Anti-crispr proteins

Abstract

Provided herein are compositions, systems, and methods comprising anti-CRISPR proteins and uses thereof. A system comprising an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, an effector protein or a nucleic acid encoding the effector protein, and an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid is provided. Also provided are inhibitor-associated proteins or a nucleic acids encoding the inhibitor-associated proteins.

Description

ANTI-CRISPR PROTEINSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 515,979, filed July 27, 2023, which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This specification describes technologies generally relating to polypeptides, such as anti-CRISPR proteins, compositions of such polypeptides, and methods of using such polypeptides and compositions, including inhibiting or inactivating effector protein activity.BACKGROUND

[0003] Programmable nucleases are proteins that bind and cleave nucleic acids in a sequence-specific manner. A programmable nuclease may bind a target region of a nucleic acid and cleave the nucleic acid within the target region or at a position adjacent to the target region. In some instances, a programmable nuclease is activated when it binds a target region of a nucleic acid to cleave regions of the nucleic acid that are near, but not adjacent to the target region. A programmable nuclease, such as a CRISPR-associated (Cas) protein, may be coupled to a guide nucleic acid that imparts activity or sequence selectivity to the programmable nuclease. In general, guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid. In some embodiments, guide nucleic acids comprise a trans activating crRNA (tracrRNA), at least a portion of which interacts with the programmable nuclease. In some embodiments, guide nucleic acids comprise a repeat region or a handle region, wherein, in some aspects, at least a portion of which interacts with the programmable nuclease, wherein in some aspects a handle region comprises at least a portion of a repeat region. In some embodiments, a tracrRNA or intermediary RNA is provided separately from the guide nucleic acid. The tracrRNA, repeat region, handle region, or any combination thereof may hybridize to a portion of the guide nucleic acid that does not hybridize to the target nucleic acid.

[0004] Programmable nucleases may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). Programmable nucleases may provide cis cleavage activity, trans cleavage activity, nickaseactivity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide RNA (a dual nucleic acid system or sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide RNA. Trans cleavage activity (also referred to as transcollateral cleavage) comprises cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide RNA. Trans cleavage activity is triggered by the hybridization of guide RNA to the target nucleic acid. Nickase activity is the selective cleavage of one strand of a dsDNA molecule. While certain programmable nucleases may be used to edit and detect nucleic acid molecules in a sequence specific manner, challenging biological sample conditions (e.g., high viscosity, metal chelating) may limit their accuracy and effectiveness. There is thus a need for systems and methods that employ programmable nucleases having specificity and efficiency across a wide range of sample conditions.SUMMARY

[0005] Polypeptides, compositions, and methods of use thereof including anti-CRISPR proteins (Acrs) are provided in the present disclosure.

[0006] Additionally, the present disclosure provides compositions, systems and methods for using anti-CRISPR proteins, for instance in CRISPR-Cas systems.

[0007] The presently disclosed anti-CRISPR proteins, compositions, systems, and methods are useful in various practical applications including, but not limited to, clinical applications (e.g., diagnostic, research, and / or experimental assays for determining pathogenesis, transmission risk, treatment response, diagnosis, and / or epidemiology of diseases) and / or genome engineering. Additionally, the presently disclosed anti-CRISPR proteins, compositions, systems, and methods provide improvements to the various technological fields described above, including, but not limited to, clinical analysis, genome engineering, and the rapid detection and identification of target nucleic acids in samples.

[0008] One aspect of the present disclosure provides systems comprising an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, an effector protein or a nucleic acid encoding the effector protein, and an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1.

[0009] In some embodiments, the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0010] In some embodiments, systems further include an inhibitor-associated protein or a nucleic acid encoding the inhibitor-associated protein. In some embodiments, the inhibitor- associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0011] Another aspect of the present disclosure provides compositions comprising any of the systems disclosed herein, or a component thereof.

[0012] Another aspect of the present disclosure provides pharmaceutical compositions, comprising any of the systems disclosed herein, or a component thereof; and a pharmaceutically acceptable excipient.

[0013] Another aspect of the present disclosure provides methods of editing a target nucleic acid comprising contacting the target nucleic acid with any of the systems disclosed herein, any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0014] Another aspect of the present disclosure provides methods of detecting a target nucleic acid in a sample, comprising contacting the sample with a system disclosed herein, and detecting the detectable signal.

[0015] Another aspect of the present disclosure provides cells comprising any of the systems or compositions disclosed herein.

[0016] Another aspect of the present disclosure provides expression vectors comprising a nucleic acid encoding an effector protein. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1. In some embodiments, expression vectors further comprise a nucleic acid encoding an anti-CRISPR protein. In some embodiments, the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0017] Another aspect of the present disclosure provides methods of modifying a target nucleic acid, comprising contacting a cell with any of the expression vectors disclosed herein.

[0018] Another aspect of the present disclosure provides methods of treating a disease, comprising administering to a subject in need any of the expression vectors disclosed herein.

[0019] Another aspect of the present disclosure provides methods of treating a disease, comprising administering to a subject in need a pharmaceutical composition disclosed herein.

