt mass spectrometer as described elsewhere (Light et al.2018). Primers, Acr candidates, CRISPR/Cas effectors, and sgRNAs [00366] FIG.19A-19C present Table 9, which provides primers used for amplification of DNA targets, cloning, and in vitro transcription (IVT) of sgRNAs. [00367] FIG.20A-20B present Table 10, which provides amino acid sequences and accession numbers of Acr candidates. [00368] FIG.21A-21D present Table 11, which provides amino acid sequences of CRISPR/Cas effector polypeptides used in this study. [00369] FIG.22 presents Table 12, which provides nucleotide sequences of sgRNAs used for in vitro cleavage assays. [00370] FIG.23A-23B present Table 13, which provides nucleotide sequences of DNA targets used for in vitro cleavage assays. Target sequences are shown in italics (GATGGTAAGCCCTCCCGTAT; SEQ ID NO:46)) (Streptococcus pyogenes, Streptococcus iniae); bold and italics (ACGTTTTCCAATGATGAGCACTTT; SEQ ID NO:47) (Acidaminococcus sp.) or bold (TATCGTAGTTATCTACACG; SEQ ID NO:48) (Staphylococcus aureus). RESULTS Cross-validation by single proteome omission [00371] In this work, a machine learning model, AcRanker, was developed, that accepts a proteome as input and ranks its constituent proteins in decreasing order of their expected Acr character. EXtreme Gradient Boosting (XGBoost) based ranking (Chen and Guestrin 2016) has been used with 1, 2 and 3-mer amino acid composition as input features (Saidi et al.2010) to train on a dataset comprised of 20 experimentally verified Acrs taken from the anti-CRISPRdb (Dong et al.2018) (FIG.1, providing Table 1) and their source proteomes. To evaluate the performance of AcRanker, leave-one-out cross-validation was performed as well as testing over an independent set of proteins. Out of the 20 known Acr proteomes tested individually, it was observed that the ranking-based model ranked seven Acrs higher than other proteins in their respective proteomes (FIG.5; providing Table 4). In total, 14 out of the 20 known Acrs are ranked within the top 5% in their respective proteomes. [00372] FIG.5 (Table 4). Results for leave-one-out cross-validation. Each each row of the table indicates which Acr was excluded from the training dataset and used as a test dataset, and each number displayed is the ranking of the known Acr received from the indicated test proteome using either the blastp search against all other known Acrs (BLAST) or AcRanker. The Acrs from bacterial proteomes - AcrIF6, AcrIF9, AcrIF10, AcrIIA1, AcrIIA2, and AcrIIA4 - were also ranked using only the subset of proteins predicted to reside within prophages as predicted by PHASTER(Arndt et al.2016). Two Acrs from bacterial proteomes did not occur in the predicted prophages and are indicated by dash placeholders. Prophage proteome subset fields have been left empty for Acrs from phage proteomes. [00373] Generally, it was observed that the machine learning rankings for Acrs contained in phage proteomes are much better than those contained in bacterial proteomes, likely due to their smaller size. To test if the relative rankings of the known Acrs found within bacterial proteomes would improve in the context of only prophage-derived proteins, which proteins in the bacterial proteomes were found within prophages were identified using PHASTER (Arndt et al.2016) and used only that subset to test both models. With the prophage subsets, a higher ranking for the known Acrs was observed due to the removal of higher-ranking proteins not found in the predicted prophages. [00374] As a baseline, the rankings obtained from the machine learning model were also compared to a blastp (Baulcombe et al.1998) comparison (FIG.5, presenting Table 4). For each excluded Acr in the leave-one-out train/test cycles, the excluded Acr’s proteome was used as a query set to BLAST against the 19 other Acrs used for training and the resulting e-values ranked from lowest to highest. The BLAST search method, however, only