Effector proteins and uses thereof

Abstract

Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins generally comprise one or more nuclease domains. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification, the detection, and engineering of nucleic acids.

Description

EFFECTOR PROTEINS AND USES THEREOFCROSS-REFERENCE[1] This application claims benefit of U.S. Provisional Application No. 63 / 497,182, filed on April 19, 2023, U.S. Provisional Application No. 63 / 515,815, filed on July 26, 2023, and of U.S. Provisional Application No. 63 / 508,361, filed on June 15, 2023, which are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING[2] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 203477-782601_PCT_SL.xml, which was created on April 1, 2024 and is 1,427,740 bytes in size, is hereby incorporated by reference in its entirety.FIELD[3] The present disclosure relates generally to polypeptides, such as effector proteins, compositions of such polypeptides and guide nucleic acids, systems, and methods of using such polypeptides and compositions, including detecting and editing target nucleic acids.BACKGROUND[4] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins), sometimes referred to as a CRISPR / Cas system, were first identified in certain bacterial species and are now understood to form part of a prokaryotic acquired immune system. CRISPR / Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequence-specific manner. Native systems contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets. The discovery of CRISPR / Cas systems and similar RNA guided nuclease systems has revolutionized the field of genomic manipulation and engineering, and therapeutic applications of these systems are being explored.SUMMARY[5] The present disclosure provides for polypeptides, such as effector proteins, compositions, systems, and methods comprising the same, and uses thereof. In some instances, compositions, systems, and methods comprise guide nucleic acids or uses thereof. Compositions, systems, and methods disclosed herein may leverage nucleic acid modification activities, such as nucleic acid editing. Editing may comprise: insertion, deletion, substitution, or a combination thereof of one or more nucleotides or amino acids. Editing may also comprise cleavage activity, such as cis cleavage activity, nickase activity, and / or nuclease activity. In some instances, compositions, systems and methods are useful for the editing the sequence of target nucleic acids. In some instances, compositions, systems and methods areuseful for the treatment of a disease or disorder. The disease or disorder may be associated with a target nucleic acid. The disease or disorder may be associated with one or more mutations in the target nucleic acid.Certain Embodiments[6] The present disclosure provides compositions, systems, devices, kits, and methods comprising effector proteins and uses thereof. Compositions, systems, devices, kits, and methods disclosed herein leverage nucleic acid modifying activities (e.g., cis cleavage activity and trans cleavage activity) of these effector proteins for the modification, detection, and engineering of target nucleic acids.[7] Provided herein are compositions comprising: a) an effector protein, or a nucleic acid encoding the effector protein, comprising an amino acid sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-161, and 391-847; and b) an engineered guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, comprising a protein binding sequence and a spacer sequence. In some embodiments, the protein binding sequence comprises a nucleotide sequence that is 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% identical to SEQ ID NO: 178-373, 375-390, and 848-1168, listed in TABLE 2. In some embodiments, the composition comprises a combination of the effector protein and the engineered guide nucleic acid comprising a protein binding sequence as described in TABLE 2. In some embodiments, the spacer sequence is 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% identical, complementary, or reverse complementary to a eukaryotic sequence. In some embodiments, the spacer sequence is linked to a 5’ end of the protein binding sequence. In some embodiments, the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, or about 540 contiguous amino acids of the sequence selected from SEQ ID NO: 1-161, and 391- 847. In some embodiments, the compositions comprising an effector protein described herein wherein a length of the effector protein is less than 600, less than 580, less than 560, less than 540, less than 520, less than 500, less than 480, less than 460, less than 440, less than 420, or less than 400 contiguous amino acids; and at least 300, at least 320, at least 340, at least 360, or at least 380 contiguous amino acids. In some embodiments, the amino acid sequence is less than 100% identical to SEQ ID NO: 1- 161 and 391-847, and wherein not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the amino acid sequence are non-conservative amino acid substitutions relative to a respective sequence selected from SEQ ID NO: 1-161 and 391-847. In some embodiments, the effector protein comprises at least one amino acid substitution in a RuvC domain, an HNH domain, or a combination thereof. In some embodiments, the at least one amino acid substitution is a non-conservative amino acid substitution. In some embodiments, the at least one amino acid substitution replaces a catalytic residuein the RuvC domain, the HNH domain, or a combination thereof. In some embodiments, the effector protein has nickase activity. In some embodiments, the effector protein is linked to a nuclear localization signal. In some embodiments, the compositions described herein, further comprising a donor nucleic acid. In some embodiments, the compositions described herein, further comprising an effector partner linked to the effector protein. In some embodiments, the effector partner comprises a polypeptide selected from a deaminase, a reverse transcriptase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof. In some embodiments, the effector protein comprises at least one mutation that reduces its nuclease activity relative to the effector protein without the at least one mutation as measured in a cleavage assay, optionally wherein the effector protein is a catalytically inactive nuclease. In some embodiments, the compositions described herein, further comprising a lipid nanoparticle containing the effector protein or the nucleic acid encoding the effector protein, the engineered guide nucleic acid, or a combination thereof. In some embodiments, the effector protein or the nucleic acid encoding the effector protein, and the engineered guide nucleic acid or the nucleic acid encoding the engineered guide nucleic acid are provided in separate compositions. In some embodiments, the nucleic acid encoding the effector protein, the nucleic acid encoding the engineered guide nucleic acid, or a combination thereof is an expression vector. In some embodiments, the expression vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.[8] Also provided herein are pharmaceutical compositions comprising any one of the compositions described herein, and a pharmaceutically acceptable excipient.[9] Also provided herein are systems comprising: (a) an effector protein, or a nucleic acid encoding the effector protein, comprising an amino acid sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-161, and 391-847; and (b) an engineered guide nucleic acid, or a nucleic acid encoding a guide nucleic acid, comprising a protein binding sequence and a spacer sequence. In some embodiments, the protein binding sequence comprises a nucleotide sequence that is 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% identical to SEQ ID NOs: 178-373, 375-390, and 848-1168, listed in TABLE 2. In some embodiments, the system comprises a combination of the effector protein and the engineered guide nucleic acid comprising a protein binding sequence as described in TABLE 2. In some embodiments, the spacer sequence is 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% identical, complementary, or reverse complementary to a eukaryotic sequence. In some embodiments, the spacer sequence is linked to a 5’ end of the protein binding sequence. In some embodiments, the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about520, about 540 contiguous amino acids of the sequence selected from SEQ ID NOs: 1-161, and 391- 847.

[0010] Provided herein are methods of detecting a target nucleic acid in a sample comprising: a) contacting the sample with: i) a composition, a pharmaceutical composition, or a system disclosed herein and ii) a reporter nucleic acid comprising a detectable moiety that produces a detectable signal in the presence of the target nucleic acid and the composition, pharmaceutical composition, or system and b) detecting the detectable signal.

[0011] Also provided herein are methods of modifying a target nucleic acid comprising contacting the target nucleic acid with a composition, a pharmaceutical composition, or a system disclosed herein, thereby modifying the target nucleic acid.

[0012] Also provided herein are cells comprising a composition disclosed herein. In some embodiments, the cells are modified by a composition, a pharmaceutical composition, or a system disclosed herein.

[0013] Also provided herein are cells comprising a modified target nucleic acid, where the modified target nucleic acid is a target nucleic acid modified as disclosed herein.

[0014] Also provided herein are methods of treating a disease comprising administering to a subject in need thereof any one of: a composition, a pharmaceutical composition, the components of a system, and a cell as disclosed herein.INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety for any purpose and to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF DRAWINGS

[0016] FIG. 1 depicts a phylogenetic tree with clusters that comprise proteins described herein.Clusters and respective protein are further described herein.DETAILED DESCRIPTION OF THE INVENTION

[0017] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.

[0018] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0019] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Definitions

[0020] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0021] The terms, “a,” “an,” and “the,” as used herein, include plural references unless the context clearly dictates otherwise.

[0022] The terms, “or” and “and / or,” as used herein, include any and all combinations of one or more of the associated listed items.

[0023] The terms, “including,” “includes,” “included,” and other forms, are not limiting.

[0024] The terms, “comprise” and its grammatical equivalents, as used herein, 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.

[0025] The term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0026] The terms, “% identical,” “% identity,” “percent identity,” and grammatical equivalents thereof, as used herein, in the context of an amino acid sequence or nucleotide sequence, refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include AUIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Uipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BEAST (Altschul er al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al. , Nucleic Acids Res. 1984 Jan 11;12(1 Pt l):387-95).

[0027] The terms, “% complementary”, “% complementarity”, “percent complementary”, “percent complementarity” and grammatical equivalents thereof, as used interchangeably herein, in the context of two or more nucleic acid molecules, refer to the percent of nucleotides in two nucleotide sequences in said nucleic acid molecules of equal length that can undergo cumulative base pairing at two or more individual corresponding positions in an antiparallel orientation. Accordingly, the terms include nucleic acid sequences that are not completely complementary over their entire length, which indicates that the two or more nucleic acid molecules include one or more mismatches. A “mismatch” is present at any position in the two opposed nucleotides that are not complementary. The % complementary is calculated by dividing the total number of the complementary residues by the total number of the nucleotides in one of the equal length sequences, and multiplying by 100. Complete or totalcomplementarity describes nucleotide sequences in 100% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. “Partially complementarity” describes nucleotide sequences in which at least 20%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some instances, at least 50%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some instances, at least 70%, 80%, 90% or 95%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. “Noncomplementary” describes nucleotide sequences in which less than 20% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.

[0028] The term, “percent similarity,” or “% similarity,” as used herein, in the context of an amino acid sequence, refers to a value that is calculated by dividing a similarity score by the length of the alignment. The similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA., 89: 10915-10919 (1992)) that is transformed so that any value > I is replaced with +1 and any value < 0 is replaced with 0. For example, an He (I) to Leu (L) substitution is scored at +2.0 by the BLOSUM62 similarity matrix, which in the transformed matrix is scored at +1. This transformation allows the calculation of percent similarity, rather than a similarity score. Alternately, when comparing two full protein sequences, the proteins can be aligned using pairwise MUSCLE alignment. Then, the % similarity can be scored at each residue and divided by the length of the alignment. For determining % similarity over a protein domain or motif, a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved. In calculating the similarity of a domain or motif, the second and third levels of the multilevel sequence are treated as equivalent to the top level. Additionally, if a substitution could be treated as conservative with any of the amino acids in that position of the multilevel consensus sequence, +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence QIQ would receive three points. This is because in the transformed BLOSUM62 matrix, each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1. For each position, the highest score is used when calculating similarity. The % similarity can also be calculated using commercially available programs, such as the Geneious Prime software given the parameters matrix = BLOSUM62 and threshold > 1.

[0029] For the purposes of calculating % identity and % complementarity, thymine (T) may be considered the same as uracil (U).

[0030] The terms, “amplification,” “amplifying,” and grammatical equivalents thereof, as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.

[0031] The terms, “bind,” “binding,” “interact” and “interacting,” as used herein, refer to a non- covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptideand a nucleic acid; between a polypeptide / guide nucleic acid complex and a target nucleic acid; and the like). While in a state of noncovalent 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). Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. 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.

[0032] The term, “base editor,” as used herein, refers to a polypeptide or fusion protein comprising a base editing activity. The polypeptide with base editing activity may be referred to as an effector partner. The base editor can differ from a naturally occurring base editing enzyme. It is understood that any reference to a base editor herein also refers to a base editing enzyme variant. The base editor is functional when the effector protein is coupled to a guide nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein). Also, by way of non-limiting example, the base editing enzyme may comprise deaminase activity.

[0033] The term, “catalytically inactive effector protein,” as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity. The naturally-occurring effector protein may be a wildtype protein. In some instances, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein.

[0034] The term, “cis 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 (e.g. , an RNP complex), 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.

[0035] The term, “codon optimized,” as used herein, refers to a mutation of a nucleotide sequence encoding a polypeptide, such as a nucleotide sequence encoding an effector protein, to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded polypeptide remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized nucleotide sequence encoding an effector protein could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized nucleotide sequence encoding an effector protein could be generated. As another non-limiting example, if the intended host cell were a eukaryotic cell, then a eukaryote codon- optimized nucleotide sequence encoding an effector protein could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell, then a prokaryote codon-optimized nucleotide sequence encoding an effector protein could be generated. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp / codon.

[0036] The terms, “complementary” and “complementarity,” as used herein, in the context of a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that can undergo cumulative base pairing with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid in antiparallel orientation. For example, when every nucleotide in a polynucleotide or a specified portion thereof forms a base pair with every nucleotide in an equal length sequence of a reference nucleic acid, that polynucleotide is said to be 100% complementary to the sequence of 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 can be referred to as its complementary nucleotide. The complementarity of modified or artificial base pairs can be based on other types of hydrogen bonding and / or hydrophobicity of bases and / or shape complementarity between bases.

[0037] The term, “cleavage assay,” as used herein, refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some instances, the cleavage activity may be cis cleavage activity.

[0038] The terms, “cleave,” “cleaving” and “cleavage,” as used herein, in the context of 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 side of 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 another organism.

[0040] The term, “conservative substitution,” as used herein, refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, the term “non-conservative substitution” as used herein refers to thereplacement of one amino acid residue for another that does not have a related side chain. Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), lie (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gin (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).

[0041] The term, “detectable product,” as used herein, refers to a unit produced after the cleavage of a reporter that is capable of being discovered, identified, perceived or noticed. A detectable product can comprise a detectable label and / or moiety that emits a detectable signal. A detectable product may include other components that are not capable of being readily discovered, identified, perceived or noticed at the same time as the detectable signal. For example, a detectable product may comprise remnants of the reporter. Accordingly, in some instances, the detectable product comprises RNA and / or DNA.

[0042] The term, “detectable signal,” as used herein, refers to an act, event, physical quantity or impulse that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.

[0043] The term, “diseased cell,” as used herein, refers to a cell comprising pathway conditions or pathway systems that are not conducive to cell survival, tissue survival, systemic survival, or organism survival.

[0044] The term, “edited target nucleic acid,” as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone an editing, for example, after contact with an effector protein. In some instances, the editing is an alteration in the sequence of the target nucleic acid. In some instances, the edited target nucleic acid comprises an insertion, deletion, or substitution of one or more nucleotides compared to the unedited target nucleic acid.

[0045] The term, “effector protein,” as used herein, refers to a protein, polypeptide, or peptide that is capable of interacting with a nucleic acid, such as a guide nucleic acid, to form a complex (e.g. , a RNP complex), wherein the complex interacts with a target nucleic acid.

[0046] The term, “effector partner,” as used herein, refers to a protein, polypeptide or peptide that can, in combination with an effector protein, impart some function or activity that can be used to effectuate modification(s) of a target nucleic acid described herein and / or change expression of the target nucleic acid or other nucleic acids associated with the target nucleic acid, when used in connection with compositions, systems and methods described herein.

[0047] The term, “engineered modification,” as used herein, refers to a structural change of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence. The engineered modifications of a nucleotide sequence can include chemical modification of one or more nucleobases, or a chemical change to the phosphate backbone, a nucleotide, a nucleobase or a nucleoside. The engineered modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in w / ro-transcription. cloning, enzymatic, or chemical cleavage, etc. In some instances, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can exist at various positions within the nucleic acid.

[0048] The term, “functional domain,” as used herein, refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid modifying, nucleic acid cleaving, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity.

[0049] The term, “functional fragment,” as used herein, refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, nucleic acid editing, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity. A functional fragment may be a recognized functional domain, e.g., a catalytic domain. In some instances, the catalytic domain comprises a RuvC domain.