[0020] Another aspect of the present disclosure provides methods of treating a disease, comprising administering to a subject in need any of the cells disclosed herein.

[0021] In some embodiments, the inhibitor-associated protein is an anti-CRISPR- associated (aca) protein.

[0022] Various embodiments of systems, methods and devices within the scope of the appended claims each have several aspects, no single one of which is solely responsible for the desirable attributes described herein. Without limiting the scope of the appended claims, some prominent features are described herein. After considering this discussion, and particularly after reading the section entitled “Detailed Description” one will understand how the features of various embodiments are used.INCORPORATION BY REFERENCE

[0023] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.DETAILED DESCRIPTION

[0024] Introduction

[0025] Effector proteins play a role in various clinical applications, including diagnostic assays for rapid detection and identification of target nucleic acids in biological samples, such as target loci involved in disease. Such identification can be used to determine, e.g., pathogenesis, transmission risk, treatment response, diagnosis, and / or epidemiology of pathogens and infectious diseases, as well as tumor DNA and / or cancer-related viruses. As described elsewhere herein, effector proteins include Cas proteins that can be employed inCRISPR-Cas systems. In addition to the applications set forth above, Cas proteins are particularly advantageous for genome engineering.

[0026] The identification and utilization of anti-CRISPR proteins can provide a valuable post-translational “off-switch” for effector proteins such as Cas proteins. Advantageously, this can add a layer of regulation on this powerful system that can be applied in eukaryotic systems to control genome engineering. This new addition to the CRISPR toolbox could enable new applications, such as specifically reducing or abolishing the effects of Cas proteins binding to a genomic locus, or limiting the amount of time that Cas proteins are active in the nucleus to reduce off-target gene editing. As described in Example 1 below, Acrs present potentially valuable components for CRISPR toolkits in that plasmids expressing Acr proteins can prevent self-inactivation in AAV-producing cells. It will be important to continue to exploit the abundant tools provided to us from the phage-bacteria arms race as the search for inhibitor proteins expands. See, for example, Rauch BJ, Silvis MR, Hultquist JF, et al. Inhibition of crispr-cas9 with bacteriophage proteins. Cell. 2017;168(l-2): 150-158.el0; Ibraheim R, Tai PWL, Mir A, et al. Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo. Nat Commun. 2021;12(l):6267; Forsberg, Anti-crispr discovery: using magnets to find needles in haystacks. Journal of Molecular Biology. 2023; 435(7): 167952; and Suresh SK, Murugan K, Sashital DG. Enzymatic anti-CRISPRs improve the bacteriophage arsenal. Nat Struct Mol Biol. 2019;26(4):250-251, each of which is hereby incorporated herein by reference in its entirety.

[0027] Advantageously, the present disclosure provides anti-CRISPR protein, compositions, systems, and methods for use in various practical applications including, but not limited to, clinical applications (e.g., diagnostic, research, and / or experimental assays for determining pathogenesis, transmission risk, treatment response, diagnosis, and / or epidemiology of diseases) and / or genome engineering. Additionally, the present disclosure provides improvements to the various technological fields described above, including, but not limited to, clinical analysis, genome engineering, and the rapid detection and identification of target nucleic acids in samples.

[0028] One aspect of the present disclosure provides a system comprising: an anti- CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, an effector protein or a nucleic acid encoding the effector protein, and an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid. In some embodiments, the effector proteincomprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1. In some embodiments, the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2. In some embodiments, the system further comprises an inhibitor-associated protein or a nucleic acid encoding the inhibitor- associated protein. In some embodiments, the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0029] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one of ordinary skill in the art that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, circuits, and networks have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.

[0030] Definitions

[0031] It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first subject could be termed a second subject, and, similarly, a second subject could be termed a first subject, without departing from the scope of the present disclosure. The first subject and the second subject are both subjects, but they are not the same subject.

[0032] The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” when used inthis specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0033] As used herein, the term “if’ may be construed to mean “when” or “upon” or “in response to determining” or “in response to detecting,” depending on the context. Similarly, the phrase “if it is determined” or “if [a stated condition or event] is detected” may be construed to mean “upon determining” or “in response to determining” or “upon detecting (the stated condition or event (” or “in response to detecting (the stated condition or event),” depending on the context.

[0034] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0035] As used herein, the terms “% identical,” “% identity,” and “percent identity,” or grammatical equivalents thereof, refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” can refer to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence that are identical to the residues of the sequence disclosed in SEQ ID NO: Y. In some embodiments, the percent identity value is a single numeric score determined for a pair of aligned sequences. It measures the number of identical residues (“matches”) in relation to the length of the alignment. Gaps that must be introduced into either sequence in the pair of aligned sequences contributes to the length of the alignment and thus decreases the percent identity. For instance, in the pair of sequences:1 : GCPVS - SPN 2 : GCPYGCDPEGCPxx-xPxone of the residues (a cysteine) in the second sequence does not have a corresponding mate in the first sequence and thus the position has been marked by a dash. The percentage of identity for this sequence alignment is 4 / 9, or 44%, and not 4 / 8 (50%), because the one residue insertion in the alignment contributes to the length of the alignment. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Set USA. 1988 Apr;85(8):2444-8; Pearson, Methods EnzymoL 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res . 1997 Sep 1;25(17):3389-40), BLASTP, BLASTN, or GCG.