returned the highest rank for the AcrIF6 family, but only because three distant homologs (using the <40% identity threshold) were included in the training dataset. Interestingly, it was also observed that the BLAST method gave a higher rank than AcRanker for AcrIIA1, which contains a motif (helix-turn-helix) that is found in some other known Acrs (Rauch et al.2017; Osuna et al.2019; Hynes et al.2018; Watters et al.2019). The rankings of all other Acrs fell outside of the top 5%, demonstrating the diversity of Acr families and the difficulty of predicting new Acrs de novo. Independent set validation [00375] To validate AcRanker, an independent testing dataset of 10 recently discovered Acrs not a part of the training dataset was used (FIG.2, presenting Table 2) (Hwang and Maxwell 2019). Of these 10 Acrs, one is found in a phage (AcrIF14) and four (AcrIE4-F7, AcrIF11, AcrIF11.1, and AcrIF11.2) were predicted to be in a prophage region using PHASTER. For the proteins predicted to be in a prophage both the complete bacterial and phage proteome was ranked with AcRanker, otherwise only the complete proteome was ranked (FIG.6; providing Table 5). The results from the complete bacterial proteomes did not perform well (FIG.6; providing Table 5) with AcrIE5 and AcrIF12 receiving ranks within the top 10. As shown in FIG.6, 10 proteomes containing non-redundant (<40% sequence identity) Acrs from bacterial and phage sources were ranked using AcRanker. Bacterial proteomes that had Acrs within PHASTER-predicted prophages were also tested with a subset of the proteome containing only the prophage proteins. However, of the five proteins found within a phage/prophage, AcRanker ranked three within the top five, including one with the highest rank (FIG.7; providing Table 6). [00376] FIG.6 (Table 5). Independent testing set validation results. Ten proteomes containing non-redundant (<40% sequence identity) Acrs from bacterial and phage sources were ranked using AcRanker. Bacterial proteomes that had Acrs within PHASTER-predicted prophages were also tested with a subset of the proteome containing only the prophage proteins. [00377] FIG.7 (Table 6). Independent testing set validation results. Five proteomes containing non-redundant (<40% sequence identity) Acrs from bacterial proteomes that had Acrs within PHASTER-predicted prophages, as well as their AcRanker results. Anti-CRISPR candidate selection [00378] AcRanker was applied to predict novel anti-CRISPRs from self-targeting genomes. Given the ubiquity of Streptococcus pyogenes Cas9 (SpyCas9) in gene editing and the inclusion of known SpyCas9 Acrs in the machine learning training dataset (AcrIIA1, AcrIIA2, AcrIIA4, AcrIIA5), it was decided to focus specifically on Streptococcus species containing Cas9 proteins homologous to SpyCas9. [00379] A list of Streptococcus genomes was generated containing at least one self-targeting type II-A CRISPR system using Self-Target Spacer Searcher, which has been previously described (Watters et al.2018).385 instances of self-targeting were found from type II-A CRISPR arrays occurring within 241 Streptococcus genome assemblies, six of which contained known Acrs. Of these 241 self-targeting arrays, instances where the target sequence was flanked by the 3′ NRG protospacer adjacent motif (PAM) characteristic of SpyCas9 were sought and it was observed that it was present in 20 genomes. These 20 self-targeting arrays would be expected to be lethal for close homologs of SpyCas9, suggesting that other factors, such as the presence of Acrs (Watters et al.2018), are preventing CRISPR self-targeting and cell death (FIG.8A-8D; providing Table 7). During the original search of these 20 genomes, Streptococcus iniae strain UEL-Si1 was the only one that contained a previously discovered Acr, AcrIIA3 (Rauch et al. 2017), providing a large proteome space to search for novel acr genes. [00380] FIG.8A-8D (Table 7). List of