[0050] The term, “functional protein,” as used herein, refers to protein that retains at least some if not all activity relative to the wildtype protein. A functional protein can also include a protein having enhanced activity relative to the wildtype protein. Assays are known and available for detecting and quantifying protein activity, e.g., colorimetric and fluorescent assays. In some instances, a functional protein is a wildtype protein. In some instances, a functional protein is a functional portion of a wildtype protein.

[0051] The term, “fused,” as used herein, refers to at least two sequences that are connected together, such as by a covalent bond (e.g., an amide bond or a phophodiester bond) or by a linker. The covalent bond can be formed by conjugation (e.g., chemical conjugation or enzymatic conjugation) reaction.

[0052] The term, “fusion protein,” as used herein, refers to a protein comprising at least two polypeptides. The fusion protein may comprise one or more effector proteins and effector partners. Insome instances, an effector protein and effector partner are not found connected to one another as a native protein or complex that occurs together in nature.

[0053] The term, “genetic disease,” as used herein, refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.

[0054] The term, “guide nucleic acid,” as used herein, refers to a nucleic acid that, when in a complex with one or more polypeptides described herein (e.g. , an RNP complex) can impart sequence selectivity to the complex when the complex interacts with a target nucleic acid. A guide nucleic acid may be referred to interchangeably as a guide RNA, however it is understood that guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0055] The term, “heterologous,” as used herein, refers to at least two different polypeptide sequences that are not found similarly connected to one another in a native nucleic acid or protein. A protein that is heterologous to the effector protein is a protein that is not covalently linked by an amide bond to the effector protein in nature. In some instances, a protein is heterologous when the protein is not encoded by a species that encodes the effector protein. A guide nucleic acid may comprise “heterologous” sequences, which means that it includes a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked by a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid. A heterologous system comprises at least one component that is not naturally occurring together with remaining components of the heterologous system.

[0056] The terms, “hybridize,” “hybridizable” and grammatical equivalents thereof, refer to a nucleotide sequence that is able to noncovalently interact, i.e. form Watson-Crick base pairs and / or G / U base pairs, or anneal, to another nucleotide sequence in a sequence-specific, antiparallel, manner (i. e. , a nucleotide sequence specifically interacts to a complementary nucleotide sequence) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) for both DNA and RNA. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.) guanine (G) can also base pair with uracil (U). For example, G / U base-pairing is at least partially responsible for the degeneracy (i.e. , redundancy) of the genetic code in the context of tRNA anti -codon base-pairing with codons in mRNA. Thus, a guanine (G) can be considered complementary to both an uracil (U) and to an adenine (A). Accordingly, when a G / U base-pair can be made at a given nucleotide position, the position is not considered to be non-complementary, but is instead considered to becomplementary. While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, hybridizable, partially hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. , a bulge, a loop structure or hairpin structure, etc.). The conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables which are well known in the art. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches may become important (see Sambrook et al. , supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Any suitable in vitro assay may be utilized to assess whether two sequences “hybridize”. One such assay is a melting point analysis where 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. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. Hybridization and washing conditions are well known and exemplified in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001); and in Green, M. and Sambrook, J., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2012).

[0057] The term, “indel,” as used herein, refers to an insertion-deletion or indel mutation, which is a type of genetic mutation that results from the insertion and / or deletion of one or more nucleotide in a target nucleic acid. An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected by any suitable method, including sequencing.

[0058] The term, “indel percentage,” as used herein, refers to a percentage of sequencing reads that show at least one nucleotide has been edited from the insertion and / or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides edited. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given effector protein.

[0059] The term, “in vitro,” as used herein, refers to describing something outside an organism. An in vitro system, composition or method may take place in a container for holding laboratory reagents suchthat it is separated from the biological source from which a material in the container is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. The term “in vivo” is used to describe an event that takes place within an organism. The term “ex vivo” is used to describe an event that takes place in a cell that has been obtained from an organism. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.

[0060] The terms, “length” and “linked” as used herein, refer to a nucleic acid (polynucleotide) or polypeptide, may be expressed as “kilobases” (kb) or “base pairs (bp),”. Thus, a length of 1 kb refers to a length of 1000 linked nucleotides, and a length of 500 bp refers to a length of 500 linked nucleotides. Similarly, a protein having a length of 500 linked amino acids may also be simply described as having a length of 500 amino acids.

[0061] The term, “linker,” as used herein, refers to a molecule that links a first polypeptide to a second polypeptide (e.g., by an amide bond) or a first nucleic acid to a second nucleic acid (e.g., by a phosphodiester bond).

[0062] The term, “mutation,” as used herein, refers to an alteration that changes an amino acid residue or a nucleotide as described herein. Such an alteration can include, for example, deletions, insertions, and / or substitutions. The mutation can refer to a change in structure of an amino acid residue or nucleotide relative to the starting or reference residue or nucleotide. A mutation of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. A mutation of a nucleotide includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. A mutation of a nucleotide base may result in a structural and / or functional alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue of the peptide, polypeptide or protein. A mutation of a nucleotide base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation. Methods of mutating an amino acid residue or a nucleotide are well known.

[0063] The terms, “mutation associated with a disease” and “mutation associated with a genetic disorder,” as used herein, refer to the co-occurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation.

[0064] The term, “nickase,” as used herein, refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid.

[0065] The term, “nickase activity,” as used herein, refers to catalytic activity that results in single stranded nucleic acid cleavage of a double stranded nucleic acid.

[0066] The terms, “non-naturally occurring” and “engineered,” as used herein, refer to indicate involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule. 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 may 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.

[0067] The terms, “nuclease” and “endonuclease” as used herein, refer to an enzyme which possesses catalytic activity for nucleic acid cleavage.

[0068] The term, “nuclease activity,” as used herein, refers to catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[0069] The term, “nucleic acid,” as used herein, refers to a polymer of nucleotides. A nucleic acid may comprise ribonucleotides, deoxyribonucleotides, combinations thereof, and modified versions of the same. A nucleic acid may be single- stranded or double-stranded, unless specified. Non-limiting examples of nucleic acids are double stranded DNA (dsDNA), single stranded (ssDNA), messenger 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. Accordingly, nucleic acids as described herein may comprise one or more mutations, one or more engineered modifications, or both.

[0070] The term, “nucleic acid expression vector,” as used herein, refers to a plasmid that can be used to express a nucleic acid of interest.

[0071] The term, “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g. , peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[0072] The terms, “nucleotide(s)” and “nucleoside(s)”, as used herein, in the context of a nucleic acid molecule having multiple residues, refer to describing the sugar and base of the residue contained in the nucleic acid molecule. Similarly, a skilled artisan could understand that linked nucleotides and / or linked nucleosides, as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule. When referringto a “nucleobase(s)”, or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides. A person of ordinary skill in the art when referring to nucleotides, nucleosides, and / or nucleobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'- AXG where X is any modified uridine, such as pseudouridine, Nl-methyl pseudouridine, or 5- methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5' -CAU).

[0073] The term, “pharmaceutically acceptable excipient, carrier or diluent,” as used herein, refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system. Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility. The selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by suitable methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0074] The terms, “polypeptide” and “protein,” as used herein, refer to a polymeric form of amino acids. A polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more engineered modifications, or both. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some instances, when a heterologous peptide, such as an effector partner, protein tag or NLS, is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient forinitiating translation (e.g., Methionine (M) or a Valine (V)) of the effector protein may be removed or absent.

[0075] The term, “prime editing enzyme”, as used herein, refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the editing (insertion, deletion, or base-to-base conversion) of a target nucleotide or nucleotide sequence in a nucleic acid.

[0076] The terms, “promoter” and “promoter sequence,” as used herein, refer to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3’ direction) coding or non-coding sequence. A transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase, can also be found in a promoter region. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure.

[0077] The terms, “protospacer adjacent motif’ and “PAM,” as used herein, refer to a nucleotide sequence found in a target nucleic acid that directs an effector protein to edit the target nucleic acid at a specific location. In some instances, a PAM is required for a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex) to hybridize to and edit the target nucleic acid. In some instances, the complex does not require a PAM to edit the target nucleic acid.

[0078] The term, “recombinant,” as used herein, in the context of proteins, polypeptides, peptides and nucleic acids, refers to proteins, polypeptides, peptides and nucleic acids that are products of various combinations of cloning, restriction, 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.

[0079] The term, “regulatory element,” used herein, refers 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., a guide nucleic acid) or a coding sequence (e.g., effector proteins, fusion proteins, and the like) and / or regulate translation of an encoded polypeptide.

[0080] The terms, “reporter,” “reporter nucleic acid,” and “reporter molecule,” as used herein, are used interchangeably and refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.

[0081] The term “retRNA” as used herein, refers to a nucleic acid comprising: a primer binding sequence and a template sequence. It is understood that retRNAs may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). In some instances, the template RNA is linked to a guide RNA. In some instances, the template RNA is not linked to a guide RNA.

[0082] The terms, “ribonucleotide protein complex” and “RNP” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes“ribonucleotides” it is understood that the one or more nucleic acid may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.

[0083] The terms, “RuvC” and “RuvC domain,” as used herein, refer to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain instances, of processing a pre-crRNA. In some instances, the RuvC domain is located near the C-terminus of the effector protein. A single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (https: / / www.ebi.ac.uk / interpro / ). For example, a RuvC-like domain may be a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons.

[0084] The term, “sample,” as used herein, refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.

[0085] The term, “single nucleic acid system,” as used herein, refers to a system that uses a guide nucleic acid complexed with one or more polypeptides described herein, wherein the complex is capable of interacting with a target nucleic acid in a sequence specific manner, and wherein the guide nucleic acid is capable of non-covalently interacting with the one or more polypeptides described herein, and wherein the guide nucleic acid is capable of hybridizing with a target sequence of the target nucleic acid. A single nucleic acid system lacks a duplex of a guide nucleic acid as hybridized to a second nucleic acid, wherein in such a duplex the second nucleic acid, and not the guide nucleic acid, is capable of interacting with the effector protein.

[0086] The term, “spacer sequence,” as used herein, refers to a nucleotide sequence in a guide nucleic acid that is capable of, at least partially, hybridizing to an equal length portion of a sequence (e.g., a target sequence) of a target nucleic acid.

[0087] The term, “subject,” as used herein, refers to an animal. The subject may be a mammal. The subject may be a human. The subject may be diagnosed or at risk for a disease.

[0088] The term, “sufficiently complementary,” as used herein, refers to a first nucleotide sequence that is partially complementarity to a second nucleotide sequence while still allowing the first nucleotide sequence to hybridize to the second nucleotide sequence with enough affinity to permit a biological activity to occur. Depending on the context, a biological activity may be the formation of a complex between two or more components described herein, such as an effector protein and a guide nucleic acid. A biological activity may also be bringing one or more components described herein into proximity ofanother component, such as bringing an effector protein-guide nucleic acid complex into proximity of a target nucleic acid. A biological activity may additionally be permitting a component described herein to act on another component described herein, such as permitting an effector protein to cleave a target nucleic acid. In some instances, sequences are said to be sufficiently complementary when at least 85% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.

[0089] The term, “syndrome,” as used herein, refers to a group of symptoms which, taken together, characterize a condition.

[0090] The term, “target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for editing, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g. , single-stranded RNA or singlestranded DNA) or double-stranded (e.g., double-stranded DNA).

[0091] The term, “target sequence,” as used herein, in the context of a target nucleic acid, refers to a nucleotide sequence found within a target strand of a target nucleic acid. Such a nucleotide sequence can, for example, hybridize to a respective length portion of a guide nucleic acid.

[0092] The terms, “target strand (TS)” and “non-target strand (NTS)” are used herein to differentiate between the strands of a double stranded DNA molecule to which a guide nucleic acid does or does not hybridize. The term, “target strand (TS),” as used herein, in the context of a target nucleic acid, refers to a strand of a double stranded DNA molecule that comprises a target sequence, to which at least a portion of a guide nucleic acid (e.g., a spacer sequence) hybridizes. The term, “non-target strand (NTS),” as used herein, in the context of a target nucleic acid, refers to a strand of a double stranded DNA molecule to which a guide nucleic acid does not hybridize. Reference may be made to a target sequence of the TS and a target sequence of the NTS. A guide nucleic acid spacer sequence is generally complementary to the target sequence of the TS and generally identical to the target sequence of the NTS.

[0093] The term, "trans cleavage,” as used herein, in the context of cleavage (e.g., hydrolysis of a phosphodiester bond) of one or more target nucleic acids or non-target nucleic acids, or both, by an effector protein that is complexed with a guide nucleic acid and the target nucleic acid. Trans cleavage activity may be triggered by the hybridization of a guide nucleic acid to a target nucleic acid. Trans cleavage of the target nucleic acid may occur away from (e.g., not within or directly adjacent to) the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.

[0094] The term, “transcriptional activator,” as used herein, refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule.

[0095] The term, “transcriptional repressor,” as used herein, refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid.

[0096] The term, “transgene,” as used herein, refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell. A transgene is meant to include (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced. The cell in which transgene expression occurs can be a target cell, such as a host cell.

[0097] The terms, “treatment” and “treating,” as used herein, refer 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 may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved 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 may 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 may undergo treatment, even though a diagnosis of this disease may not have been made.

[0098] The term, “variant,” as used herein, refers to a form or version of a protein that differs from the wild-type protein. A variant may have a different function or activity relative to the wild-type protein.

[0099] The term, “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle.Introduction

[0100] Disclosed herein are compositions, systems, and methods comprising at least one of: (1) an effector protein or a nucleic acid encoding the effector protein; and (2) an engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid. The term, “system,” may be used herein for embodiments in which the effector protein or a nucleic acid encoding the effector protein; and the engineered guide nucleic acid or a nucleic acid encoding the engineered guide nucleic acid are not provided in the same container or solution. In some embodiments, these system components are packaged separately, e.g., to be administered separately.

[0101] Effector proteins described herein may bind and cleave (e.g., nick) nucleic acids in a sequencespecific manner. Effector proteins described herein may also cleave (e.g., nick) the target nucleic acidwithin a target sequence or at a position adjacent to the target sequence. An effector protein may be similar to a CRISPR-associated (Cas) protein in that it may bind a guide nucleic acid that imparts activity or sequence selectivity to the polypeptide. However, unlike typical Cas proteins, effector proteins described herein are not found near a CRISPR array, which includes direct repeats flanking short spacer sequences. Typically, Cas proteins are found within ~5kb of such an array. In contrast, effector proteins described herein may not have a CRISPR array within 5kb of their corresponding genomic locus. In some embodiments, effector proteins described herein are similar to an IscB protein.

[0102] In some embodiments, compositions, systems, and methods comprising guide nucleic acids comprise a first region or sequence, at least a portion of which interacts with a polypeptide. In some embodiments, compositions, systems, and methods comprising guide nucleic acids comprise a second sequence that is at least partially complementary to a target nucleic acid, and which may be referred to as a spacer sequence.

[0103] Effector proteins disclosed herein may bind and cleave (e.g., nick) nucleic acids, including double stranded RNA (dsRNA), single-stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). Polypeptides disclosed herein may provide cis cleavage activity, trans cleavage activity, binding activity, nickase activity, or a combination thereof.

[0104] The compositions, systems, and methods described herein are non-naturally occurring. In some embodiments, compositions, systems, and methods comprise an engineered guide nucleic acid (also referred to herein as a guide nucleic acid) or a use thereof. In some embodiments, compositions, systems, and methods comprise an engineered protein or a use thereof. In some embodiments, compositions, systems, and methods comprise an isolated polypeptide or a use thereof. In general, compositions, methods, and systems described herein are not found in nature. In some embodiments, compositions, methods, and systems described herein comprise at least one non-naturally occurring component. For example, disclosed compositions, methods, and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally- occurring guide nucleic acid.