[0036] The term, “cz cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur within or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.

[0037] The terms “complementary” and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T or U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is, in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement" sequence or the ‘reverse complementary" sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0038] The terms “cleave,” “cleaving,” and “cleavage,” as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one sideof a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[0039] The term “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen.

[0040] The terms “CRISPR RNA” or “crRNA,” as used herein, refer to a type of guide nucleic acid, where the nucleic acid is RNA comprising a first sequence, often referred to herein as a spacer sequence, that hybridizes to a target sequence of a target nucleic acid, and a second sequence that is capable of connecting crRNA to an effector protein by either a) hybridizing to a portion of a tracrRNA or b) being non-covalently bound by an effector protein. In a dual nucleic acid system, where a crRNA and a tracrRNA forms a complex with an effector protein, a crRNA includes the first sequence that hybridizes to the target sequence of the target nucleic acid and the second sequence hybridizes to a portion of the tracrRNA.

[0041] The term “effector protein,” as used herein, refers to a polypeptide or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, where at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. A complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein. In some instances, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some instances, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid. A non-limiting example of an effector protein modifying a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout.

[0042] The term “guide nucleic acid,” as used herein, refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of connecting an effector protein to the nucleic acid by either a) hybridizing to a portion of an additional nucleic acid that is bound by an effector protein (e.g., a tracrRNA) or b) being non-covalently bound by an effector protein. In some embodiments, the first sequence is referred to herein as a spacer sequence. In some embodiments, the second sequence is referred to herein as a repeat sequence. In some embodiments, the second sequence is referred to herein as a handle sequence. In some embodiments, the handle sequence comprises a portion of, or all of a repeat sequence. In some embodiments, the first sequence is located 5’ of the second nucleotide sequence. In some embodiments, the first sequence is located 3’ of the second nucleotide sequence. In some embodiments, in a single guide nucleic acid system, also referred to as a single guide RNA (sgRNA), the second sequence is a handle sequence.

[0043] The term “handle sequence,” as used herein, in the context of an sgRNA, refers to a portion of the sgRNA that is capable of being non-covalently bound by an effector protein. The nucleotide sequence of a handle sequence can contain or be derived from a tracrRNA sequence. For example, in some aspects, a handle sequence can include a portion of a tracrRNA sequence that is capable of being non-covalently bound by an effector protein, but does not include all or a part of the portion of a tracrRNA sequence that hybridizes to a portion of a crRNA as found in a dual nucleic acid system. In some aspects, a handle sequence can include a portion of a tracrRNA sequence, as well as a portion of a repeat sequence, which can optionally be connected by a linker. In some aspects, a handle sequence in the context of an sgRNA can also be described as the portion of the sgRNA that does not hybridize to a target sequence in a target nucleic acid (e.g., a spacer sequence).

[0044] The term “linker,” as used herein, refers to a bond or molecule that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid. The molecule may comprise one or more amino acids in the case of linking polypeptides. The molecule may comprise one or more nucleotides in the case of linking nucleic acids.

[0045] The terms “non-naturally occurring” and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and / orcontains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition can include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0046] The term “nuclease activity,” as used herein, refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids; the term “endonuclease activity” refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bond within a polynucleotide chain. An enzyme with nuclease activity can be referred to as a “nuclease.” In some instances, nuclease activity results in nicking (cleaving a single strand of) dsDNA.

[0047] As used herein, the term “protein” means two or more amino acids or residues linked by a peptide bond. The terms “protein” and “polypeptide” may be used interchangeably herein.

[0048] In some embodiments, a polypeptide or protein is not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. A protein can be an enzyme. In some embodiments, a protein is an effector protein, including Cas proteins disclosed herein. In some embodiments, a protein sequence is a sequence of the identities of each residue in a protein, such as the amino acid sequence of the protein.

[0049] A protein in accordance with some embodiments of the disclosed systems and methods can also have any number of post-expression and / or posttranslational modifications. Thus, a protein includes those that are modified by acylation, alkylation, amidation, biotinylation, formylation, y-carboxylation, glutamyl ati on, glycosylation, glycylation, hydroxylation, iodination, isoprenylation, lipoylation, cofactor addition (for example, of a heme, flavin, metal, etc.), addition of nucleosides and their derivatives, oxidation, reduction, pegylation, phosphatidylinositol addition, phosphopantetheinylation, phosphorylation,pyroglutamate formation, racemization, addition of amino acids by tRNA (for example, arginylation), sulfation, selenoylation, ISGylation, SUMOylation, ubiquitination, chemical modifications (for example, citrullination and deamidation), and treatment with other enzymes (for example, proteases, phosphotases and kinases). Other types of posttranslational modifications are known in the art and are also included.

[0050] As used herein, the term “reference sequence” refers to a sequence of nucleotide bases. In some embodiments, a reference sequence is a reference genome.