expected lethal self-targeting Streptococcus genomes obtained with Self-Target Spacer Searcher (STSS). Searching Streptococcus assemblies from NCBI with STSS returned 385 cases of self-targeting derived from type II-A arrays representing 241 individual genomes. Of those genomes, 20 contained at least one spacer with the characteristic NRG 3′ PAM for SpyCas9, shown in Table 7. Only Streptococcus iniae strain UEL-Si1 contains a previously discovered anti-CRISPR (AcrIIA3). Also shown in Table 7 are the self-targeting spacers for Listeria monocytogenes strain R2-502, which was also ranked with AcRanker. [00381] To identify new acr gene candidates, PHASTER (Arndt et al.2016) was first used to predict all of the prophages residing within the 20 self-targeting Streptococcus genomes as well as an additional Listeria monocytogenes genome (strain R2-502) containing a type II-A self- targeting CRISPR system (with six self-targets) and three well-known AcrIIA genes (Rauch et al.2017). The Listeria strain was included to determine if the known Acrs within it were returned as the top ranked genes, and if not, test the higher ranking genes as potential additional Acrs within a known Acr-harboring strain. Lists were created of the annotated proteins found within each genome’s set of prophages. These protein lists were then ranked with AcRanker to predict the 10 highest ranked genes most likely to be an acr (FIG.9A-9E; providing Table 8). [00382] FIG.9A-9E (Table 8). Top Acr gene candidates within each genome ranked by AcRanker. The proteins found within the prophages of 20 Streptococcus genomes were ranked using AcRanker; up to the top 10 highest ranking genes are listed in ascending order. Known Acr genes and the 10 genes synthesized for biochemical testing are indicated in the rightmost column. Genomes with fewer than 10 listed have very few annotated proteins found within predicted prophages. [00383] Of the approximately 200 genes returned, a subset was selected as the most likely to be undiscovered acr genes for further biochemical testing, based on previous observations that many Acrs are: 1) encoded in operons along other acrs 2) typically short genes, and 3) often have transcripts driven by strong promoters and ribosome binding sites that frequently end with intrinsic terminator sequences (Watters et al.2018; Rauch et al.2017; Watters et al.2019) (FIG. 10). [00384] As with the previous testing dataset, it was observed that the known acr genes were highly ranked within the test proteomes. Interestingly, other proteins contained in the same, or overlapping, transcripts as the known Acrs ranked higher with AcRanker (ML1 and ML2). These candidates as well as nine others (ML3-ML10) containing the features described above, were selected (FIG.10). [00385] FIG.10. Acr candidates selected for biochemical testing. Ten Acr candidates were selected from manual inspection for further biochemical testing (blue). Each candidate is shown in its genomic context with its assigned rank from AcRanker noted in red. Homologous proteins share the same color border (green, blue). Homologs of AcrIIA3 (orange border) and AcrIIA1 (red border) are indicated. While testing the ML candidates, ML3 (yellow) has been identified as a specific inhibitor of LmoCas9 (Osuna et al.2019). Biochemical validation of novel Acrs identified by AcRanker [00386] To determine if the identified proteins were inhibitors of SpyCas9, each candidate was purified and their ability to directly inhibit DNA targeting in vitro was tested. Of the ten candidate inhibitors, nine were successfully cloned, expressed and purified (FIG.4A-4B). To assess inhibition of DNA targeting in vitro, the ability of SpyCas9 to cleave double stranded (ds) DNA when incubated in the presence of a 50-fold excess of each candidate Acr was assayed (FIG.11A). [00387] While SpyCas9 was capable of complete DNA target cleavage, the generation of DNA cleavage products was