[0105] In some embodiments, compositions, systems, and methods comprise at least two components that do not naturally occur together. For example, disclosed compositions, systems, and methods may comprise a guide nucleic acid comprising a first region, at least a portion of which, interacts with a polypeptide, and a second region that is at least partially complementary to a target sequence in a target nucleic acid, wherein the first region and second region do not naturally occur together and / or are heterologous to each other. Also, by way of non-limiting example, disclosed compositions, systems, and methods may comprise a guide nucleic acid and an effector protein that do not naturally occur together. Likewise, by way of non-limiting example, disclosed compositions, systems, and methods may comprise a ribonucleotide-protein (RNP) complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. Conversely, and for clarity, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes effector proteins andguide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.

[0106] In some embodiments, the guide nucleic acid comprises a non-natural nucleotide sequence. In some embodiments, the non-natural nucleotide sequence is a nucleotide sequence that is not found in nature. The non-natural nucleotide sequence may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some embodiments, the guide nucleic acid comprises two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, compositions and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid that do not occur together in nature. In some embodiments, compositions and systems comprise at least two components that do not occur together in nature, wherein the at least two components comprise at least one of an effector protein, an effector partner and a guide nucleic acid. Guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. A guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3 ’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. In some embodiments, the guide nucleic acid comprises two heterologous sequences arranged in an order or proximity that is not observed in nature. Therefore, compositions and systems described herein are not naturally occurring.

[0107] In some embodiments, compositions, systems, and methods described herein comprise a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof) that is similar to a naturally occurring polypeptide. The polypeptide may lack a portion of the naturally occurring polypeptide. The polypeptide may comprise a mutation relative to the naturally-occurring polypeptide, wherein the mutation is not found in nature. The polypeptide may also comprise at least one additional amino acid relative to the naturally-occurring polypeptide. In some embodiments, the polypeptide may comprise a heterologous peptide. For example, the polypeptide may comprise an addition of a nuclear localization signal relative to the natural occurring polypeptide. In some embodiments, a nucleotide sequence encoding the polypeptide is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.Effector Proteins

[0108] Provided herein, in certain embodiments, are compositions that comprise one or more effector proteins and uses thereof. Also provided herein are compositions that comprise a nucleic acid, wherein the nucleic acid encodes any of one the effector proteins described herein. The nucleic acid may be a nucleic acid expression vector. By way of non-limiting example, the nucleic acid expression vector may be a viral vector, such as an AAV vector.

[0109] An effector protein provided herein interacts with a guide nucleic acid to form a complex. In some embodiments, the complex interacts with a target nucleic acid, a non-target nucleic acid, or both. In some embodiments, an interaction between the complex and a target nucleic acid, a non-target nucleic acid, or both comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid and / or the non-target nucleic acid by the effector protein, or combinations thereof. In some embodiments, recognition of a PAM sequence within a target nucleic acid may direct the modification activity of an effector protein.[HO] In some embodiments, the effector protein described herein may bind and, optionally, modify nucleic acids in a sequence-specific manner. Effector proteins described herein may also modify the target nucleic acid within a target sequence or at a position adjacent to the target sequence. In some embodiments, an effector protein is activated when it binds a certain sequence of a nucleic acid described herein, allowing the effector protein to modify a region of a target nucleic acid that is near, but not adjacent to the target sequence. Effector proteins may modify a nucleic acid by cis cleavage or trans cleavage. Alternatively or additionally, effector proteins described herein modify a non-target nucleic acid by trans cleavage on the non-target nucleic acid. In some embodiments, effector proteins may have nickase activity or nuclease activity. Effector proteins disclosed herein may modify nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). The modification of the target nucleic acid generated by an effector protein, as a nonlimiting example, may result in modulation of the expression of the nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g., inactivation of a protein binding to an RNA molecule or hybridization).[Hl] In some embodiments, effector proteins described herein comprise modification activities. In some embodiments, the modification activity of the polypeptide described herein may be nickase activity for a double stranded nucleic acid, binding activity, insertion activity, substitution activity, chemical modification activity, or a combination thereof. In some embodiments, the modification activity of the polypeptide may result in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, chemical modification of one or more nucleotides of a target nucleic acid to an alternative nucleotide, or combinations thereof. In some embodiments, the cleavage activity is a nicking activity.

[0112] An effector protein may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). In some embodiments, an effector protein, when functioning in a multiprotein complex, may have differing and / or complementary functional activity to other effector proteins in the multiprotein complex. Aneffector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g., modifying a target nucleic acid). The first and second effector proteins may be the same. The first and second effector proteins may be different. The sequences of the first and second effector proteins may be 15% to 20% identical, 20% to 25% identical, 25% to 30% identical, 30% to 35% identical, 35% to 40% identical, 40% to 45% identical, 45% to 50% identical, 50% to 55% identical, 55% to 60% identical, 60% to 65% identical, 65% to 70% identical, 70% to 75% identical, 75% to 80% identical, 80% to 85% identical, 85% to 90% identical, 90% to 95% identical, 95% to 99.9% identical, or 100% identical.

[0113] An effector protein may be a modified effector protein having increased modification activity (e.g., catalytic activity) and / or increased substrate binding activity (e.g., substrate selectivity, specificity, and / or affinity). An effector protein may be a modified effector protein having reduced modification activity (e.g., a catalytically defective effector protein) or no modification activity (e.g., a catalytically inactive effector protein). Accordingly, an effector protein as used herein encompasses a modified or programmable nuclease that does not have nuclease activity.

[0114] In some embodiments, effector proteins described herein comprise one or more functional domains. Effector protein functional domains can include a protospacer adjacent motif (PAM)- interacting domain, an oligonucleotide-interacting domain, one or more recognition domains, a nontarget strand interacting domain, and HNH domain, a RuvC domain, or a combination thereof. A PAM interacting domain can be a target strand PAM interacting domain (TPID) or a non-target strand PAM interacting domain (NTPID). In some embodiments, a PAM interacting domain, such as a TPID or a NTPID, on an effector protein describes a region of an effector protein that interacts with target nucleic acid. In some embodiments, effector proteins comprise one or more recognition domain (REC domain) with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. An effector protein may comprise a zinc finger domain.

[0115] In some embodiments, the effector protein comprises an HNH domain. In some embodiments, the effector protein comprises an HNH domain and a RuvC domain. In some embodiments, the effector protein may have a mutation in a nuclease domain. In some embodiments, the nuclease domain is a RuvC domain. In some embodiments, the nuclease domain is an HNH domain. An HNH domain may be characterized as comprising two antiparallel (3-strands connected with a loop of varying length, and flanked by an a-helix, with a metal (divalent cation) binding site between the two (3-strands. A RuvC domain may be characterized by a six-stranded beta sheet surrounded by four alpha helices, with three conserved subdomains contributing catalytic to the activity of the RuvC domain. In some embodiments, a RuvC domain comprises with substrate binding activity, catalytic activity, or both. In some embodiments, the RuvC domain may be defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multiple RuvC subdomains, which may combineto generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary amino acid sequence of the effector protein but form a RuvC domain once the protein is produced and folds.

[0116] When describing a mutation that changes an amino acid residue or a nucleotide as described herein, such a change or changes can include, for example, deletions, insertions, and / or substitutions. The mutation can refer to a change in structure of an amino acid residue or nucleotide relative to the starting or reference residue or nucleotide. A mutation of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein. A mutation of a nucleotide includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine. A mutation of a nucleotide base may result in a structural and / or functional alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue of the peptide, polypeptide or protein. A mutation of a nucleotide base may not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation.

[0117] Described herein are illustrative amino acid sequences of effector proteins modified for use in the compositions, systems and methods described herein. In general, an effector protein described herein does not comprise an amino acid sequence that is identical to any one of the amino acid sequences selected from SEQ ID NOs: 1-161 and 391-847.

[0118] In some embodiments, compositions, systems and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least 200 contiguous amino acids or more of the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847, wherein the amino acid sequence of the effector protein is not identical to any one of the amino acid sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the amino acid sequence of an effector protein provided herein comprises at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, at least 380, at least 400 contiguous amino acids, at least 420 contiguous amino acids, at least 440 contiguous amino acids, at least 460 contiguous amino acids, at least 480 contiguous amino acids, at least 500 contiguous amino acids of a sequence selected from SEQ ID NOs: 1-161 and 391-847.

[0119] In some embodiments, the amino acid sequence of an effector protein provided herein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540 contiguous amino acids of a sequenceselected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the length of the effector protein is less than 600, less than 580, less than 560, less than 540, less than 520, less than 500, less than 480, less than 460, less than 440, less than 420, or less than 400 contiguous amino acids; and at least 300, at least 320, at least 340, at least 360, at least 380 contiguous amino acids of a sequence selected from SEQ ID NOs: 1-161 and 391-847.

[0120] In some embodiments, compositions, systems and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises a portion of the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the effector protein comprises a portion of the amino acid selected from SEQ ID NOs: 1-161 and 391-847, wherein the portion does not comprise at least the first 10 amino acids, at least the first 20 amino acids, at least the first 40 amino acids, at least the first 60 amino acids, at least the first 80 amino acids, at least the first 100 amino acids, at least the first 120 amino acids, at least the first 140 amino acids, at least the first 160 amino acids, at least the first 180 amino acids, or at least the first 200 amino acids of the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the effector protein comprises a portion of the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847, wherein the portion does not comprise the last 10 amino acids, the last 20 amino acids, the last 40 amino acids, the last 60 amino acids, the last 80 amino acids, the last 100 amino acids, the last 120 amino acids, the last 140 amino acids, the last 160 amino acids, the last 180 amino acids, or the last 200 amino acids of the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847.

[0121] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the 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 97%, at least 98%, at least 99%, but less than 100% identical to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 70% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391- 847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 75% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% but less than 100% identical to the aminoacid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391- 847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% but less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is less than 100% identical to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847.

[0122] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391- 847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1- 161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is 100% similar, but not the same, to the amino acid sequence as selected from SEQ ID NOs: 1-161 and 391-847.

[0123] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more alterations comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more amino acid alterations relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more alterations comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, sixteen to twenty, or more amino acid alterations relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more amino acid alterations comprises substitutions (e.g., conservative substitutions, nonconservative substitutions), deletions, or combinations thereof. In some embodiments, an effector protein or a nucleic acid encoding the effector protein comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations or more relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391- 847.

[0124] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more substitutions relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more substitutions relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, sixteen to twenty, or more substitutions relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more substitutions comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more amino acid substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more substitutions relative to the amino acid sequence selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues. In some embodiments, the positively charged amino acid residue is independently selected from Lys (K), Arg (R), or His (H). In some embodiments, the one or more substitutions comprise one or more conservative substitutions, one or more non-conservative substitutions, or combinations thereof.

[0125] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprisesone or more conservative substitutions relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more conservative substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more conservative substitutions relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more conservative substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty conservative substitutions relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more conservative substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more conservative substitutions relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847.

[0126] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more non-conservative substitutions relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more conservative substitutions comprises not more than one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the amino acid sequence relative to any one of the sequences selected from SEQ ID NOs: 1-161 and 391-847. In some embodiments, the one or more non-conservative substitutions are in a RuvC domain, HNH domain, or both. In some embodiments, the non-conservative substitution(s) replaces a catalytic residue(s) of the domain(s).

[0127] In some embodiments, the one or more amino acid alterations may result in a change in activity of the effector protein relative to a naturally-occurring counterpart. In some embodiments, the one or more amino acid alterations does not result in a change in activity of the effector protein relative to a naturally-occurring counterpart. For example, and as described in further detail below, the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a naturally-occurring counterpart. In another example, the one or more amino acid alteration increases or decreases binding activity of the effector protein relative to a naturally-occurring counterpart.Catalytically Inactive Proteins

[0128] An effector protein that has decreased catalytic activity may be referred to as catalytically or enzymatically inactive, catalytically or enzymatically dead, as a dead protein or a dCas protein. In some embodiments, such a protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain). For example, a nuclease domain (e.g., RuvC domain or HNH domain) of an effector protein may be deleted or mutated relative to a wildtype counterpart so that it is no longer functional or comprises reduced nuclease activity. In some embodiments, a catalytically inactive effector protein may bind to a guide nucleic acid and / or a target nucleic acid but does not cleave the target nucleic acid. Insome embodiments, a catalytically inactive effector protein may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid. In some embodiments, a catalytically inactive effector protein is fused to an effector partner that confers an alternative activity to an effector protein activity. Such fusion proteins are described herein and throughout.Effector Protein Activity

[0129] Effector proteins of the present disclosure may show an enhanced activity (e.g., nucleic acid binding activity, nuclease activity), when measured in a cleavage assay or a reporter assay, under certain conditions relative to a control condition. For example, the effector proteins of the present disclosure may have variable levels of activity based on a buffer formulation, a pH level, temperature, or salt. Buffers consistent with the present disclosure include phosphate buffers, Tris buffers, and HEPES buffers. In some embodiments, the effector proteins may show optimal activity in one or more of phosphate buffers, Tris buffers, and HEPES buffers.

[0130] In some embodiments, engineered proteins comprise modifications to exhibit optimal activity at lower salinity and viscosity than the protoplasm of their bacterial cell of origin. Also, by way of nonlimiting example, bacteria often comprise protoplasmic salt concentrations greater than 250 mM and room temperature intracellular viscosities above 2 centipoise, whereas engineered proteins exhibit optimal activity (e.g., cis cleavage activity) at salt concentrations below 150 mM and viscosities below 1.5 centipoise. The present disclosure leverages these dependencies by providing engineered proteins in solutions optimized for their activity and stability.

[0131] In some embodiments, effector proteins may also exhibit varying levels of activity at different pH levels. For example, enhanced nuclease activity may be observed between pH 7 and pH 9. In some embodiments, the effector proteins exhibit enhanced cleavage at about pH 7, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9, from pH 7 to 7.5, from pH 7.5 to 8, from pH 8 to 8.5, from pH 8.5 to 9, or from pH 7 to 8.5.

[0132] In some embodiments, effector proteins of the present disclosure exhibit activity or enhanced activity at a temperature of 25°C to 50°C in the presence of a target nucleic acid. For example, the effector proteins may exhibit enhanced cleavage of an ssDNA-FQ reporter at about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, from 30°C to 40°C, from 35°C to 45°C, or from 35°C to 40°C.

[0133] In some embodiments, effector proteins of the present disclosure may exhibit activity or enhanced activity under a salt concentration from 25 nM salt to 200 mM salt. Non-limiting examples of such salts are NaCl and KC1. The effector proteins may be active at salt concentrations ranging from25 nM to 500 nM, from 500 nM to 1000 nM, from 1000 nM to 2000 nM, from 2000 nM to 3000 nM, from 3000 nM to 4000 nM, from 4000 nM to 5000 nM, from 5000 nM to 6000 nM, from 6000 nM to 7000 nM, from 7000 nM to 8000 nM, from 8000 nM to 9000 nM, from 9000 nM to 0.01 mM, from 0.01 mM to 0.05 mM, from 0.05 mM to 0. 1 mM, from 0. 1 mM to 10 mM, from 10 mM to 100 mM, or from 100 mM to 500 mM. The effector proteins may exhibit cleavage activity independent of the salt concentration in a sample.

[0134] In some embodiments, effector proteins of the present disclosure may exhibit activity or enhanced activity in a solution at a room temperature viscosity of less than about 15 centipoise, less than about 12 centipoise, less than about 10 centipoise, less than about 8 centipoise, less than about 6 centipoise, less than about 5 centipoise, less than about 4 centipoise, less than about 3 centipoise, less than about 2 centipoise, or less than about 1.5 centipoise.