[0051] In some embodiments, the term “region” as used herein is used to describe a portion of or all of a corresponding sequence, for example, a spacer region is understood to comprise a portion of or all of a spacer sequence.

[0052] The term “subject,” as used herein, refers to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. In some implementations, the subject is diagnosed with or suspected of being at high risk for a disease. In some implementations, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0053] The term “target sequence,” as used herein, when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to an equal length portion of a guide nucleic acid. In some implementations, hybridization of the guide nucleic acid to the target sequence brings an effector protein into contact with the target nucleic acid.

[0054] The term “trans-activating RNA (tracrRNA),” as used herein, refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by an effector protein. In some implementations, tracrRNAs comprise a second sequence that hybridizes to a portion of a crRNA, which can be referred to as a repeat hybridization sequence.

[0055] The terms “treatment” or “treating,” as used herein, are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can beachieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made.

[0056] The implementations described herein provide various embodiments and technical solutions for anti-CRISPR protein or polynucleotides encoding the same that guide inhibition and / or inactivation of effector protein systems, compositions thereof, and systems and methods of obtaining the same. Details of implementations are now described.

[0057] Certain embodiments

[0058] One aspect of the present disclosure comprises a system comprising: an anti- CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, an effector protein or a nucleic acid encoding the effector protein, and an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

[0059] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1

[0060] In some embodiments, the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0061] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% identical to one or more sequences comprising a predicted target of CasM.265466 recited in TABLE 2.

[0062] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 2, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.2390217 recited in TABLE 2.

[0063] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 3, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.19522 recited in TABLE 2.

[0064] In some embodiments, the system further includes an inhibitor-associated protein or a nucleic acid encoding the inhibitor-associated protein. In some embodiments, the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0065] In some embodiments, the system further includes a plurality of inhibitor- associated proteins or a nucleic acid encoding the plurality of inhibitor-associated proteins.

[0066] In some embodiments, the length of the effector protein is about 350 to about 450 linked amino acids, about 380 to about 430 linked amino acids, about 395 to about 410 linked amino acids, or about 300 to about 500 linked amino acids.

[0067] In some embodiments, the amino acid sequence of the effector protein comprises one or more amino acid alterations relative to a sequence selected from TABLE 1.

[0068] In some embodiments, the one or more amino acid alterations comprises: a) 1 to 5, 1 to 10, or 1 to 20 non-conservative amino acid substitutions; b) 1 to 5, 1 to 10, or 1 to 20 conservative amino acid substitutions; or c) a combination thereof.

[0069] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: K58W, T84H, T84K, T84R, K105R, K105H, I80K, I80R, I80H, N193K, C202K, C202R, C202H, S209F, G210R, G210K, G210H, A218R, A218K, A218H, D220H, D220K, D220R, E225R, E225K, E225H, C246K, C246R, C246H, N286K, M295W, M298L, Y315M, A306K, Q360R, Q360K, and Q360H.

[0070] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: D220R / A306K and D220R / K250N.

[0071] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: D237A, D418A, D418N, E335A, and E335Q.

[0072] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions selected from the group consisting of: K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, and Q360, and the amino acid substitutions are independently selected from arginine, lysine, and histidine.

[0073] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from TABLE 4.

[0074] In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten amino acids of the effector protein are substituted with a positively charged amino acid residue relative to a sequence recited in TABLE 1. In some embodiments, the positively charged amino acid residue for each of the one, two, three, four, five, six, seven, eight, nine, or ten amino acids is independently selected from arginine, lysine, and histidine. In some embodiments, the positively charged amino acid residue is arginine.

[0075] In some embodiments, the engineered guide nucleic acid is a single guide RNA (sgRNA).

[0076] In some embodiments, the effector protein is fused to a fusion partner. In some embodiments, the fusion partner comprises a protein selected from a polymerase, deaminase, a reverse transcriptase, a transcriptional repressor, and a transcriptional activator.

[0077] In some embodiments, the system further includes a lipid nanoparticle.

[0078] In some embodiments, the nucleic acid encoding the effector protein is a messenger RNA.

[0079] In some embodiments, the effector protein is capable of forming a complex with a guide nucleic acid, and where the complex is capable of binding a target nucleic acid.

[0080] In some embodiments, the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid are in separate compositions.

[0081] In some embodiments, the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid are in a single composition.

[0082] In some embodiments, the system further includes at least one detection reagent for detecting a target nucleic acid, where the at least one detection reagent is selected from a reporter nucleic acid, a detection moiety, amplification reagent, and a combination thereof. In some embodiments, the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof.

[0083] Another aspect of the present disclosure comprises a composition comprising the system of any one of the embodiments disclosed herein, or a component thereof.

[0084] Another aspect of the present disclosure comprises a pharmaceutical composition, comprising the system of any one of the embodiments disclosed herein, or a component thereof; and a pharmaceutically acceptable excipient.

[0085] Another aspect of the present disclosure comprises a method of editing a target nucleic acid comprising contacting the target nucleic acid with the system of any one of the embodiments disclosed herein, the composition of the embodiments disclosed herein, or the pharmaceutical composition of the embodiments disclosed herein.