attenuated in the presence of the positive control inhibitor AcrIIA4 and the candidates ML1 or ML8. To determine the potency of inhibition, the ability of SpyCas9 to cleave the DNA target in the presence of a dilution series of ML1 or ML8 was tested (FIG.11B). [00388] In contrast to AcrIIA4, an established potent inhibitor of SpyCas9 (Rauch et al.2017), both ML1 and ML8 inhibited SpyCas9 with around a 10-fold lower potency. It was wondered if the high concentration of ML1 or ML8 required to completely inhibit Cas9 might represent an in vitro concentration-dependent artefact. To explore this, SpyCas9 DNA cleavage against a titration series of either non-target DNA competitor, BSA, ML2, or ML3 was assayed and no significant inhibition of SpyCas9 was observed, even with a 100-fold excess (FIG.12B-12D). Taken together, these data indicated that both ML1 and ML8 weakly inhibit SpyCas9 DNA cleavage in vitro. [00389] Next, the ability of the AcRanker-generated candidates to inhibit Staphylococcus aureus (SauCas9), another Cas9 commonly used for gene editing (Ran et al.2015; Yourik et al.2019) was tested to determine whether any of the candidates identified from self-targeting Streptococcus genomes had broader Cas9 inhibition activity. At a 25-fold excess relative to the SauCas9 RNP complex, ML3 and ML8 were able to inhibit SauCas9 dsDNA cleavage (FIG. 11C). To determine potency, a dilution series of either ML3 or ML8 with SauCas9 was incubated before the addition of the DNA target. However, in comparison to AcrIIA5, an established strong inhibitor of SauCas9 (Hynes et al.2017; Watters et al.2019), both Acr candidates inhibited SauCas9 with approximately 50-fold lower potency (FIG.11D and FIG. 13A-13B), an activity that was confirmed not to be due to a false positive from the high concentration of protein in the assay (FIG.14A-14B). [00390] Given the relatively weak inhibition of both SpyCas9 and SauCas9, specificity of each inhibitor was tested by assaying their ability to block DNA targeting by either AsCas12a or the restriction enzyme AlwNI. Neither AcrIIA4, ML1, ML3, nor ML8 were able to inhibit DNA targeting by AlwNI, consistent with them being anti-CRISPRs (FIG.14A-14B). Inhibition of AsCas12a was only observed with ML1 and ML8 at a 100-fold excess (FIG.14C). Taken together, the data is consistent with ML1, ML3, and ML8 being low potency inhibitors of SpyCas9 (ML1 and ML8) or SauCas9 (ML3 and ML8). Interestingly, while testing ML1-ML10 for Acr activity, Osuna, et al. described AcrIIA12, a specific inhibitor of LmoCas9 in plaque assays, which shares the same sequence as ML3 (Osuna et al.2019). [00391] FIG.11A-11D. Inhibition of SpyCas9 and SauCas9 by newly discovered Acr candidates. (A) In vitro cleavage assay using linear dsDNA template as a target. Presence of uncleaved DNA target indicates the ability of the Acr candidate to inhibit SpyCas9 at 50-fold excess. AcrllA4 is used as a positive control. (B) In vitro cleavage assay with an increasing concentration of AcrIIA4, ML1 and ML8 (Acr:RNP 0.1-, 1-, 2- ,10-, 50- and 100-fold excess from left to right) showing the ability of ML1 and ML8 to inhibit SpyCas9 at high concentrations. (C) In vitro cleavage assay used to test the inhibition of SauCas9 by Acr candidates. (D) Dilution series of AcrllA5 (Acr:RNP 0.1-, 1-, 2- ,4-, 8- and 10-fold excess from left to right, positive control), ML3 and ML8 (Acr:RNP 0.1-, 1-, 2- ,4-, 8- and 10-fold excess from left to right) showing the ability of MLAcr3 and MLAcr8 to inhibit SauCas9 at high concentrations. [00392] FIG.12A-12D. In vitro cleavage assay with SpyCas9. (A) AcrllA4 (positive control): The same controls and dilution series are used in all of the cleavage assays unless otherwise indicated. (B) Cleavage assay with DNA mimic and BSA as negative controls. (C) Representative gels of ML1, ML2, ML3 and ML8. (D) Quantified band intensities of the in vitro cleavage assays. Fraction of dsDNA