[0135] In some embodiments, effector proteins of the present disclosure may exhibit activity or enhanced activity in a solution comprising an ionic strength of less than about 500 mM, less than about 400 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 150 mM, less than about 100 mM, less than about 80 mM, less than about 60 mM, or less than about 50 mM. In some embodiments, effector proteins may exhibit activity or enhanced activity with an assay excipient, which may stabilize a reagent or product, prevent aggregation or precipitation, or enhance or stabilize a detectable signal (e.g., a fluorescent signal). Examples of assay excipients include, but are not limited to, saccharides and saccharide derivatives (e.g., sodium carboxymethyl cellulose and cellulose acetate), detergents, glycols, polyols, esters, buffering agents, alginic acid, and organic solvents (e.g., DMSO).

[0136] In some embodiments, effector proteins of the present disclosure may exhibit activity or enhanced activity in the presence of a co-factor. In some embodiments, the co-factor allows the effector proteins to perform a function. In some embodiments, the function is pre-crRNA processing and / or target nucleic acid cleavage. As discussed in Jiang F. and Doudna J.A. (Annu. Rev. Biophys. 2017. 46:505-29), Cas9 uses divalent metal ions as co-factors. The suitability of a divalent metal ion as a cofactor can easily be assessed, such as by methods based on those described by Sundaresan et al. (Cell Rep. 2017 Dec 26; 21(13): 3728-3739). In some embodiments, the co-factor is a divalent metal ion. Non-limiting exemplary divalent metal ions include: Mg2+, Mn2+, Zn2+, Ca2+, and Cu2+. In some embodiments, the effector protein forms a complex with a divalent metal ion. In some embodiments, the effector protein forms a complex with Mg2+, Mn2+, Zn2+, Ca2+, or Cu2+.Synthesis, Isolation and Assaying

[0137] Effector proteins of the present disclosure may be synthesized, using any suitable method. In some embodiments, the effector proteins may be produced in vitro or by eukaryotic cells or by prokaryotic cells. In some embodiments, the effector proteins may be further processed by unfolding (e.g., heat denaturation, dithiothreitol reduction, etc.) and may be further refolded, using any suitablemethod. In some embodiments, the nucleic acid(s) encoding the effector proteins described herein, the recombinant nucleic acid(s) described herein, the vectors described herein may be produced in vitro or in vivo by eukaryotic cells or by prokaryotic cells.

[0138] Any suitable method of generating and assaying the effector proteins described herein may be used. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)). One non-limiting example of a method for preparing an effector protein is to express recombinant nucleic acids encoding the effector protein in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art. Exemplary methods are also described in the Examples provided herein.

[0139] In some embodiments, an effector protein provided herein is an isolated effector protein. In some embodiments, the effector proteins may be isolated and purified for use in compositions, systems, and / or methods described herein. In some embodiments, methods described here may include the step of isolating effector proteins described herein. Any suitable method to provide isolated effector proteins described herein may be used in the present disclosure, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

[0140] In some embodiments, compositions, systems, and methods described herein may further comprise a purification tag that can be attached to an effector protein, or a nucleic acid encoding the purification tag that can be attached to a nucleic acid encoding the effector protein as described herein. In some embodiments, the purification tag may be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which may be its biological source, such as a cell lysate. Attachment of the purification tag may be at the N or C terminus of the effector protein. Furthermore, an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease, such as TEV protease or the HRV3C protease may be inserted between the purification tag and the effector protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag. Purification and / or isolation may be performed through highperformance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Non-limiting examples of purification tags are as described herein.

[0141] In some embodiments, effector proteins described herein are isolated from cell lysate. In some embodiments, the compositions described herein may comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, or 99.5% or more by weight of an effector protein, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages may be upon total protein content in relation to contaminants. Thus, in some embodiments, the effector protein is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-engineered proteins or other macromolecules, etc.).Protospacer Adjacent Motif (PAM) Sequences

[0142] Effector proteins of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, effector proteins described herein recognize a PAM sequence. In some embodiments, recognizing a PAM sequence comprises interacting with a sequence adjacent to the PAM. In some embodiments, a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence. In some embodiments, the effector protein does not require a PAM to bind and / or cleave a target nucleic acid.

[0143] In some embodiments, a target nucleic acid is a single stranded target nucleic acid comprising a target sequence . Accordingly, in some embodiments, the single stranded target nucleic acid comprises a PAM sequence described herein that is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) or directly adjacent to the target sequence. In some embodiments, an RNP cleaves the single stranded target nucleic acid.

[0144] In some embodiments, a target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, the PAM sequence is located on the target strand. In some embodiments, the PAM sequence is located on the non-target strand. In some embodiments, the PAM sequence described herein is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) to the target sequence on the target strand or the non-target strand. In some embodiments, the PAM sequence is located 5’ of the target sequence on the non-target strand. In some embodiments, such a PAM described herein is directly adjacent to the target sequence on the target strand or the non-target strand. In some embodiments, an RNP cleaves the target strand or the non-target strand. In some embodiments, the RNP cleaves both, the target strand and the non-target strand. In some embodiments, an RNPrecognizes the PAM sequence, and hybridizes to a target sequence of the target nucleic acid. In some embodiments, the RNP cleaves the target nucleic acid, wherein the RNP has recognized the PAM sequence and is hybridized to the target sequence.

[0145] In some embodiments, an effector protein described herein, or a multimeric complex thereof, recognizes a PAM on a target nucleic acid. In some embodiments, multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid. In some embodiments, at least two of the multiple effector proteins recognize the same PAM sequence. In some embodiments, at least two of the multiple effector proteins recognize different PAM sequences. In some embodiments, only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid.

[0146] An effector protein of the present disclosure, or a multimeric complex thereof, may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence.Effector Partners

[0147] Provided herein are compositions, systems, and methods comprising one or more effector partners or uses thereof. In some embodiments, the effector partner is a heterologous protein an effector protein described herein. In some embodiments, the effector partner is not an effector protein as described herein. In some embodiments, the effector partner is capable of imparting a function or activity that is not provided by an effector protein as described herein. In some embodiments, the effector partner comprises a second effector protein or a multimeric form thereof.

[0148] In some embodiments, an effector partner imparts a function or activity to a fusion protein comprising an effector protein that is not provided by the effector protein, including but not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g., pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity, modification of a polypeptide associated with target nucleic acid (e.g., a histone), and / or signaling activity.

[0149] In some embodiments, the effector partner is fused or linked to an effector protein described herein. In some embodiments, the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein directly or by a linker. In some embodiments, the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein directly or by a linker. In some embodiments, the effector partner may be functional when the effector protein is coupled to aguide nucleic acid. In some embodiments, the effector partner may be functional when the effector protein is coupled to a target nucleic acid. In some embodiments, the guide nucleic acid imparts sequence specific activity to the effector partner. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein) when fused or linked to an effector partner.

[0150] In some embodiments, the effector partner may directly or indirectly edit a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid. In some embodiments, the effector partner may interact with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid. In other embodiments, the effector partner may modify proteins associated with a target nucleic acid. In some embodiments, an effector partner may modulate transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid. In yet another example, an effector partner may directly or indirectly inhibit, reduce, activate or increase expression of a target nucleic acid.Reverse Transcriptase (RT) Editing System

[0151] In some embodiments, systems and methods comprise components or uses of an RT editing system to modify a target nucleic acid. RT editing may also be referred to as prime editing or precise nucleobase editing. In some embodiments, an RT editing system comprises an effector protein and an effector partner comprising an RT editing enzyme. In some embodiments, the RT editing enzyme may be referred to as a reverse DNA dependent polymerase (RDDP). Non-limiting examples of RDDPs are described in W02024 / 040202, which are incorporated herein by reference. In some embodiments, the effector protein that is linked to the RT editing enzyme. In some embodiments, an RT editing enzyme comprises a polymerase. In some embodiments, an RT editing enzyme comprises a reverse transcriptase. A non-limiting example of a reverse transcriptase is an M-MLV RT enzyme and variants thereof having polymerase activity. In some embodiments, the M-MLV RT enzyme comprises at least one mutation selected from D200N, L603W, T330P, T306K, and W313F relative to wildtype M-MLV RT enzyme. In some embodiments, systems and methods comprise an RT editing enzyme, wherein the RT editing enzyme is not fused or linked to the effector protein. In some embodiments, the RT editing enzyme comprises a recruiting moiety that recruits the RT editing enzyme to the target nucleic acid. By way of non-limiting example, the RT editing enzyme may comprise a peptide that binds an aptamer, wherein the aptamer is located on a guide RNA, template RNA, or combination thereof. Also, by way of non-limiting example, the RT editing enzyme may be linked to a protein that binds to (or is bound by) the effector protein or a protein linked / fused to the effector protein. In some embodiments, an RT editing enzyme may require an RT editing guide RNA (pegRNA) to catalyze editing. Such a pegRNA may be capable of identifying a target nucleotide or target sequence in a target nucleic acid to be edited and encoding a new genetic information that replaces the target nucleotide or target sequence in the target nucleic acid. An RT editing enzyme may require a pegRNA and a guide RNA, such as asingle guide RNA, to catalyze the editing. In some embodiments, the RT editing system comprises a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule except for at least one nucleotide. In some embodiments, the template RNA is covalently linked to a guide RNA. In some embodiments, the template RNA is not covalently linked to a guide RNA. In some embodiments, at least a portion of the template RNA hybridizes to the target nucleic acid. In some embodiments, the target nucleic acid is a dsDNA molecule. In some embodiments, at least a portion of the template RNA hybridizes to a first strand of the target nucleic acid and at least a portion of the guide RNA hybridizes to a second strand of the target nucleic acid. In some embodiments, the pegRNA comprises: a guide RNA comprising a second region that is bound by the effector protein, and a first region comprising a spacer sequence that is complementary to a target sequence of the dsDNA molecule; and a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule with the exception of at least one nucleotide. In some embodiments, the at least one nucleotide is incorporated into the target nucleic acid by activity of the RT editing enzyme, thereby modifying the target nucleic acid. In some embodiments, the spacer sequence is complementary to the target sequence on a target strand of the dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on a non-target strand of the dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the non-target strand of the dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the dsDNA molecule. In some embodiments, the target strand is cleaved. In some embodiments, the non- target strand is cleaved.

[0152] In some embodiments, the present disclosure provides a split RT editing system, comprising a first polynucleotide comprising a spacer sequence and a repeat sequence (e.g., a gRNA) and a second polynucleotide comprising a primer binding sequence and a template sequence (e.g., a retRNA), and an effector protein and RT, or fusion protein thereof described herein, or nucleic acids encoding the same.

[0153] In some embodiments, the first polynucleotide comprises a spacer sequence and a repeat sequence. In some embodiments, the first polynucleotide is a crRNA, as described above. In some embodiments, the first polynucleotide comprises a spacer sequence and protein binding sequence. In some embodiments, the first polynucleotide is a single guide RNA (sgRNA), as described above.

[0154] In some embodiments, the second polynucleotide comprises a primer binding sequence and a template sequence (e.g., a retRNA). In some embodiments, the second polynucleotide further comprises an aptamer that is recognized by a biological tether protein linked to an RT. In some embodiments, the aptamer is an MS2 aptamer (See Said et al (November 2009). "In vivo expression and purification of aptamer-tagged small RNA regulators". Nucleic Acids Research. 37 (20): el33; and Johansson et al(1997). "RNA recognition by the MS2 phage coat protein". Seminars in Virology. 8 (3): 176-185). The MS2 aptamer is an exemplary sequence and an RNA sequence with the appropriate secondary structure may also be used instead of the aptamer.

[0155] In some embodiments, the second polynucleotide comprises, from 5’ to 3’, an aptamer sequence, a template sequence, and a primer binding sequence. In some embodiments, the second polynucleotide comprises, from 5 ’ to 3 ’, template sequence, a primer binding sequence, and an aptamer sequence.Nucleic Acid Modification Activity

[0156] In some embodiments, effector partners have enzymatic activity that modifies a nucleic acid, such as a target nucleic acid. In some embodiments, the target nucleic acid may comprise or consist of a ssRNA, dsRNA, ssDNA, or a dsDNA. Examples of enzymatic activity that modifies the target nucleic acid include, but are not limited to: nuclease activity, which comprises the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids, such as that provided by a restriction enzyme, or a nuclease (e.g., FokI nuclease); methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c- methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1); DNA repair activity; DNA damage (e.g., oxygenation) activity; deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1); dismutase activity; alkylation activity; depurination activity; oxidation activity; pyrimidine dimer forming activity; integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase); transposase activity; recombinase activity such as that provided by a recombinase (e.g. , catalytic domain of Gin recombinase); polymerase activity; ligase activity; helicase activity; photolyase activity; and glycosylase activity.

[0157] In some embodiments, effector partners target a ssRNA, dsRNA, ssDNA, or a dsDNA. In some embodiments, effector partners target ssRNA. Non-limiting examples of effector partners for targeting ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and / or C to U editing enzymes); helicases; and RNA-binding proteins.

[0158] It is understood that an effector partner may include an entire protein, or in some embodiments, may include a fragment of the protein (e.g., a functional domain). In some embodiments, the functional domain binds or interacts with a nucleic acid, such as ssRNA, including intramolecular and / orintermolecular secondary structures thereof (e.g., hairpins, stem-loops, etc.). The functional domain may interact transiently or irreversibly, directly, or indirectly. In some embodiments, a functional domain comprises a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid mutating, nucleic acid modifying, nucleic acid cleaving, protein binding or combinations thereof. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity.

[0159] Accordingly, effector partners may comprise a protein or domain thereof selected from: endonucleases (e.g., RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus); SMG5 and SMG6; domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFIIm); exonucleases such as XRN-1 or Exonuclease T; deadenylases such as HNT3; protein domains responsible for nonsense mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRml60); protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (e.g., CI DI and terminal uridylate transferase); and other suitable domains that affect nucleic acid modifications.

[0160] In some embodiments, an effector partner may comprise a chromatin-modifying enzyme. In some embodiments, the effector partner chemically modifies a target nucleic acid, for example by methylating, demethylating, or acetylating the target nucleic acid in a sequence specific or non-specific manner.Base Editing Enzymes

[0161] In some embodiments, effector partners edit a nucleobase of a target nucleic acid. Such a effector partner may be referred to as a base editing enzyme. In some embodiments, a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant.

[0162] In some embodiments, a base editor is a system comprising an effector protein and a base editing enzyme. In some embodiments, the base editor comprises a base editing enzyme and an effector protein as independent components. In some embodiments, the base editor comprises a fusion protein comprising a base editing enzyme fused or linked to an effector protein. In some embodiments, the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein by the linker. In some embodiments, the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein by the linker. The base editor may be functional when the effector protein is coupled to a guide nucleic acid. The base editor may be functional when the effector protein is coupled to a target nucleic acid. The guide nucleic acid imparts sequence specific activity to the base editor. By way of non-limiting example, the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein). Also, byway of non-limiting example, the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.

[0163] In some embodiments, base editing enzymes are capable of catalyzing editing (e.g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded). In some embodiments, a base editing enzyme, and therefore a base editor, is capable of converting an existing nucleobase to a different nucleobase, such as: an adenine (A) to guanine (G); cytosine (C) to thymine (T); cytosine (C) to guanine (G); uracil (U) to cytosine (C); guanine (G) to adenine (A); hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC). In some embodiments, base editing enzymes edit a nucleobase on a ssDNA. In some embodiments, base editing enzymes edit a nucleobase on both strands of dsDNA. In some embodiments, base editing enzymes edit a nucleobase of an RNA.