[0086] In some embodiments, the method further includes contacting the target nucleic acid with the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid sequentially.

[0087] In some embodiments, the method further includes contacting the target nucleic acid with the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid simultaneously.

[0088] In some embodiments, the method further includes contacting a cell comprising the target nucleic acid with the system or the composition.

[0089] Another aspect of the present disclosure comprises a method of detecting a target nucleic acid in a sample, comprising: contacting the sample with the system disclosed herein; and detecting the detectable signal.

[0090] Another aspect of the present disclosure comprises a cell comprising the system of any one of the embodiments disclosed herein or the composition of the embodiments disclosed herein.

[0091] Another aspect of the present disclosure comprises a cell modified by the system of any one of the embodiments disclosed herein, the composition of the embodiments disclosed herein, or the pharmaceutical composition of the embodiments disclosed herein.

[0092] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a stem cell.

[0093] Another aspect of the present disclosure comprises an expression vector comprising: a nucleic acid encoding an effector protein, where the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and a nucleic acid encoding an anti-CRISPR protein, where the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0094] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% identical to one or more sequences comprising a predicted target of CasM.265466 recited in TABLE 2.

[0095] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 2, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.2390217 recited in TABLE 2.

[0096] In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 3, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.19522 recited in TABLE 2.

[0097] In some embodiments, the expression vector further includes a nucleic acid encoding an inhibitor-associated protein, where the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0098] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: K58W, T84H, T84K, T84R, K105R, K105H, I80K, I80R, I80H, N193K, C202K, C202R, C202H, S209F, G210R, G210K, G210H, A218R, A218K, A218H, D220H, D220K, D220R, E225R, E225K, E225H, C246K, C246R, C246H, N286K, M295W, M298L, Y315M, A306K, Q360R, Q360K, and Q360H.

[0099] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: D220R / A306K and D220R / K250N.

[0100] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from the group consisting of: D237A, D418A, D418N, E335A, and E335Q.

[0101] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions selected from the group consisting of: K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, and Q360, and the amino acid substitutions are independently selected from arginine, lysine, and histidine.

[0102] In some embodiments, the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, where the one or more amino acid substitutions is selected from TABLE 4.

[0103] In some embodiments, the expression vector further includes an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is a self- complementary AAV vector.

[0104] Another aspect of the present disclosure comprises a method of modifying a target nucleic acid, comprising contacting a cell with the expression vector of any one of the embodiments disclosed herein. In some embodiments, the method further includes transducing the cell by contacting the cell with a virus containing the expression vector.

[0105] Another aspect of the present disclosure comprises a method of treating a disease, comprising administering to a subject in need the expression vector of any one of the embodiments disclosed herein.

[0106] Another aspect of the present disclosure comprises a method of treating a disease comprising administering to a subject in need thereof the pharmaceutical composition according to any one of the embodiments disclosed herein.

[0107] Another aspect of the present disclosure comprises a method of treating a disease comprising administering to a subject in need thereof the cell of any one of the embodiments disclosed herein.

[0108] Another aspect of the present disclosure comprises a method of modifying a target nucleic acid, comprising: contacting a cell comprising the target nucleic acid with an effectorprotein or a nucleic acid encoding the effector protein, and contacting the cell with an anti- CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, where the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0109] In some embodiments, the method further includes contacting the cell with an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

[0110] In some embodiments, the method further includes contacting the cell with an inhibitor-associated protein or a nucleic acid encoding an inhibitor-associated protein, where the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0111] Another aspect of the present disclosure comprises a method of treating a disease comprising: administering to a subject in need thereof an effector protein or a nucleic acid encoding the effector protein, and administering to the subject an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, where the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

[0112] In some embodiments, the method further includes administering to the subject an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

[0113] In some embodiments, the method further includes administering to the subject an inhibitor-associated protein or a nucleic acid encoding an inhibitor-associated protein, where the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0114] In some embodiments, the genome of the subject comprises a target nucleic acid or a polynucleotide encoding the same.

[0115] Another aspect of the present disclosure comprises a method of treating a condition in a subject, the method comprising: administering to the subject an effector protein or a nucleic acid encoding the effector protein, and administering to the subject an anti- CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, where the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2, thereby producing a therapeutic effect in the subject.

[0116] In some embodiments, the method further includes administering to the subject an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

[0117] In some embodiments, the method further includes administering to the subject an inhibitor-associated protein or a nucleic acid encoding an inhibitor-associated protein, where the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

[0118] In some embodiments, the condition is a disease selected from the group consisting of cancer, ophthalmological diseases, neurological and neurodegenerative diseases, blood disorders, metabolic diseases, genetic disorders, and infection.

[0119] In some embodiments, the method further includes modifying a target nucleic acid contained in a genome of the subject, where the target nucleic acid is a gene associated with the disease.

[0120] In some embodiments, the inhibitor-associated protein is an anti-CRISPR- associated (aca) protein.