cleaved (vertical axis) is plotted against the Acr to SpyCas9 RNP ratio (horizontal axis). AcrllA4, BSA, ML1 and ML8 were run in triplicates. [00393] FIG.13A-13B. In vitro cleavage assay with SauCas9. (A) In vitro cleavage assay with a dilution series of AcrllA5 (positive control) and BSA (negative control). (B) Dilution series of ML3 and ML8 showing their ability to inhibit SauCas9. [00394] FIG.14A-14C. Control experiments for the in vitro cleavage assay. (A) Cleavage assay with the restriction enzyme AlwN1 and AcrllA4, ML1, ML3 and ML8 showing that none of the Acrs is able to inhibit AlwN1. (B) Different dilution series with ML8 showing that even 10,000- fold excess of Acr does not lead to AlwN1 inhibition. (C) Cleavage assay with AsCas12a and AcrVA1 (positive control), ML1, ML3 and ML8. ML1: a potent inhibitor of SinCas9 [00395] ML1 was identified in the Streptococcus iniae (Sin) genome. Previous studies have reported anti-CRISPRs can exhibit either selective or broad-spectrum inhibition of divergent Cas effectors (Knott et al.2019; Harrington et al.2017). Given that SinCas9 is 70.10% identical to SpyCas9 and only 25.58% identical to SauCas9 it was wondered if ML1 might be a more potent inhibitor of SinCas9. To explore this, SinCas9 protein was cloned, expressed, and purified for use in in vitro DNA targeting assays. Like SpyCas9, SinCas9 was capable of cleaving dsDNA targets proximal to an NGG PAM using a sgRNA derived from a fusion of the tracrRNA and crRNA (FIG.15A, FIG.16A-16B). [00396] Similar to SpyCas9, both ML1 and ML8 inhibited DNA cleavage by SinCas9. Using a titration of ML1, the potency of SinCas9 inhibition was assayed again (FIG.15B). Strikingly, in contrast to the weak inhibition of SpyCas9, ML1 was able to potently inhibit DNA cleavage by SinCas9 (FIG.15B). [00397] To investigate at which step ML1 inactivates SinCas9 function, in vitro cleavage assays were carried out where ML1 was incubated with SinCas9 before and after the addition of sgRNA (FIG.17C). In both cases the DNA cleavage activity of SinCas9 was potently inhibited, suggesting that ML1 inhibits activity after sgRNA binding to Cas9. [00398] A number of reported AcrIIA inhibit their cognate Cas9 by competing with target DNA through PAM mimicry (Yang and Patel 2017; Jiang et al.2019). It was noted that SinCas9 was susceptible to inhibition by AcrIIA4 (FIG.18A) and AcrIIA2 (FIG.17D), both PAM mimics that inhibit PAM recognition by SpyCas9 (Shin et al.2017; Jiang et al.2019). Like these established PAM mimics, ML1 is a small protein with a predicted negatively charged surface potential (isoelectric point of 4.3), suggesting that it too might compete with negatively charged DNA. To explore this idea, a competition binding experiment was developed to assay if the association of ML1 with SinCas9 might prevent the binding of AcrIIA2 (FIG.18A). First, either AcrIIA2 or ML1 was incubated with the SinCas9-sgRNA complex and observed a stable SinCas9-sgRNA-Acr complex on a gel filtration column (FIG.18B) with the complex components all resolvable on a protein gel (FIG.18C). To determine if ML1 binding to the SinCas9 RNP could prevent AcrIIA2 binding, the SinCas9-sgRNA-ML1 complex was formed and then incubated with AcrIIA2 before resolving over a column. Incubating ML1 with the SinCas9 RNP before adding AcrIIA2 abolished AcrIIA2 co-elution with SinCas9-sgRNA (FIG. 18C), suggesting that ML1 might occupy the same site on SinCas9. Collectively, these data are consistent with a model where ML1 directly binds to the SinCas9-sgRNA complex at a site shared by AcrIIA2. [00399] FIG.15A-15B. ML1 and ML8 also inhibit SinCas9 with ML1 showing very high potency. (A) In vitro cleavage assay showing the inhibition of SinCas9 by ML1 and ML8. (B) Dilution series of ML1 shows potent inhibition even at sub stoichiometric ratio. [00400] FIG.16A-16B. SinCas9 works with a similar sgRNA as SpyCas9. (A) Schematic of the pre-crRNA:tracrRNA