[0164] In some embodiments, a base editing enzyme itself may or may not bind to the nucleic acid molecule containing the nucleobase. In some embodiments, upon binding to its target locus in the target nucleic acid (e.g., a DNA molecule), base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop”. In some embodiments, DNA bases within the R-loop are edited by the base editing enzyme having the deaminase enzyme activity. In some embodiments, base editing systems for improved efficiency in eukaryotic cells comprise a base editing enzyme, and a catalytically inactive effector protein that may generate a nick in the non-edited strand and induce repair of the non-edited strand using the edited strand as a template.

[0165] In some embodiments, a base editing enzyme comprises a deaminase enzyme. Exemplary deaminases are described in US20210198330, WO2021041945, W0202I05057IAI, and WO2020123887, all of which are incorporated herein by reference in their entirety. Exemplary deaminase domains are described WO 2018027078 and W02017070632, and each are hereby incorporated in its entirety by reference. Also, additional exemplary deaminase domains are described in Komor et al., Nature, 533, 420-424 (2016); Gaudelli et al., Nature, 551, 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-1, which are hereby incorporated by reference in their entirety. In some embodiments, the deaminase functions as a monomer. In some embodiments, the deaminase functions as heterodimer with an additional protein. In some embodiments, base editing enzymes comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)). In some embodiments, the effector partner is a deaminase, e.g., ADAR1 / 2, ADAR-2, AID, or any functional variant thereof.

[0166] In some embodiments, the base editor is a cytosine base editor (CBE), wherein the base editing enzyme is a cytosine base editing enzyme. In some embodiments, the cytosine base editing enzyme, and therefore CBE, may convert a cytosine to a thymine. In some embodiments, a cytosine base editing enzyme may accept ssDNA as a substrate but may not be capable of cleaving dsDNA, wherein the CBE comprises a catalytically inactive effector protein. In some embodiments, when bound to its cognateDNA, the catalytically inactive effector protein of the CBE may perform local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single-stranded bubble. In some embodiments, the catalytically inactive effector protein generated ssDNA R-loop may enable the CBE to perform efficient and localized cytosine deamination in vitro. In some embodiments, deamination activity is exhibited in a window of about 4 to about 10 base pairs. In some embodiments, the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which may enable the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies. In some embodiments, the CBE is capable of mediating RNA-programmed deamination of target cytosines in vitro or in vivo. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2018) Nature Biotechnology 36:848-846; Komor et al. (2016) Nature 533:420-424; Koblan et al. (2021) “Efficient C*G-to-G*C base editors developed using CRISPRi screens, targetlibrary analysis, and machine learning,” Nature Biotechnology; Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al. (2021) Nature Communications 12: 1384, all incorporated herein by reference.

[0167] In some embodiments, the effector partner comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the CBE described herein comprises UGI. Base excision repair (BER) of U*G in DNA is initiated by a uracil N-glycosylase (UNG), which recognizes a U*G mismatch and cleaves the glycosidic bond between a uracil and a deoxyribose backbone of DNA. BER results in the reversion of the U*G intermediate created by the cytosine base editing enzyme back to a C*G base pair. Accordingly, in some embodiments, the UNG may be inhibited by fusion of a UGI to the effector protein. In some embodiments, the UGI is a small protein from bacteriophage PBS. In some embodiments, the UGI is a DNA mimic that potently inhibits both human and bacterial UNG. In some embodiments, the UGI inhibitor is any protein or polypeptide that inhibits UNG.

[0168] In some embodiments, the CBE described herein may mediate efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C*G base pair to a T«A base pair through a U*G intermediate. In some embodiments, the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.

[0169] In some embodiments, the CBE described herein nicks a non-edited DNA strand. In some embodiments, the non-edited DNA strand nicked by the CBE biases cellular repair of a U*G mismatch to favor a U*A outcome, elevating base editing efficiency.

[0170] In some embodiments, a base editor described herein comprising one or more base editing enzymes (e.g., APOBEC1, UGI) that efficiently edits in mammalian cells, while minimizing frequency of non-target indels. In some embodiments, base editors do not comprise a functional fragment of the base editing enzyme. In some embodiments, base editors do not comprise a function fragment of a UGI,where such a fragment may be capable of excising a uracil residue from DNA by cleaving an N- glycosidic bond.

[0171] In some embodiments, the effector partner comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI). In some embodiments, the npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG. In some embodiments, the npUGI is a small molecule derived from uracil. Examples of small molecule non-protein uracil- DNA glycosylase inhibitors, fusion proteins, and Cas-CRISPR systems comprising base editing activity are described in WO2021087246, which is incorporated by reference in its entirety.

[0172] In some embodiments, the base editor is a cytosine base editor, wherein the based editing enzyme is a cytosine base editing enzyme. In some embodiments, the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the base editor comprising the cytidine deaminase is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety. Non-limiting exemplary cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE1 (APOBECl-XTEN-dCas9), BE2 (APOBECl-XTEN-dCas9-UGI), BE3 (APOBECl-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, and saBE4-Gam as described in WO2021163587, WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.

[0173] In some embodiments, a base editor is a cytosine to guanine base editor (CGBE), wherein the base editing enzyme is a cytosine to guanine base editing enzyme. In some embodiments, the cytosine to guanine base editing enzyme, and therefore the CGBE, may convert a cytosine to a guanine.

[0174] In some embodiments, a base editor is an adenine base editor (ABE), wherein the base editing enzyme is an adenine base editing enzyme. In some embodiments, the adenine base editing enzyme, and therefore the ABE, may convert an adenine to a guanine. In some embodiments, the adenine base editing enzyme converts an A«T base pair to a G*C base pair. In some embodiments, the adenine base editing enzyme converts a target A«T base pair to G*C in vivo or in vitro. In some embodiments, the adenine base editing enzymes provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations. In some embodiments, the adenine base editing enzymes provided herein enable correction of pathogenic SNPs (-47% of disease-associated point mutations). In some embodiments, the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine. In some embodiments, inosine exhibits the base-pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon is capable of pairing with A, U, or C in mRNA during translation. Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2. Non-limiting exemplary ABEs suitable for useherein include: ABE7, ABE8.1m, ABE8.2m, ABE8.3m, ABE8.4m, ABE8.5m, ABE8.6m, ABE8.7m, ABE8.8m, ABE8.9m, ABE8.10m, ABE8.11m, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8.18m, ABE8.19m, ABE8.20m, ABE8.21m, ABE8.22m, ABE8.23m, ABE8.24m, ABE8.1d, ABE8.2d, ABE8.3d, ABE8.4d, ABE8.5d, ABE8.6d, ABE8.7d, ABE8.8d, ABE8.9d, ABE8.10d, ABE8.11d, ABE8.12d, ABE8.13d, ABE8.14d, ABE8.15d, ABE8.16d, ABE8.17d, ABE8.18d, ABE8.19d, ABE8.20d, ABE8.21d, ABE8.22d, ABE8.23d, and ABE8.24d. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The CRISPR Journal 4:2: 169-177, incorporated herein by reference. In some embodiments, the adenine deaminase is an adenine deaminase described by Koblan et al. (2018) Nature Biotechnology 36:848-846, incorporated herein by reference. In some embodiments, the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871.

[0175] In some embodiments, the ABE described herein is capable of targeting polyA signals, splice site acceptors, and start codons. In some embodiments, the ABE cannot create stop codons for knockdown.

[0176] In some embodiments, an adenine base editing enzyme is an adenosine deaminase. Nonlimiting exemplary adenosine base editors suitable for use herein include ABE9. In some embodiments, the ABE comprises an engineered adenosine deaminase enzyme capable of acting on ssDNA. The engineered adenosine deaminase enzyme may be an adenosine deaminase variant that differs from a naturally occurring deaminase. Relative to the naturally occurring deaminase, the adenosine deaminase variant may comprise one or more amino acid alteration, including a V82S alteration, a T166R alteration, a Y147T alteration, a Y147R alteration, a Q154S alteration, a Y123H alteration, a Q154R alteration, or a combination thereof.

[0177] In some embodiments, the base editor comprises an adenine deaminase (e.g., TadA). In some embodiments, the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA*9). In some embodiments, the adenosine deaminase is a TadA* 8 variant (e.g., any one of TadA* 8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24 as described in WO2021163587 and W02021050571, which are each hereby incorporated by reference in its entirety). In some embodiments, the base editor comprises TadA.

[0178] In some embodiments, a base editing enzyme is a deaminase dimer. In some embodiments, the ABE comprises the effector protein, the adenine base editing enzyme and the deaminase dimer. In some embodiments, the deaminase dimer comprises an adenosine deaminase. In some embodiments, the deaminase dimer comprises TadA and a suitable adenine base editing enzyme including an: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), BtAPOBEC2, and variants thereof. Insome embodiments, the adenine base editing enzyme is fused to amino-terminus or the carboxyterminus of TadA.

[0179] In some embodiments, a base editor is an RNA base editor, wherein the base editing enzyme is an RNA base editing enzyme. In some embodiments, the RNA base editing enzyme comprises an adenosine deaminase. In some embodiments, ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine. In some embodiments, RNA base editors comprise an effector protein that is activated by or binds RNA.

[0180] In some embodiments, base editing enzymes, and therefore base editors, are used for treating a subject having or a subject suspected of having a disease related to a gene of interest. In some embodiments, base editing enzymes, and therefore base editors, are useful for treating a disease or a disorder caused by a point mutation in a gene of interest. In some embodiments, compositions, systems, and methods described herein comprise a base editor and a guide nucleic acid, wherein the base editor comprises an effector protein and a base editing enzyme, and wherein the guide nucleic acid directs the base editor to a sequence in a target gene.Protein Modification Activity

[0181] In some embodiments, an effector partner provides enzymatic activity that modifies a protein associated with a target nucleic acid. The protein may be a histone, an RNA binding protein, or a DNA binding protein. Examples of such protein modification activities include: methyltransferase activity, such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1); demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID 1 C / SMCX, JARID1D / SMCY, UTX, JMJD3); acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, M0Z / MYST3, M0RF / MYST4, HBO1 / MYST2, HM0F / MYST1, SRC1, ACTR, P160, CLOCK); deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11); kinase activity; phosphatase activity; ubiquitin ligase activity; deubiquitinating activity; adenylation activity; deadenylation activity; SUMOylating activity; deSUMOylating activity; ribosylation activity; deribosylation activity; myristoylation activity; and demyristoylation activity.Transcription Modulator Fusions

[0182] In some embodiments, effector partners include, but are not limited to, a protein that directly and / or indirectly provides for increased or decreased transcription and / or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription and / or translation regulator, a translation-regulating protein, etc.). In some embodiments, effector partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.

[0183] In some embodiments, effector partners activate or increase expression of a target nucleic acid. In some embodiments, effector partners increase expression of the target nucleic acid relative to its expression in the absence of the effector partners. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR. In some embodiments, effector partners comprise a transcriptional activator. In some embodiments, the transcriptional activators may promote transcription by: recruitment of other transcription factor proteins; modification of target DNA such as demethylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones; or a combination thereof.

[0184] Non-limiting examples of effector partners that promote or increase transcription include: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, M0Z / MYST3, M0RF / MYST4, SRC1, ACTR, Pl 60, CLOCK; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1; and functional domains thereof. Other non-limiting examples of suitable effector partners include: proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for stimulation of RNA splicing (e.g., Serine / Arginine-rich (SR) domains); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat).

[0185] In some embodiments, effector partners inhibit or reduce expression of a target nucleic acid. In some embodiments, effector partners reduce expression of the target nucleic acid relative to its expression in the absence of the effector partners. Relative expression, including transcription and RNA levels, may be assessed, quantified, and compared, e.g., by RT-qPCR. In some embodiments, effector partners may comprise a transcriptional repressor. In some embodiments, the transcriptional repressors may inhibit transcription by: recruitment of other transcription factor proteins; modification of target DNA such as methylation; recruitment of a DNA modifier; modulation of histones associated withtarget DNA; recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones; or a combination thereof.

[0186] Non-limiting examples of effector partners that decrease or inhibit transcription include: transcriptional repressors such as the Kriippel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID 1 C / SMCX, JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11; DNA methylases such as Hhal DNA m5c- methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and periphery recruitment elements such as Lamin A, and Lamin B; and functional domains thereof. Other non-limiting examples of suitable effector partners include: proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for repression of RNA splicing (e.g., PTB, Sam68, and hnRNP Al); proteins and protein domains responsible for reducing the efficiency of transcription (e.g., FUS (TLS)).

[0187] In some embodiments, fusion proteins are targeted by a guide nucleic acid (e.g., guide RNA) to a specific location in a target nucleic acid and exert locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and / or changes a local chromatin status (e.g., when a fusion sequence is used that edits the target nucleic acid or modifies a protein associated with the target nucleic acid). In some embodiments, the modifications are transient (e.g., transcription repression or activation). In some embodiments, the modifications are inheritable. For example, epigenetic modifications made to a target nucleic acid, or to proteins associated with the target nucleic acid, e.g., nucleosomal histones, in a cell, can be observed in a successive generation.

[0188] In some embodiments, effector partner comprises an RNA splicing factor. The RNA splicing factor may be used (in whole or as fragments thereof) for modular organization, with separate sequencespecific RNA binding modules and splicing effector domains. In some embodiments, the RNA splicing factors comprise members of the Serine / Arginine-rich (SR) protein family containing N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. In some embodiments, a hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C- terminal Glycine-rich domain. In some embodiments, the RNA splicing factors may regulate alternative use of splice site (ss) by binding to regulatory sequences between two alternative sites. For example, in some embodiments, ASF / SF2 may recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al may bind to ESSs and shift splicing towards the use of intron distal sites. Oneapplication for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. Long splicing isoform Bcl- xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. Short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). A ratio of the two Bcl-x splicing isoforms is regulated by multiple co'j-clcmcnts that are located in either core exon region or exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see W02010075303, which is hereby incorporated by reference in its entirety.Recombinases

[0189] In some embodiments, effector partners comprise a recombinase. In some embodiments, provided herein is a recombinase system comprising effector proteins described herein and the recombinase. In some embodiments, the effector proteins have reduced nuclease activity or no nuclease activity. In some embodiments, the recombinase is a site-specific recombinase.

[0190] In some embodiments, the recombinase system comprises a catalytically inactive effector protein, wherein the recombinase can be a site-specific recombinase. Such systems can be used for site- directed transgene insertion. Non-limiting examples of site-specific recombinases include a tyrosine recombinase (e.g., Cre, Flp or lambda integrase), a serine recombinase (e.g., gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase and integrase), or mutants or variants thereof. In some embodiments, the recombinase is a serine recombinase. Non-limiting examples of serine recombinases include gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase, and IS607 integrase. In some embodiments, the site-specific recombinase is an integrase. Non-limiting examples of integrases include:Bxbl, wBeta, BL3, phiR4, Al 18, TGI, MR11, phi370, SPBc, TP90I-I, phiRV, FC1, K38, phiBTl, and phiC31. Further discussion and examples of suitable recombinase effector partners are described in US 10,975,392, which is incorporated herein by reference in its entirety. In some embodiments, the fusion protein comprises a linker that links the recombinase to the effector protein. In some embodiments, the linker is The-Ser.Linkers

[0191] In some embodiments, a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide. Accordingly, in some embodiments, effector proteins, effector partners, or combinations thereof are connected by one or more linkers. The linker may comprise or consist of a covalent bond. The linker may comprise or consist of a chemical group. In some embodiments, the linker comprises an amino acid. In some embodiments, a peptide linker comprises at least two amino acids linked by an amide bond. In general, the linker connects a terminus of the effector protein to aterminus of the effector partner. In some embodiments, carboxy terminus of the effector protein is linked to the amino terminus of the effector partner. In some embodiments, carboxy terminus of the effector partner is linked to the amino terminus of the effector protein. In some embodiments, the effector protein and the effector partner are directly linked by a covalent bond.