[0121] TABLE 1 provides illustrative amino acid sequences of effector proteins.TABLE 1: Exemplary Amino Acid Sequences of Effector Proteins

[0122] TABLE 2 provides illustrative amino acid sequences for anti-CRISPR proteins.TABLE 2: Exemplary Amino Acid Sequences of Anti-CRISPR proteins*Inhibitor-associated proteins are indicated by ACA ID NO; sequences for inhibitor- associated proteins provided in TABLE 3.

[0123] TABLE 3 provides illustrative amino acid sequences for inhibitor-associated proteins.TABLE 3: Exemplary Amino Acid Sequences of Inhibitor- Associated Proteins

[0124] TABLE 4 provides illustrative variants for the CasM.265466 effector protein.TABLE 4: Exemplary Variants of CasM.265466 Effector Protein

[0125] EXAMPLES

[0126] EXAMPLE 1 - Determining CasM.265466 anti-CRISPR proteins.

[0127] Anti-CRISPR proteins (Acrs) generally refer to small proteins encoded by phages and other mobile genetic elements (MGEs) that inhibit at least one CRISPR-Cas system.Typically, they are found to act on Type II, V, and VI CRISPR systems via at least three different mechanisms. One of these is direct inhibition, where the Acr interacts with the Cas protein and prevents DNA binding or nuclease activity. Typical characteristics of competitiveinhibitors include a small size (50-120 amino acids), an overall negative charge, and a position within an open reading frame on a prophage. In some implementations, Acrs present potentially valuable components for CRISPR toolkits in that plasmids expressing Acr proteins can prevent self-inactivation in AAV-producing cells.

[0128] Spacers encoded in CRISPR arrays are used to identify and excise previously recognized phages. For instance, if a genome contains that spacer elsewhere (outside an array), Cas machinery can recognize and destroy it, leading to cell death. Presence of both a self-targeting spacer and a Cas protein suggests that there may be some inhibition of CRISPR activity. Self-targeting spacers provide clues to the presence of an anti-CRISPR protein.

[0129] A self-targeted protospacer search process was performed including the following steps:

[0130] 1. Identify CasM.265466 homologs on isolate genomes, MAGs, and long metagenomic contigs (e.g., greater than or equal to 50kb).

[0131] 2 Predict CRISPR arrays on these genomes and / or contigs.

[0132] 3. Search whether the spacers from these arrays are located elsewhere in the genome as self-targeted (ST) protospacers.

[0133] 4. Determine whether these ST protospacers are on or near a predicted prophage.

[0134] 58 self-targeted protospacer candidates on 22 genomes were identified using the self-targeted protospacer search. To determine putative Acrs from self-targets, a set of Acr protein candidate criteria was applied to the 58 protospacer candidates. These Acr protein candidate criteria included:

[0135] 1. 50 < ORF length < 120

[0136] 2 Close to prophage a. within lOkb of prophage, or b. within 30kb of protospacer, and / or

[0137] 3. Isoelectric point (pl) < 7.

[0138] 415 anti-CRISPR candidates were determined and were further prioritized by a set of Acr candidate prioritization criteria, including:

[0139] 1. Homolog % identity to nanocaps,

[0140] 2. Protospacer % identity to spacer,

[0141] 3 Candidates where the protospacer is on an ORF,

[0142] 4. ORFs closest to an HTH domain-containing ORF, and

[0143] 5. ORFs closest to the protospacer.

[0144] Prioritization resulted in a filtered set of approximately 208 Acr candidates from 22 genomes (28 contigs) for further downstream screening and analysis. This process was repeated for genomes containing CasM.19952 and CasM.2390217 homologs, resulting in the identification of 406 additional self-targeted protospacer candidates. Applying Acr candidate criteria and prioritizing the resulting proteins yielded 661 additional Acr candidates. These candidates, along with those predicted to inhibit CasM.265466, were clustered and deduplicated to obtain 403 unique anti-CRISPR candidates from 98 source genomes.

[0145] CONCLUSION

[0146] Plural instances may be provided for components, operations or structures described herein as a single instance. Finally, boundaries between various components, operations, and data stores are somewhat arbitrary, and particular operations are illustrated in the context of specific illustrative configurations. Other allocations of functionality are envisioned and may fall within the scope of the implementation(s). In general, structures and functionality presented as separate components in the example configurations may be implemented as a combined structure or component. Similarly, structures and functionality presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements fall within the scope of the implementation(s).

[0147] The foregoing description included example systems, methods, techniques, instruction sequences, and computing machine program products that embody illustrative implementations. For purposes of explanation, numerous specific details were set forth in order to provide an understanding of various implementations of the inventive subject matter. It will be evident, however, to those skilled in the art that implementations of the inventive subject matter may be practiced without these specific details. In general, well-known instruction instances, protocols, structures and techniques have not been shown in detail.

[0148] The foregoing description, for the purpose of explanation, has been described with reference to specific implementations. However, the illustrative discussions above are not intended to be exhaustive or to limit the implementations to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. Theimplementations were chosen and described in order to best explain the principles and their practical applications, to thereby enable others skilled in the art to best utilize the implementations and various implementations with various modifications as are suited to the particular use contemplated.