complex. (B) Schematic of the single-guide RNA.20-bp spacer sequence is shown in blue, tracrRNA is shown in red and the direct repeat sequence in grey. [00401] FIG.17A-17D. In vitro cleavage assay with SinCas9. (A) In vitro cleavage assay with a dilution series of ML1. (B) In vitro cleavage assay with a dilution series of ML8. (C) In vitro cleavage assay where MLAcr1 is incubated with SinCas9 before and after the incubation with sgRNA. (D) In vitro cleavage assay with AcrIIA2. [00402] FIG.18A-18E. ML1 competes with AcrIIA2 for the same binding site. (A) Flowchart for the competition binding experiment between ML1 and AcrIIA2. Binding of the Acr to the SinCas9-sgRNA RNP was reconstituted using size-exclusion chromatography (SEC). (B) Size- exclusion chromatogram of SinCas9-sgRNA in the presence of either ML1, AcrIIA2 or both Acrs with AcrIIA2 added after ML1. (C) Coomassie-stained polyacrylamide gel showing the co- purification of ML1 (I) respectively AcrIIA2 (II) with the sgRNA-bound SinCas9. 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Doudna.2018. “Systematic Discovery of Natural CRISPR-Cas12a Inhibitors.” Science 362 (6411): 236–39. [00431] Watters, Kyle E., Haridha Shivram, Christof Fellmann, Rachel J. Lew, Blake McMahon, and Jennifer A. Doudna.2019. “Potent CRISPR-Cas9 Inhibitors from Staphylococcus Genomes.” bioRxiv. https://doi.org/10.1101/799403. [00432] Yang, Hui, and Dinshaw J. Patel.2017. “Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.” Molecular Cell 67 (1): 117–27.e5. [00433] Yourik, Paul, Ryan T. Fuchs, Megumu Mabuchi, Jennifer L. Curcuru, and G. Brett Robb.2019. “Staphylococcus Aureus Cas9 Is a Multiple-Turnover Enzyme.” RNA 25 (1): 35– 44. [00434] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is: 1. A fusion polypeptide comprising: a) anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a heterologous fusion partner.
2. The fusion polypeptide of claim 1, wherein the heterologous fusion partner is a nuclear localization sequence.
3. The fusion polypeptide of claim 1, wherein the heterologous fusion partner is an epitope tag.
4. The fusion polypeptide of any one of claims 1-3, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence set forth in FIG.20A-20B; and wherein the Acr polypeptide has a length of from 30 amino acids to 64 amino acids.
5. The fusion polypeptide of any one of claims 1-3, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence set forth in FIG.20A-20B; and wherein the Acr polypeptide has a length of from 50 amino acids to 114 amino acids.
6. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 1-4.
7. The nucleic acid of claim 6, wherein the nucleotide sequence is operably linked to a promoter.
8. A recombinant expression vector comprising the nucleic acid of claim 6 or claim 7.
9. A cell comprising the nucleic acid of claim 6 or claim 7, or the recombinant expression vector of claim 8.
10. The cell of claim 9, wherein the cell is a eukaryotic cell.
11. The cell of claim 9 or claim 10, wherein the cell is in vitro.
12. The cell of claim 9 or claim 10, wherein the cell is in vivo.
13. A modified anti-CRISPR (Acr) polypeptide comprising: a) an Acr polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; and b) a non-peptidic moiety covalently linked to the Acr polypeptide.
14. The modified Acr polypeptide of claim 13, wherein the non-peptidic moiety provides for one or more of an increase in in vivo half-life, in vivo stability, and bioavailability of the Acr polypeptide, compared to the unmodified Acr polypeptide.
15. The modified Acr polypeptide of claim 13 or claim 14, wherein the non-peptidic moiety comprises poly(ethylene glycol).
16. A recombinant expression vector comprising a nucleotide sequence encoding an anti- CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10.
17. The recombinant expression vector of claim 16, wherein the Acr polypeptide-encoding nucleotide sequence is operably linked to a promoter.