[0192] In some embodiments, linkers comprise one or more amino acids. In some embodiments, linker is a protein. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through an amide bond. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through a peptide bond. In some embodiments, linkers comprise an amino acid. In some embodiments, linkers comprise a peptide. In some embodiments, an effector protein is coupled to a effector partner by a linker protein. In some embodiments, the linker may have any of a variety of amino acid sequences. In some embodiments, the linker may comprise a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof. In some embodiments, the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. In some embodiments, linked amino acids described herein comprise at least two amino acids linked by an amide bond.

[0193] Linkers may be produced by using synthetic, linker-encoding oligonucleotides to couple proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein (e.g., an effector protein coupled to an effector partner). In some embodiments, the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 10 to 27 amino acids in length. In some embodiments, linker proteins include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ ID NO: 1169), GGSGGSn (SEQ ID NO: 1170), and GGGSn (SEQ ID NO: 1171), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. In some embodiments, linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 1172), GGSGG (SEQ ID NO: 1173), GSGSG (SEQ ID NO: 1174), GSGGG (SEQ ID NO: 1175), GGGSG (SEQ ID NO: 1176), and GSSSG (SEQ ID NO: 1177). In some embodiments, the linker comprises one or more repeats a tri-peptide GGS. In some embodiments, the linker is an XTEN linker. In some embodiments, the XTEN linker is an XTEN80 linker. In some embodiments, the XTEN linker is an XTEN20 linker. In some embodiments, the XTEN20 linker has an amino acid sequence of GSGGSPAGSPTSTEEGTSESATPGSG (SEQ ID NO: 1178).

[0194] In some embodiments, linkers do not comprise an amino acid. In some embodiments, linkers do not comprise a peptide. In some embodiments, linkers comprise a nucleotide, a polynucleotide, a polymer, or a lipid. In some embodiments, linker may be a polyethylene glycol (PEG), polypropyleneglycol (PPG), co-poly(ethylene / propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker.

[0195] In some embodiments, a linker is recognized and cleaved by a protein described herein. In some embodiments, a linker comprises a recognition sequence that may be recognized and cleaved by the protein. In some embodiments, a guide nucleic acid comprises an aptamer, which may serve s a similar function as a linker, bringing an effector protein and an effector partner protein into proximity. The aptamer can functionally connect two proteins (e.g., effector protein, effector partner) by interacting non-covalently with both, thereby bringing both proteins into proximity of the guide nucleic acid. In some embodiments, the first protein and / or the second protein comprise or is covalently linked to an aptamer binding moiety. In some embodiments, the aptamer is a short single stranded DNA (ssDNA) or RNA (ssRNA) molecule capable of being bound be the aptamer binding moiety. In some embodiments, the aptamer is a molecule that is capable of mimicking antibody binding activity and may be classified as a chemical antibody. In some instances, the aptamer described herein refers to artificial oligonucleotides that bind one or more specific molecules. In some embodiments, aptamers exhibit a range of affinities (KD in the pM to pM range) with little or no off-target binding.Engineered Proteins

[0196] In some embodiments, proteins (e.g., effector protein or effector partner) described herein have been modified (also referred to as an engineered protein). In some embodiments, a modification of the proteins may include addition of one or more amino acids, deletion of one or more amino acids, substitution of one or more amino acids, or combinations thereof. In some embodiments, the proteins disclosed herein are engineered proteins. Unless otherwise indicated, reference to the proteins throughout the present disclosure include engineered proteins thereof.

[0197] In some embodiments, proteins (e.g., effector protein or effector partner) described herein can be modified with the addition of one or more heterologous peptides. In some embodiments, the protein modified with the addition of one or more heterologous peptides may be referred to herein as a fusion protein. Such fusion proteins are described herein and throughout.

[0198] In some embodiments, a heterologous peptide comprises a subcellular localization signal. In some embodiments, a subcellular localization signal can be a nuclear localization signal (NLS). In some embodiments, the NLS facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment. TABLE 1 lists exemplary NLS sequences. In some embodiments, the subcellular localization signal is a nuclear export signal (NES), a sequence to keep the protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like. In some embodiments, the protein described herein is not modified witha subcellular localization signal so that the protein is not targeted to the nucleus, which can be advantageous depending on the circumstance (e.g., when the target nucleic acid is an RNA that is present in the cytosol).

[0199] In some embodiments, a heterologous peptide comprises a chloroplast transit peptide (CTP), also referred to as a chloroplast localization signal or a plastid transit peptide, which targets the protein to a chloroplast. Chromosomal transgenes from bacterial sources may require a sequence encoding a CTP sequence fused to a sequence encoding an expressed protein (e.g., effector protein, effector partner) if the expressed protein is to be compartmentalized in the plant plastid (e.g., chloroplast). The CTP may be removed in a processing step during translocation into the plastid. Accordingly, localization of the protein to a chloroplast is often accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous protein.

[0200] In some embodiments, the heterologous peptide is an endosomal escape peptide (EEP). An EEP is an agent that quickly disrupts the endosome in order to minimize the amount of time that a delivered molecule, such protein, spends in the endosome-like environment, and to avoid getting trapped in the endosomal vesicles and degraded in the lysosomal compartment.

[0201] In some embodiments, the heterologous peptide is a cell penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD). A CPP or PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.

[0202] Further suitable heterologous peptides include, but are not limited to, proteins (or fragments / domains thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc ).

[0203] In some embodiments, a heterologous peptide comprises a protein tag. In some embodiments, the protein tag is referred to as purification tag or a fluorescent protein. The protein tag may be detectable for use in detection of the protein and / or purification of the protein. Accordingly, in some embodiments, compositions, systems and methods comprise a protein tag or use thereof. Any suitable protein tag may be used depending on the purpose of its use. Non-limiting examples of protein tags include a fluorescent protein, a histidine tag, e.g., a 6XHis tag (SEQ ID NO: 1179); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and maltose binding protein (MBP). In some embodiments, the protein tag is a portion of MBP that can be detected and / or purified. Non-limiting examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato.

[0204] A heterologous peptide may be located at or near the amino terminus (N-terminus) of the protein (e.g., effector protein, effector partner) disclosed herein. A heterologous peptide may be located at or near the carboxy terminus (C-terminus) of the proteins disclosed herein. In some embodiments, aheterologous peptide is located internally in the protein described herein (i.e., is not at the N- or C- terminus of the protein described herein) at a suitable insertion site.

[0205] In some embodiments, protein (e.g., effector protein, effector partner) described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the N-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the C-terminus, or a combination of these (e.g., one or more heterologous peptides at the amino-terminus and one or more heterologous peptides at the carboxy terminus). When more than one heterologous peptide is present, each may be selected independently of the others, such that a single heterologous peptide may be present in more than one copy and / or in combination with one or more other heterologous peptides present in one or more copies. In some embodiments, a heterologous peptide is considered near the N- or C-terminus when the nearest amino acid of the heterologous peptide is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.

[0206] In some embodiments, proteins (e.g., effector protein, effector partner) described herein are encoded by a codon optimized nucleic acid. In some embodiments, a nucleic acid sequence encoding the protein described herein, is codon optimized. In some embodiments, the proteins described herein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the effector protein is codon optimized for a human cell.

[0207] For example, proteins (e.g., effector protein, effector partner) may comprise one or more modifications that may provide increased activity as compared to a naturally-occurring counterpart. As another example, proteins may provide increased catalytic activity (e.g., nickase, nuclease, binding, activity) as compared to a naturally-occurring counterpart. Proteins may provide enhanced nucleic acid binding activity (e.g., enhanced binding of a guide nucleic acid, and / or target nucleic acid) as compared to a naturally-occurring counterpart. Protein may have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, or more, increase of the activity of a naturally-occurring counterpart.

[0208] In some embodiments, proteins (e.g., effector protein, effector partner) may comprise one or more modifications that reduce an activity of the effector proteins relative to a naturally occurring nuclease. In some embodiments, the activity is nucleic acid binding activity. In some embodiments, the activity is a catalytic activity. In some embodiments, the catalytic activity is nuclease activity. In some embodiments, the catalytic activity is cis cleavage activity. In some embodiments, the effector protein comprises at least one amino acid substitution relative to the respective sequence selected from SEQ ID NOs: 1-161 and 391-847, wherein the amino acid substitution is located in a RuvC domain and / or HNH domain of the effector protein, and wherein the amino acid substitution reduces an activity of the domain(s), thereby providing the effector protein with nickase activity. In some embodiments, an effector protein has 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less activity than a naturally occurring counterpart.Fusion Proteins

[0209] In some embodiments, compositions, systems, and methods comprise a fusion protein or uses thereof. A fusion protein generally comprises an effector protein and an effector partner. In some embodiments, the effector partner comprises a polypeptide or peptide that is fused or linked to the effector protein. In some embodiments, the effector partner is fused to the N-terminus of the effector protein. In some embodiments, the effector partner is fused to the C-terminus of the effector protein.

[0210] In such embodiments, the fusion protein can comprise at least two effector proteins that are same. In some embodiments, the fusion protein comprises at least two effector proteins that are different. In some embodiments, the multimeric form is a homomeric form. In some embodiments, the multimeric form is a heteromeric form. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins comprising the effector protein described herein and an effector partner.[2H] In some embodiments, the effector partner is a heterologous protein capable of imparting some function or activity that is not provided by an effector protein. In some embodiments, the effector partner is capable of cleaving or modifying the target nucleic acid, a non-target nucleic acid, or both. In some embodiments, the fusion protein disclosed herein may provide cleavage activity, such as cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, other activity, or a combination thereof. Fusion proteins disclosed herein may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). In some embodiments, fusion proteins cleave the target nucleic acid at the target sequence or adjacent to the target sequence. [In some embodiments, fusion proteins cleave the non-target nucleic acid.

[0212] In some embodiments, the fusion protein complexes with a guide nucleic acid and the complex interacts with the target nucleic acid, a non-target nucleic acid, or both. In some embodiments, the interaction comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid and / or the non-target nucleic acid by the fusion protein, or combinations thereof. In some embodiments, recognition of a PAM sequence within a target nucleic acid may direct the modification activity of a fusion protein.

[0213] Modification activity of a fusion protein described herein may be cleavage activity, binding activity, insertion activity, substitution activity, and the like. Modification activity of an effector protein may result in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or any combination thereof. In some embodiments, an ability of a fusion protein to edit a target nucleic acid may depend upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or combinations thereof.

[0214] In some embodiments, the fusion protein described herein comprises a fusion protein, wherein the fusion protein affects formation of a multimeric complex of the fusion protein. By way of nonlimiting example, the fusion protein may comprise an effector protein described herein and an effector partner comprising a Calcineurin A tag, wherein the fusion protein dimerizes in the presence of Tacrolimus (FK506). Also, by way of non-limiting example, the fusion protein may comprise an effector protein described herein and a SpyTag configured to dimerize or associate with another effector protein in a multimeric complex. Multimeric complex formation is further described herein.Guide Nucleic Acids

[0215] The compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid. Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid. Guide nucleic acids are also referred to herein as “guide RNA.” A guide nucleic acid, as well as any components thereof (e.g., spacer sequence, protein binding sequence, linker nucleotide sequence, handle sequence, intermediary sequence etc.) may comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more engineered modifications as described herein), or any combinations thereof. Such nucleotide sequences described herein may be described as a nucleotide sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid, such as a nucleotide sequence described herein for a vector. Similarly, disclosure of the nucleotide sequences described herein also discloses the complementary nucleotide sequence, the reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which can be a nucleotide sequence for use in a guide nucleic acid as described herein. In some embodiments, a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion. In some embodiments, guide nucleic acids comprise one or more nucleotide sequences as described herein (e.g., TABLE 2)

[0216] A guide nucleic acid may comprise a naturally occurring sequence. A guide nucleic acid may comprise a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, may be different from the sequence of a naturally occurring guide nucleic acid. A guide nucleic acid of the present disclosure comprises one or more of the following: a) a single nucleic acid molecule; b) a DNA base; c) an RNA base; d) a modified base; e) a modified sugar; f) a modified backbone; and the like. Modifications are described herein and throughout the present disclosure (e.g.,in the section entitled “Engineered Modifications”). A guide nucleic acid may be chemically synthesized or recombinantly produced by any suitable methods. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.

[0217] In some embodiments, compositions, systems and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins. In some embodiments, a first region (FR1) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In some embodiments, a second region (FR2) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In general, the FR1 is located 5’ of the FR2.

[0218] In some embodiments, the guide nucleic acid comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked nucleosides. In general, a guide nucleic acid comprises at least linked nucleosides. In some embodiments, a guide nucleic acid comprises at least 25 linked nucleosides. A guide nucleic acid may comprise 10 to 50 linked nucleosides. In some embodiments, the guide nucleic acid comprises or consists essentially of about 12 to about 80 linked nucleosides, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19 , about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleosides. In some embodiments, the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleosides.

[0219] In some embodiments, the compositions, systems, and methods of the present disclosure comprise two ormore guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 9, 10 or more guide nucleic acids), and / or uses thereof. Multiple guide nucleic acids may target an effector protein to different locations in the target nucleic acid by hybridizing to different target sequences. In some embodiments, a first guide nucleic acid may hybridize within a location of the target nucleic acid that is different from where a second guide nucleic acid may hybridize the target nucleic acid. In some embodiments, the first loci and the second loci of the target nucleic acid may be located at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides apart. In some embodiments, the first loci and the second loci of the target nucleic acid may be located between 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900 or 900 and 1000 nucleotides apart. In some embodiments, the first loci and / or the second loci of the target nucleic acid are located in an intron of a gene. In some embodiments, the first loci and / or the second loci of the target nucleic acid are located in an exon of a gene. In some embodiments, the first loci and / or the second loci of the target nucleic acid span an exon-intron junction of a gene. In someembodiments, the first portion and / or the second portion of the target nucleic acid are located on either side of an exon and cutting at both sites results in deletion of the exon. In some embodiments, composition, systems, and methods comprise a donor nucleic acid that may be inserted in replacement of a deleted or cleaved sequence of the target nucleic acid. In some embodiments, compositions, systems, and methods comprising multiple guide nucleic acids or uses thereof comprise multiple effector proteins, wherein the effector proteins may be identical, non-identical, or combinations thereof.

[0220] In some embodiments, a guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. Such a eukaryotic sequence is a nucleotide sequence that is present in a host eukaryotic cell. Such a nucleotide sequence is distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses. Said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, a non-coding (e.g., promoter or enhancer) region, a selectable marker, tag, signal, and the like. In some embodiments, a target sequence is a eukaryotic sequence.

[0221] In some embodiments, a length of a guide nucleic acid is about 30 to about 120 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, or about 125 linked nucleotides.

[0222] In some embodiments, guide nucleic acids comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid. Such elements may be one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.).