Claims

What is claimed is:

1. A system comprising: an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, an effector protein or a nucleic acid encoding the effector protein, and an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1.

2. The system of claim 1, wherein the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

3. The system of claim 1 or 2, wherein: the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.265466 recited in TABLE 2.

4. The system of claim 1 or 2, wherein: the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 2, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.2390217 recited in TABLE 2.

5. The system of claim 1 or 2, wherein:the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 3, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.19522 recited in TABLE 2.

6. The system of any one of claims 1-5, further comprising an inhibitor-associated protein or a nucleic acid encoding the inhibitor-associated protein.

7. The system of claim 6, wherein the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

8. The system of any one of claims 1-7, further comprising a plurality of inhibitor-associated proteins or a nucleic acid encoding the plurality of inhibitor-associated proteins.

9. The system of any one of claims 1-8, wherein the length of the effector protein is about 350 to about 450 linked amino acids, about 380 to about 430 linked amino acids, about 395 to about 410 linked amino acids, or about 300 to about 500 linked amino acids.

10. The system of any one of claims 1-9, wherein the amino acid sequence of the effector protein comprises one or more amino acid alterations relative to a sequence selected from TABLE 111. The system of claim 10, wherein the one or more amino acid alterations comprises: a) 1 to 5, 1 to 10, or 1 to 20 non-conservative amino acid substitutions; b) 1 to 5, 1 to 10, or 1 to 20 conservative amino acid substitutions; or c) a combination thereof.

12. The system of any one of claims 1-11, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acidsubstitutions is selected from the group consisting of: K58W, T84H, T84K, T84R, K105R, K105H, I80K, I80R, I80H, N193K, C202K, C202R, C202H, S209F, G210R, G210K, G210H, A218R, A218K, A218H, D220H, D220K, D220R, E225R, E225K, E225H, C246K, C246R, C246H, N286K, M295W, M298L, Y315M, A306K, Q360R, Q360K, and Q360H.

13. The system of any one of claims 1-11, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from the group consisting of: D220R / A306K and D220R / K250N.

14. The system of any one of claims 1-11, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from the group consisting of: D237A, D418A, D418N, E335A, and E335Q.

15. The system of any one of claims 1-11, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions selected from the group consisting of: K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, and Q360, and the amino acid substitutions are independently selected from arginine, lysine, and histidine.

16. The system of any one of claims 1-15, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from TABLE 4.

17. The system of any one of claims 1-16, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids of the effector protein are substituted with a positively charged amino acid residue relative to a sequence recited in TABLE 1.

18. The system of claim 17, wherein the positively charged amino acid residue for each of the one, two, three, four, five, six, seven, eight, nine, or ten amino acids is independently selected from arginine, lysine, and histidine.

19. The system of claim 17 or 18, wherein the positively charged amino acid residue is arginine.

20. The system of any one of claims 1-19, wherein the engineered guide nucleic acid is a single guide RNA (sgRNA).

21. The system of any one of claims 1-20, wherein the effector protein is fused to a fusion partner.

22. The system of claim 21, wherein the fusion partner comprises a protein selected from a polymerase, deaminase, a reverse transcriptase, a transcriptional repressor, and a transcriptional activator.

23. The system of any one of claims 1-22, comprising a lipid nanoparticle.

24. The system of any one of claims 1-23, wherein the nucleic acid encoding the effector protein is a messenger RNA.

25. The system of any one of claims 1-24, wherein the effector protein is capable of forming a complex with a guide nucleic acid, and wherein the complex is capable of binding a target nucleic acid.

26. The system of any one of claims 1-25, wherein the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti- CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid are in separate compositions.

27. The system of any one of claims 1-25, wherein the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti- CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid are in a single composition.

28. The system of any one of claims 1-27, further comprising at least one detection reagent for detecting a target nucleic acid, wherein the at least one detection reagent is selected from a reporter nucleic acid, a detection moiety, amplification reagent, and a combination thereof.

29. The system of claim 28, wherein the reporter nucleic acid comprises a fluorophore, a quencher, or a combination thereof.

30. A composition comprising the system of any one of claims 1-29, or a component thereof.

31. A pharmaceutical composition, comprising the system of any one of claims 1-27, or a component thereof; and a pharmaceutically acceptable excipient.

32. A method of editing a target nucleic acid comprising contacting the target nucleic acid with the system of any one of claim 1-29, the composition of claim 30, or the pharmaceutical composition of claim 31.

33. The method of claim 32, comprising contacting the target nucleic acid with the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid sequentially.

34. The method of claim 32, comprising contacting the target nucleic acid with the effector protein or nucleic acid encoding the effector protein, the anti-CRISPR protein or nucleic acid encoding the anti-CRISPR protein, and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid simultaneously.

35. The method of any one of claims 32-34, comprising contacting a cell comprising the target nucleic acid with the system or the composition.

36. A method of detecting a target nucleic acid in a sample, comprising: contacting the sample with the system of claim 29; and detecting the detectable signal.

37. A cell comprising the system of any one of claim 1-27 or the composition of claim 30.

38. A cell modified by the system of any one of claim 1-27, the composition of claim 30, or the pharmaceutical composition of claim 31.