18. The recombinant expression vector of claim 17, wherein the promoter is functional in a eukaryotic cell.
19. The recombinant expression vector of claim 17 or claim 18, wherein the promoter is a regulated promoter.
20. The recombinant expression vector of claim 19, wherein the regulated promoter is an inducible promoter.
21. The recombinant expression vector of claim 20, wherein the inducible promoter is a heat-inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone-inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter.
22. The recombinant expression vector of claim 17, wherein the promoter is a tissue-specific promoter or a cell type-specific promoter.
23. The recombinant expression vector of any one of claims 16-22, further comprising a nucleotide sequence encoding a guide RNA that binds to and activates a Cas9 polypeptide.
24. The recombinant expression vector of any one of claims 15-22, wherein the recombinant expression vector is a recombinant viral vector.
25. A cell comprising the recombinant expression vector of any one of claims 16-24.
26. The cell of claim 25, wherein the cell is in vitro.
27. The cell of claim 25, wherein the cell is in vivo.
28. The cell of any one of claims 24-27, wherein the cell is a eukaryotic cell.
29. The cell of claim 28, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
30. A nucleic acid comprising: a) a first nucleotide sequence encoding the constant region of a guide RNA; b) a second nucleotide sequence encoding a Cas9 polypeptide; and c) a third nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10.
31. The nucleic acid of claim 30, comprising an insertion site for inserting a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the insertion site is 5’ of and immediately adjacent to first nucleotide sequence.
32. The nucleic acid of claim 30, comprising a nucleotide sequence encoding a guide sequence of the guide RNA, wherein the guide sequence-encoding nucleotide sequence is 5’ of and immediately adjacent to first nucleotide sequence.
33. The nucleic acid of any one of claims 30-32, wherein the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to any one of the amino acid sequences depicted in FIG.21A-21D, optionally wherein: a) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence depicted in FIG.21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; b) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; c) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; d) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or e) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.21A-21D; and wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B.
34. The nucleic acid of any one of claims 30-33, wherein the third nucleotide sequence is operably linked to a promoter.
35. The nucleic acid of claim 34, wherein the promoter is functional in a eukaryotic cell.
36. The nucleic acid of claim 33 or claim 34, wherein the promoter is an inducible promoter.
37. The nucleic acid of claim 36, wherein the inducible promoter is a heat-inducible promoter, a drug-inducible promoter, an alcohol-inducible promoter, a hormone-inducible promoter, a steroid-inducible promoter, or a metal-inducible promoter.
38. The nucleic acid of claim 34 or 35, wherein the promoter is a tissue-specific promoter or a cell type-specific promoter.
39. A recombinant expression vector comprising the nucleic acid of any one of claims 30- 38.
40. A cell comprising the nucleic acid of any one of claims 30-38 or the recombinant expression vector of claim 39.
41. The cell of claim 40, wherein the cell is in vitro.
42. The cell of claim 40, wherein the cell is in vivo.
43. The cell of any one of claims 40-42, wherein the cell is a eukaryotic cell.
44. The cell of claim 43, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
45. A nucleic acid comprising a nucleotide sequence encoding an anti-CRISPR (Acr) polypeptide that is an inhibitor of an enzymatic activity of a Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, and wherein the nucleotide sequence is optimized for expression in a mammalian cell.
46. The nucleic acid of claim 45, wherein the nucleotide sequence is operably linked to a promoter.
47. A recombinant expression vector comprising the nucleic acid of claim 45 or claim 46.
48. A kit comprising: a) a Cas9 polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to a Cas9 amino acid sequence depicted in FIG.21A-21D, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 polypeptide; and b) an anti-CRISPR (Acr) polypeptide that is an inhibitor of an activity of the Cas9 polypeptide, wherein the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10, or a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide, wherein the enzymatic activity is nucleic acid cleavage, optionally wherein: i) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Staphylococcus aureus Cas9 amino acid sequence depicted in FIG.21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; ii) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; iii) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus pyogenes Cas9 amino acid sequence depicted in FIG.21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B; iv) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML1 amino acid sequence depicted in FIG.20A-20B; or v) the Cas9 polypeptide comprises an amino acid sequence having at least 50% amino acid sequence identity to the Streptococcus iniae Cas9 amino acid sequence depicted in FIG.21A-21D; and the Acr polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the ML8 amino acid sequence depicted in FIG.20A-20B.