[0223] In some embodiments, guide nucleic acids comprise one or more linkers connecting different nucleotide sequences as described herein. A linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. A linker may be any suitable linker, examples of which are described herein.Protein Binding Sequence

[0224] In some embodiments, the compositions, systems, and methods of the present disclosure comprise an effector protein and a guide nucleic acid comprising a protein binding sequence as described herein. In some embodiments, the guide nucleic acid comprises a protein binding sequence, wherein an effector protein described herein binds the protein binding sequence. In some embodiments,the protein binding sequence does not comprise a CRISPR RNA (crRNA) or repeat sequence thereof. A crRNA may be transcribed from the CRISPR locus. In some embodiments, the protein binding sequence does not comprise a transactivating crispr RNA (tracr) sequence. A tracrRNA is partially complementary to and base pairs with at least a portion of a pre-crRNA.

[0225] Exemplary protein binding sequences are provided in TABLE 2. In some embodiments, a guide nucleic acid comprises a nucleotide 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 97%, or at least 98%, at least 99%, or 100% identical to an equal length portion of any one of the sequences in TABLE 2. In some embodiments, the guide nucleic acid comprises a nucleotide sequence is at least 85% identical to any one of sequences set forth in TABLE 2. In some embodiments, a guide nucleic acid comprises at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 1700, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, or at least 300 contiguous nucleotides of any one of the sequences recited in TABLE 2. In some embodiments, the protein binding sequence comprises a nucleotide sequence that is 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% identical to any one of the sequences in TABLE 2. In some embodiments, the protein binding sequence comprises a nucleotide sequence that is 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% identical to SEQ ID NO: 178-373, 375- 390, and 848-1168, listed in TABLE 2.

[0226] In some embodiments, the compositions, systems, and methods of the present disclosure comprise a combination of the effector protein and the engineered guide nucleic acid comprising a protein binding sequence as described in TABLE 2. Each row of TABLE 2 describes exemplary combinations of the effector protein and the protein binding sequence comprised in an engineered guide nucleic acid. In some embodiments, TABLE 2 details the effector protein that is functional with the engineered guide nucleic acid comprising the protein binding sequence. However, this is not intended to be limiting. Several of the proteins in TABLE 2 cluster phylogenetically as shown in FIG. 1 and therefore they may bind similar guide nucleic acid sequences. In particular, SEQ ID NOS: 1-26 cluster into reference cluster number 20; SEQ ID NOS: 27-51 cluster into reference cluster number 30; SEQ ID NOS: 52-68 cluster into reference cluster number 83; SEQ ID NOS: 69-80 cluster into reference cluster number 124; SEQ ID NOS: 81-98 cluster into reference cluster number 71; SEQ ID NOS: 99- 121 cluster into reference cluster number 68; SEQ ID NOS: 122-137 cluster into reference cluster number 95; and SEQ ID NOS: 138-161 cluster into reference cluster number 51. In some embodiments, a guide nucleic acid comprising a protein binding sequence of TABLE 2 is functional with a protein described in TABLE 2, wherein the protein binding sequence and the protein are not in the same row of TABLE 2. In some embodiments, a guide nucleic acid comprising a protein binding sequence of TABLE 2 is functional with a protein described in TABLE 2, wherein the protein binding sequenceand the protein are not in the same row of TABLE 2, but from rows wherein the proteins in that row belong to the same cluster as described herein.

[0227] In some embodiments, a protein binding sequence comprises one or more nucleotide alterations at one or more positions in the sequence described in TABLE 2. Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.Spacer Sequence

[0228] Guide nucleic acids described herein may comprise one or more spacer sequences. In some embodiments, a spacer sequence is capable of hybridizing to a target sequence of a target nucleic acid. In some embodiments, a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g., a target sequence) of a target nucleic acid. Exemplary hybridization conditions are described herein. In some embodiments, the spacer sequence may function to direct an RNP complex comprising the guide nucleic acid to the target nucleic acid for detection and / or modification. The spacer sequence may function to direct a RNP to the target nucleic acid for detection and / or modification. A spacer sequence may be complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein described herein.

[0229] In general, the guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence. In some embodiments, the guide nucleic acid comprises at least 10 contiguous nucleotides that are complementary to the target sequence in the target nucleic acid. In some embodiments, guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence. In some embodiments, the target sequence is a eukaryotic sequence.

[0230] The spacer sequence may comprise complementarity with (e.g., hybridize to) a target sequence of a target nucleic acid. In some embodiments, a spacer sequence comprises at least 5 to about 50, at least 5 to about 25, at least about 10 to at least about 25, or at least about 15 to about 25 linked nucleotides. In some embodiments, the spacer sequence is 15-28 linked nucleosides in length. In some embodiments, the spacer sequence is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleosides in length. In some embodiments, the spacer sequence is 18-24 linked nucleosides in length. In some embodiments, the spacer sequence is at least 15 linked nucleosides in length. In some embodiments, the spacer sequence is at least 16, 18, 20, or 22 linked nucleosides in length. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence is at least 17 linked nucleosides in length. In some embodiments, the spacer sequence is at least 18 linked nucleosides in length. In some embodiments, the spacer sequence is at least 20 linked nucleosides in length. In some embodiments, a spacer sequence comprises at least 5 to about 50 contiguous nucleotides that are complementary to atarget sequence in a target nucleic acid. In some embodiments, the spacer sequence comprises at least 15 contiguous nucleobases that are complementary to the target nucleic acid.

[0231] In some embodiments, the spacer sequence is located 5’ of the protein binding sequence. In some embodiments, the spacer sequence is located at the 5’ end of the guide nucleic acid. In some embodiments, the spacer and protein binding sequences are linked directly to one another. In some embodiments, a linker is present between the spacer and protein binding sequences. Linkers may be any suitable linker. In some embodiments, the spacer sequence and the protein binding sequence of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions.

[0232] In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% complementary to a target sequence of a target nucleic acid. A spacer sequence hybridizes to an equal length portion of a target nucleic acid (e.g., a target sequence). In some embodiments, a target nucleic acid, such as DNA or RNA, comprises a cancer gene or gene associated with a genetic disorder, or an amplicon thereof, as described herein. In some embodiments, a target nucleic acid is a gene selected from TABLE 4. In some embodiments, a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid selected from TABLE 4. In some embodiments, a target nucleic acid is a nucleic acid associated with a disease or syndrome set forth in TABLE 3. In some embodiments, a spacer sequence comprises a nucleotide 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%, or at least 100% complementary to a target sequence of a target nucleic acid associated with a disease or syndrome set forth in TABLE 3. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that hybridizes to the target sequence. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to the target sequence.

[0233] It is understood that the sequence of a spacer sequence need not be 100 % complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. Accordingly, in some embodiments, a spacer sequence may comprise a nucleotide sequence that may have partial complementarity with (e.g., hybridize to) an equal length of a target sequence of a target nucleic acid. The guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 20 of the spacer sequence that is not complementary to the corresponding nucleoside of the target sequence. The guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 9, 10 to 14, or 15 to 20 of the spacer sequence that is not complementary to the corresponding nucleoside of the target sequence. In some embodiments, the region of the target nucleic acid that is complementary to the spacer sequence comprises an epigenetic modification or a post-transcriptional modification. In some embodiments, the epigenetic modification comprises acetylation, methylation, or thiol modification.Linkers for Nucleic Acids

[0234] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises a linker connecting a protein binding sequence and a spacer sequence.

[0235] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5’- GAAA-3’.Guide Nucleic Acid Modifications

[0236] Guide nucleic acids (e.g., guide nucleic acids) described herein may comprise chemical modifications. In some embodiments, guide nucleic acids are chemically synthesized (as opposed to transcribed). Modifications may provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a base modification, a backbone modification, a sugar modification, or combinations thereof, of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.

[0237] In some embodiments, nucleic acids (e.g., engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2’0-methyl modified nucleotides, 2’ Fluoro modified nucleotides; locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7-methylguanylate cap (m7G)), 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 aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, 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 intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage; phosphorothioate and / or heteroatom intemucleoside linkages, such as -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester intemucleotide linkage is represented as -0-P(=0)(0H)-0-CH2-); morpholino linkages (formed in part from the sugar portion of anucleoside); morpholino backbones; phosphorodiamidate or other non-phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH2 component parts; and combinations thereof.Vectors and Multiplexed Expression Vectors

[0238] In some embodiments, compositions and systems provided herein comprise a vector system, wherein the vector system comprises one or more vectors. When a vector is described herein, such a vector can be used as a vehicle to introduce one or more molecules of interest into a host cell. A molecule of interest can comprise a polypeptide (e.g., an effector protein), an engineered guide or a component thereof (e.g., crRNA, tracrRNA sequence, or sgRNA), a donor nucleic acid, a nucleic acid encoding a polypeptide, a nucleic acid encoding an engineered guide or a component thereof. The vector may be part of a vector system, wherein a vector system comprises a library of vectors each encoding one or more component of a composition or system described herein. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and / or a target nucleic acid) are encoded by the same vector. In some embodiments, components described herein (e.g., an effector protein, a guide nucleic acid, and / or a target nucleic acid) are each encoded by different vectors of the system. For example, vector systems described herein can comprise one or more vectors comprising a polypeptide (e.g., an effector protein), an engineered guide (e.g., crRNA, tracrRNA sequence, or sgRNA), or encoding for, or a nucleic acid or nucleic acids encoding a polypeptide, engineered guide, a donor nucleic acid, or any combination thereof.

[0239] In some embodiments, compositions and systems provided herein comprise a vector system comprising a polypeptide (e.g., an effector protein, effector partner, or fusion protein) described herein. In some embodiments, compositions and systems provided herein comprise a vector system comprising a guide nucleic acid (e.g., crRNA, tracrRNA sequence, or sgRNA) described herein. In some embodiments, compositions and systems provided herein comprise a vector system comprising a donor nucleic acid described herein.

[0240] In some embodiments, compositions and systems provided herein comprise a vector system encoding a polypeptide (e.g., an effector protein, effector partner, or fusion protein) described herein. In some embodiments, compositions and systems provided herein comprise a vector system encoding a guide nucleic acid (e.g., crRNA, tracrRNA sequence, or sgRNA) described herein. In some embodiments, compositions and systems provided herein comprise a multi-vector system encoding an effector protein and a guide nucleic acid described herein, wherein the guide nucleic acid and the effector protein are encoded by the same or different vectors. In some embodiments, the guide nucleic acid and the effector protein are encoded by different vectors of the system. In some embodiments, anucleic acid encoding a polypeptide (e.g., an effector protein, effector partner, or fusion protein ) comprises an expression vector. In some embodiments, a nucleic acid encoding a polypeptide is a messenger RNA. In some embodiments, an expression vector comprises or encodes an engineered guide nucleic acid. In some embodiments, the expression vector encodes the crRNA or sgRNA.

[0241] In some embodiments, a vector may encode one or more effector proteins. In some embodiments, the one or more effector proteins comprise at least two effector proteins. In some embodiments, the at least two effector protein are the same. In some embodiments, the at least two effector proteins are different from each other. In some embodiments, the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell. In some embodiments, a vector may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 effector proteins. In some embodiments, a vector can encode one or more effector proteins comprising an amino acid sequence of any one of SEQ ID NOs: 1-78 and 499.

[0242] In some embodiments, a vector may encode one or more guide nucleic acids. In some embodiments, the one or more guide nucleic acids comprise at least two guide nucleic acids. In some embodiments, the at least two guide nucleic acids are the same. In some embodiments, the at least two guide nucleic acids are different from each other. In some embodiments, a vector may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 different guide nucleic acids as described herein. In some embodiments, a vector can encode one or more guide nucleic acids comprising a nucleotide sequence of any one of SEQ ID NOs: 123-227, 327-425 and 436-498. In some embodiments, a vector can encode one or more guide nucleic acids comprising a nucleotide sequence with at least 75%, 80%, 85%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 123-227, 327-425 and 436-498.

[0243] In some embodiments, a vector comprises one or more donor nucleic acids as described herein. In some embodiments, the one or more donor nucleic acids comprise at least two donor nucleic acids. In some embodiments, the at least two donor nucleic acids are the same. In some embodiments, the at least two donor nucleic acids are different from each other. In some embodiments, the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donor nucleic acids.

[0244] In some embodiments, a vector can comprise or encode one or more regulatory elements. Regulatory elements can 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 or a coding sequence and / or regulate translation of an encoded polypeptide. In some embodiments, a vector can comprise or encode for one or more additional elements, such as, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like.

[0245] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3 ' direction) coding or non-coding sequence. As used herein, a promoter can be bound at its 3' terminus to a nucleic acid the expression or transcription of which is desired, and extends upstream (5' direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. A promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest. Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.

[0246] Promotors can be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and / or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); 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), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10-fold, by 100- fold, or by 1000-fold, or more. In addition, vectors used for providing a nucleic acid encoding an engineered guide nucleic acid and / or an effector protein 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 engineered guide nucleic acid and / or an effector protein.

[0247] In general, plasmids and vectors described herein comprise at least one promoter. In some embodiments, the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides. In some embodiments, the length of the promoter is at least 100 linked nucleotides. In some embodiments, the promoters are constitutive promoters. In other embodiments, the promoters are inducible promoters. In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)- regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44. In additional embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). Exemplary promoters include, but are not limited to, CMV, 7SK, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, CaMV35S, MNDU3, MSCV and HSV TK promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is EFla. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.

[0248] In some embodiments, a vector described herein is a delivery vector. In some examples, the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle may be a non-viral vector. In some embodiments, the delivery vehicle may be a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double -stranded DNA. In some examples, the plasmid may be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory elements. In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid may be a minicircle plasmid. In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid may be formulated for delivery through injection by a needle carrying syringe. In some examples, the plasmid may be formulated for delivery via electroporation. In some examples, the plasmids may be engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.

[0249] In some embodiments, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and / or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit (3-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981); and the genome region of human growth hormone (J Immunol., Vol. 155(3), p. 1286-95, 1995).

[0250] In some embodiments, an effector protein (or a nucleic acid encoding same) and / or an engineered guide nucleic acid (or a nucleic acid encoding same) are co-administered with a donor nucleic acid. Co-administration can be contact with a target nucleic acid, administered to a cell, such as a host cell, or administered as method of nucleic acid detection, editing, and / or treatment as described herein, in a single vehicle, such as a single expression vector. In certain embodiments, an effector protein (or a nucleic acid encoding same) and / or an engineered guide nucleic acid (or a nucleic acid encoding same) are not co-administered with donor nucleic acid in a single vehicle. In certain embodiments, an effector protein (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid encoding same), and / or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.Administration of a non-viral vector

[0251] In some embodiments, the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; or cell-penetrating peptides.

[0252] In some embodiments, a vector is administered as part of a method of nucleic acid detection, editing, and / or treatment as described herein. In some embodiments, a vector is administered in a single vehicle, such as a single expression vector. In some embodiments, at least two of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid, are provided in the single expression vector. In some embodiments, components, such as a guide nucleic acid and an effector protein, are encoded by the same vector. In some embodiments, a polypeptide (e.g., an effector protein, effector partner, or fusion protein) (or a nucleic acid encoding same) and / or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle. In some embodiments, an effector protein (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and / or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.

[0253] In some embodiments, a vector may be part of a vector system. In some embodiments, the vector system comprises a library of vectors each encoding one or more components of a composition or system described herein. In some embodiments, a vector system is administered as part of a method of nucleic acid detection, editing, and / or treatment as described herein, wherein at least two vectors are co-administered. In some embodiments, the at least two vectors comprise different components. In someembodiments, the at least two vectors comprise the same component having different sequences. In some embodiments, at least one of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof is provided in a different vector. In some embodiments, the nucleic acid encoding the effector protein, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors. In some embodiments, the donor nucleic acid is encoded by a different vector than the vector encoding the effector protein and the guide nucleic acid.Lipid Particles and Non-viral Vectors

[0254] In some embodiments, compositions and systems provided herein comprise a lipid particle. In some embodiments, a lipid particle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector. In some embodiments, a lipid or a lipid nanoparticle can encapsulate the effector protein, the guide nucleic acid, the nucleic acid encoding the effector protein and / or the DNA molecule encoding the guide nucleic acid. LNPs are a non-viral delivery system for gene therapy. LNPs are effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkami et al., (2018) Nucleic Acid Therapeutics, 28(3): 146-157). In some embodiments, a method can comprise contacting a cell with an expression vector. In some embodiments, contacting can comprise electroporation, lipofection, or lipid nanoparticle (LNP) delivery of an expression vector. In some embodiments, a nucleic acid expression vector is a non-viral vector. In some embodiments, compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce an effector protein, guide nucleic acid, donor template or any combination thereof to a cell. Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, or bio- responsive polymers. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.