39. The cell of claim 37 or 38, wherein the cell is a eukaryotic cell.

40. The cell of claim 37 or 38, wherein the cell is a mammalian cell.

41. The cell of claim 37 or 38, wherein the cell is a human cell.

42. The cell of claim 37 or 38, wherein the cell is a liver cell.

43. The cell of claim 37 or 38, wherein the cell is a stem cell.

44. An expression vector comprising: a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and a nucleic acid encoding an anti-CRISPR protein, wherein the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

45. The expression vector of claim 44, wherein: the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.265466 recited in TABLE 2.

46. The expression vector of claim 44, wherein: the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 2, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% or 100% identical to one or more sequences comprising a predicted target of CasM.2390217 recited in TABLE 2.

47. The expression vector of claim 44, wherein: the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 3, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences comprising a predicted target of CasM.19522 recited in TABLE 2.

48. The expression vector of any one of claims 44-47, further comprising a nucleic acid encoding an inhibitor-associated protein, wherein the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

49. The expression vector of any one of claims 44-48, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from the group consisting of: K58W, T84H, T84K, T84R, K105R, K105H, I80K, I80R, I80H, N193K, C202K, C202R, C202H, S209F, G210R, G210K, G210H, A218R, A218K, A218H, D220H, D220K, D220R, E225R, E225K, E225H, C246K, C246R, C246H, N286K, M295W, M298L, Y315M, A306K, Q360R, Q360K, and Q360H.

50. The expression vector of any one of claims 44-49, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from the group consisting of: D220R / A306K and D220R / K250N.

51. The expression vector of any one of claims 44-50, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or moreamino acid substitutions is selected from the group consisting of: D237A, D418A, D418N, E335A, and E335Q.

52. The expression vector of any one of claims 44-51, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1 at one or more positions selected from the group consisting of: K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, and Q360, and the amino acid substitutions are independently selected from arginine, lysine, and histidine.

53. The expression vector of any one of claims 44-52, wherein the effector protein comprises one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the one or more amino acid substitutions is selected from TABLE 4.

54. The expression vector of any one of claims 44-53, further comprising an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

55. The expression vector of any one of claims 44-54, wherein the expression vector is a viral vector.

56. The expression vector of claim 55, wherein the viral vector is an adeno-associated viral (AAV) vector.

57. The expression vector of claim 56, wherein the AAV vector is a self-complementary AAV vector.

58. A method of modifying a target nucleic acid, comprising contacting a cell with the expression vector of any one of claims 44-57.

59. The method of claim 58, further comprising transducing the cell by contacting the cell with a virus containing the expression vector.

60. A method of treating a disease, comprising administering to a subject in need the expression vector of any one of claims 44-57.

61. A method of treating a disease comprising administering to a subject in need thereof the pharmaceutical composition according to claim 31.

62. A method of treating a disease comprising administering to a subject in need thereof the cell of any one of claims 37-43.

63. A method of modifying a target nucleic acid, comprising: contacting a cell comprising the target nucleic acid with an effector protein or a nucleic acid encoding the effector protein, and contacting the cell with an anti-CRISPR protein or a nucleic acid encoding the anti- CRISPR protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

64. The method of claim 63, further comprising contacting the cell with an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

65. The method of claim 63 or 64, further comprising contacting the cell with an inhibitor- associated protein or a nucleic acid encoding an inhibitor-associated protein, wherein the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

66. A method of treating a disease comprising: administering to a subject in need thereof an effector protein or a nucleic acid encoding the effector protein, and administering to the subject an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, whereinthe effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2.

67. The method of claim 65, further comprising administering to the subject an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

68. The method of claim 65 or 66, further comprising administering to the subject an inhibitor-associated protein or a nucleic acid encoding an inhibitor-associated protein, wherein the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

69. The method of any one of claims 65-67, wherein the genome of the subject comprises a target nucleic acid or a polynucleotide encoding the same.

70. A method of treating a condition in a subject, the method comprising: administering to the subject an effector protein or a nucleic acid encoding the effector protein, and administering to the subject an anti-CRISPR protein or a nucleic acid encoding the anti-CRISPR protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of the sequences recited in TABLE 1, and the anti-CRISPR protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 2, thereby producing a therapeutic effect in the subject.

71. The method of claim 70, further comprising administering to the subject an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid.

72. The method of claim 70 or 71, further comprising administering to the subject an inhibitor-associated protein or a nucleic acid encoding an inhibitor-associated protein, wherein the inhibitor-associated protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to one or more sequences recited in TABLE 3.

73. The method of any one of claims 70-72, wherein the condition is a disease selected from the group consisting of cancer, ophthalmological diseases, neurological and neurodegenerative diseases, blood disorders, metabolic diseases, genetic disorders, and infection.

74. The method of any one of claims 70-73, further comprising modifying a target nucleic acid contained in a genome of the subject, wherein the target nucleic acid is a gene associated with the disease.

75. The system of any one of claims 6-8, wherein the inhibitor-associated protein is an anti- CRISPR-associated (aca) protein.