49. The kit of claim 48, wherein component (a) and component (b) are in separate containers.
50. A method for inhibiting an activity of a Cas9 polypeptide, the method comprising contacting the Cas9 polypeptide with: a) an anti-CRISPR (Acr) polypeptide comprising an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-10; or b) the Acr fusion polypeptide of any one of claims 1-5.
51. The method of claim 50, wherein the Cas9 polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence depicted in FIG. 21A-21D.
52. The method of claim 50 or 51, wherein said contacting occurs in a living cell in vitro.
53. The method of claim 50 or 51, wherein said contacting occurs in a living cell in vivo.
54. The method of claim 52 or claim 53, comprising introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the Acr polypeptide.
55. The method of any one of claims 52-54, wherein the nucleotide sequence encoding the Acr polypeptide is operably linked to an inducible promoter.
56. The method of any one of claims 52-55, wherein the cell is a eukaryotic cell.
57. The method of claim 56, wherein the cell is a mammalian cell, an insect cell, a plant cell, an arthropod cell, a helminth cell, a protozoan cell, a reptile cell, an avian cell, an amphibian cell, a fungal cell, an algal cell, or a fish cell.
58. The method of claim 55, wherein the inducible promoter is a drug-inducible promoter, and wherein the method comprises contacting the cell with a drug that induces the drug-inducible promoter.
59. The method of any one of claims 52-55, wherein the cell is a prokaryotic cell.
60. A method of training a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity, the method comprising a) collecting sequence information for one or more anti-CRISPR (Acr) proteins known to inhibit CRISPR-Cas activity from one or more databases; b) representing each of the one or more Acr proteins as a feature vector based on amino acid composition of the Acr protein; c) collecting sequence information for one or more non-Acr proteins known not to inhibit CRISPR-Cas activity from one or more databases; d) representing each non-Acr protein as a feature vector based on amino acid composition of the protein; and e) training the machine learning model using the feature vectors representing one or more anti- CRISPR proteins and the feature vectors representing one or more non-Acr proteins.
61. The method of claim 60, wherein collecting sequence information for one or more Acr proteins comprises collecting one or more non-redundant Acr proteins.
62. The method of claim 61, wherein collecting sequence information for one or more non- Acr proteins comprises selecting one or more non-Acr proteins that are not redundant with the one or more Acr proteins.
63. The method of claim 62, wherein collecting sequence information for one or more non- Acr proteins further comprises selecting one or more non-Acr proteins that belong to the same species as one or more of the Acr proteins.
64. The method of claim 60, wherein the machine learning model is a tree-based model.
65. The method of claim 64, wherein the machine learning model utilizes pairwise ranking.
66. A method of identifying one or more proteins based on their potential to inhibit CRISPR-Cas activity, the method comprising: a) using sequence information for one or more known anti-CRISPR (Acr) proteins to train a machine learning model to rank a plurality of proteins based on each protein’s potential to inhibit CRISPR-Cas activity; b) applying the trained machine learning model to a plurality of proteins to obtain an ordered list of the proteins based on each protein’s potential to inhibit CRISPR-Cas activity relative to the other proteins; and c) identifying one or more potential Acr proteins based on each such protein’s position in the ordered list.
67. The method of claim 66, wherein the plurality of proteins belongs to the same species.
68. The method of claim 67, wherein each of the plurality of proteins is found within one or more prophage regions.
69. The method of identifying one or more proteins of claim 68, wherein identifying one or more potential anti-CRISPR proteins further comprises inspecting whether each potential anti-CRISPR protein exhibits specific features.
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