[0255] In some embodiments, a LNP comprises an outer shell and an inner core. In some embodiments, the outer shell comprises lipids. In some embodiments, the lipids comprise modified lipids. In some embodiments, the modified lipids comprise pegylated lipids. In some embodiments, the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids. In some embodiments, the LNP comprises one or more of Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide (TT3), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2- oleoylsn-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Choi), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol (DMG- PEChooo), derivatives, analogs, or variants thereof. In some embodiments, the LNP has a negative netoverall charge prior to complexation with one or more of a guide nucleic acid, a nucleic acid encoding the one or more guide nucleic acid, a nucleic acid encoding the polypeptide (e.g., effector protein, effector partner, fusion protein, or combinations thereof), and / or a donor nucleic acid. In some embodiments, the inner core is a hydrophobic core. In some embodiments, the one or more of a guide nucleic acid, the one or more nucleic acid encoding the one or more guide nucleic acid, one or more nucleic acid encoding one or more polypeptides, and / or the one or more donor nucleic acid forms a complex with one or more of the cationic lipids and the ionizable lipids. In some embodiments, the nucleic acid encoding the polypeptide or the nucleic acid encoding the guide nucleic acid is selfreplicating.

[0256] In some embodiments, a LNP comprises one or more of cationic lipids, ionizable lipids, and modified versions thereof. In some embodiments, the ionizable lipid comprises TT3 or a derivative thereof. Accordingly, in some embodiments, the LNP comprises one or more of TT3 and pegylated TT3. The publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and representative methods of delivering LNP formulations in Example 7.

[0257] In some embodiments, a LNP comprises a lipid composition targeting to a specific organ. In some embodiments, the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes).Delivery of Viral Vectors

[0258] An expression vector can be a viral vector. In some embodiments, a viral vector comprises a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or non-segmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. In some embodiments, the expression vector is an adeno-associated viral vector. There are a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. The virus may be a lentivirus. The virus may be an adenovirus. The virus may be a non-replicating virus. The virus may be an adeno- associated virus (AAV). The viral vector may be a retroviral vector. Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome.In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In some embodiments, the viral vector is a recombinant viral vector. In some embodiments, the viral vector is a chimeric viral vector. In some embodiments, the chimeric viral vector comprises viral portions from two or more viruses. In some embodiments, the viral vector corresponds to a virus of a specific serotype.

[0259] In some embodiments, the viral vector is an AAV. The AAV may be any AAV known in the art. In some embodiments, a viral particle that delivers a viral vector described herein is an AAV. In some embodiments, the viral vector corresponds to a virus of a specific serotype. In some examples, the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV 10 serotype, an AAV-rhlO serotype, an AAV 11 serotype, and an AAV 12 serotype. In some embodiments the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.

[0260] In some embodiments, the AAV vector may be a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[0261] In some embodiments, the viral vector is a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. A viral vector provided herein can be derived from or based on any such virus. Often the viral vectors provided herein are an adeno-associated viral vector (AAV vector). Generally, an AAV vector has two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. The DNA sequence in between the ITRs of an AAV vector provided herein may be referred to herein as the sequence encoding the genome editing tools. These genome editing tools can include, but are not limited to, an effector protein, effector protein modifications (e.g., nuclear localization signal (NLS), polyA tail), guide nucleic acid(s), respective promoter(s), and a donor nucleic acid, or combinations thereof. In general, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating an AAV vector that is a self-complementary AAV (scAAV) vector. In general, the sequence encoding the genome editing tools of an scAAV vector has a length of about 2 kb to about 3 kb. The scAAV vector can comprise nucleotide sequences encoding an effector protein, providing guide nucleic acids described herein, and a donor nucleic acid described herein. In some embodiments, the AAV vector provided herein is a self-inactivating AAV vector. In someembodiments, an AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector.

[0262] In some embodiments, a fusion protein as described herein is inserted into a vector. In some embodiments, the vector comprises one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and / or transcriptional terminator sequences.Producing A A V Delivery Vectors

[0263] In some embodiments, methods of producing delivery vectors herein comprise packaging a nucleic acid encoding a polypeptide (e.g., an effector protein, effector partner, fusion protein, or combination thereof) and a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging an effector protein-encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector. In some examples, the AAV vector can package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g. , a 5 ’ inverted terminal repeat and a 3 ’ inverted terminal repeat. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[0264] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes may be not the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2 / 9. In some examples, the hybrid AAV delivery vector comprises an AAV2 / 1, AAV2 / 2, AAV 2 / 4, AAV2 / 5, AAV2 / 8, or AAV2 / 9 vector.Producing A A V Particles

[0265] The AAV particles described herein can be referred to as recombinant AAV (rAAV). Often, rAAV particles are generated by transfecting AAV producing cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing cells are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian cells. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KBcell, a derivative thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell can comprise transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31(4):317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties.

[0266] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, an insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells can comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells can comprise infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Then, 1; 13(16): 1935-43; and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety.

[0267] In some embodiments, the viral particle that delivers the viral vector described herein is an AAV. AAVs are characterized by their serotype. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, scAAV, AAV-rhlO, chimeric or hybrid AAV, or any combination, derivative, or variant thereof.Target Nucleic Acids

[0268] Disclosed herein are compositions, systems, and methods for modifying and detecting target nucleic acids. In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some embodiments, the target nucleic acid is a single stranded nucleic acid. In some embodiments, the target nucleic acid is a double stranded nucleic acid that is prepared into single stranded nucleic acids before or upon contacting a reagent or sample. In some embodiments, the target nucleic acid comprises DNA. In some embodiments, the target nucleic acid comprises RNA. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA). In some embodiments, the target nucleic acid is complementary DNA (cDNA) synthesizedfrom a single-stranded RNA template in a reaction catalyzed by a reverse transcriptase. In some embodiments, the target nucleic acid is single -stranded RNA (ssRNA) or mRNA. In some embodiments, the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence. In some embodiments, where a target strand comprises a target sequence, at least a portion of the engineered guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, where the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, and wherein the target strand comprises a target sequence, at least a portion of the engineered guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, a target nucleic acid comprises a PAM as described herein that is located on the non-target strand. Such a PAM described herein, in some embodiments, is adjacent (e.g., within 1, 2, 3, 4 or 5 nucleotides) to the 3’ end of the target sequence on the non-target strand of the double stranded DNA molecule. In certain embodiments, such a PAM described herein is directly adjacent to the 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule. In some embodiments, the PAM sequence 5’ end to the target sequence. In some embodiments, the PAM sequence is directly 5’ end to the target sequence. In some embodiments, the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence. However, any target nucleic acid of interest may be generated using the methods described herein to comprise a PAM sequence, and thus be a PAM target nucleic acid. A PAM target nucleic acid, as used herein, refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by an effector protein system.

[0269] In some embodiments, target nucleic acids comprise a mutation. In some embodiments, a composition, system or method described herein can be used to modify a target nucleic acid comprising a mutation such that the mutation is modified to be a wild-type nucleotide or nucleotide sequence. In some embodiments, a composition, system or method described herein can be used to detect a target nucleic acid comprising a mutation. The mutation may be a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. The mutation can be a deletion of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. The mutation can be a deletion of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides. The mutation can be in the open reading frame of a target nucleic acid that results in the insertion of at least one amino acid in a protein encoded by the target nucleic acid. The mutation can also be in the open reading frame of a target nucleic acid that results in the deletion of at least one amino acid in a protein encoded by the target nucleic acid. The mutation can be in the open readingframe of a target nucleic acid that results in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid can result in misfolding of the protein. The mutation can result in a premature stop codon. The mutation can result in a truncation of the protein.

[0270] In some embodiments, at least a portion of a guide nucleic acid of a composition described herein hybridizes to a region of the target nucleic acid comprising the mutation. In some embodiments, at least a portion of a guide nucleic acid of a composition described herein hybridizes to a region of the target nucleic acid that is within 10 nucleotides, within 50 nucleotides, within 100 nucleotides, or within 200 nucleotides of the mutation. The mutation may be located in a non-coding region or a coding region of a gene.

[0271] In some embodiments, compositions, systems, and methods described herein comprise a target nucleic acid may be responsible for a disease, contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g., contain a unique sequence of nucleotides). In some embodiments, the target nucleic acid has undergone a modification (e.g., an editing) after contacting with an RNP. In some embodiments, the editing is a change in the sequence of the target nucleic acid. In some embodiments, the change comprises an insertion, deletion, or substitution of one or more nucleotides compared to the target nucleic acid that has not undergone any modification.

[0272] In some embodiments, the mutation is an autosomal dominant mutation. In some embodiments, the mutation is a dominant negative mutation. In some embodiments, the mutation is a loss of function mutation. In some embodiments, the mutation is a single nucleotide polymorphism (SNP). In some embodiments, the SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. The SNP, in some embodiments, is associated with altered phenotype from wild type phenotype. The SNP may be a synonymous substitution or a nonsynonymous substitution. The nonsynonymous substitution may be a missense substitution, or a nonsense point mutation. The synonymous substitution may be a silent substitution. The mutation may be a deletion of one or more nucleotides. Often, the single nucleotide mutation, SNP, or deletion is associated with a disease such as cancer or a genetic disorder. The mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell, such as a cancer cell.

[0273] In some embodiments, the target nucleic acid comprises a mutation associated with a disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, condition, or syndrome. In some examples, a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. A mutation associated with a disease can also refer to any mutation which generates transcription ortranslation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some embodiments, the mutation causes the disease.

[0274] In some embodiments, the target nucleic acid is from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. Non-limiting examples of diseases associated with genetic mutations are recited in TABLE 3.

[0275] The disease may comprise, at least in part, a cancer, an inherited disorder, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, a metabolic disorder, a genetic disorder, an infection, or a combination thereof. In some embodiments, the cancer is a solid cancer (i.e., a tumor). In some embodiments, the cancer is a blood cell cancer, including a leukemia or lymphoma. In some embodiments, the cancer is colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, bladder cancer, cancer of the kidney or ureter, lung cancer, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, brain cancer (e.g., glioblastoma), cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, breast cancer, testicular cancer, cervical cancer, stomach cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, thyroid cancer. The cancer may be a leukemia, such as, by way of non-limiting example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0276] In some embodiments, the target nucleic acid comprises a portion of a gene comprising a mutation associated with cancer, a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some embodiments, the assay may be used to detect “hotspots” in target nucleic acids that may be predictive of lung cancer. In some embodiments, the target nucleic acid comprises a portion of a nucleic acid that is associated with a hemorrhagic fever.

[0277] In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of at least one of the genes recited in TABLE 4.

[0278] In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of at least one of: ABL, ACE, AF4 / HRX, AKT-2, ALK, ALK / NPM, AML1, AML1 / MTG8, APC, ATM, AXIN2, AXL, BAP1, BARD1, BCL-2, BCL-3, BCL- 6, BCR / ABL, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, c-MYC, CASR, CCR5, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CREBBP, CTNNA1, DBL, DEK / CAN, DICER1, DIS3L2, E2A / PBX1, EGER, ENL / HRX, EPCAM, ERG / TLS, ERBB, ERBB- 2, ETS-1, EWS / FLI-1, FH, FKRP, FLCN, FMS, FOS, FPS, GATA2, GCG, GLI, GPC3, GPGSP,GREM1, HER2 / neu, HOX11, H0XB13, HRAS, HST, IL-3, INT-2, JAK1, JUN, KIT, KS3, K-SAM, LBC, LCK, LM01, IM02, L-MYC, LYL-I, LYT-10, LYT-10 / Cal, MAS, MAX, MDM-2, MEN1, MET, MITE, MLH1, MLL, MOS, MSH1, MSH2, MSH3, MSH6, MTG8 / AML1, MUTYH, MYB, MYH11 / CBFB, NBN, NEU, NFI, NF2, N-MYC, NTHL1, OST, PALB2, PAX-5, PBX1 / E2A, PCDC1, PDGFRA, PH0X2B, PIM-1, PMS2, POLDI, POLE, POTI, PPARG, PRAD-1, PRKAR1A, PTCHI, PTEN, RAD50, RAD51C, RAD51D, RAF, RAR / PML, RAS-H, RAS-K, RAS-N, RBI, RECQL4, REL / NRG, RET, RH0M1, RH0M2, ROS, RUNX1, SDHA, SDHAF, SDHAF2, SDHB, SDHC, SDHD, SET / CAN, SIS, SKI, SMAD4, SMARCA4, SMARCB1, SMARCE1, SRC, STK11, SUFU, TALI, TAL2, TAN-1, TIAM1, TERC, TERT, TIMP3, TMEM127, TNF, TP53, TRAC, TSC1, TSC2, TRK, VHL, WRN, and WT1. Non-limiting examples of oncogenes are KRAS, NRAS, BRAF, MYC, CTNNB1, and EGFR. In some embodiments, the oncogene is a gene that encodes a cyclin dependent kinase (CDK). Non-limiting examples of CDKs are Cdkl, Cdk4, Cdk5, Cdk7, Cdk8, Cdk9, Cdkll and CDK20. Non-limiting examples of tumor suppressor genes are TP 53, RBI, and PTEN. Any region of the aforementioned gene loci may be probed for a mutation or deletion using the compositions and methods disclosed herein. For example, in the EGFR gene locus, the compositions and methods for detection disclosed herein may be used to detect a single nucleotide polymorphism or a deletion.

[0279] In some embodiments, the target nucleic acid comprises a portion of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a locus of at least one of: TRAC, B2M, PD1, PCSK9, DNMT1, HPRT1, RPL32P3, CCR5, FANCF, GRIN2B, EMX1, AAVS1, ALKBH5, CLTA, CDK11, CTNNB1, AXIN1, LRP6, TBK1, BAP1, TLE3, PPM1A, BCL2L2, SUFU, RICTOR, VPS35, TOPI, SIRT1, PTEN, MMD, PAQR8, H2AX, P0U5F1, OCT4, SYS1, ARFRP1, TSPAN14, EMC2, EMC 3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1.

[0280] In some embodiments, the method for treating a disease comprises modifying at least one gene associated with the disease or modifying expression of the at least one gene such that the disease is treated. In some embodiments, the disease is Alzheimer’s disease and the gene is selected from APP, BACE-1, PSD95, MAPT, PSEN1, PSEN2. and APOEe4. In some embodiments, the disease is congenital muscular dystrophy 1A (MDC1A) and the gene is LAMA1 or LAMA2. In some embodiments, the disease is Ullrich Congenital Muscular Dystrophy (UCMD) and the gene is selected from COL6A1 , COL6A2 and COL6A3. In some embodiments, the disease is Limb Girdle Muscular Dystrophies (LGMD1B, LGMD2A, LGMD2B) and the gene is selected from LMNA. DYSF, and CAPN3. In some embodiments, the disease is Nemaline Myopathy and the gene is selected from ACTA1 , NEB, TPM2, TPM3, TNNT1, TNNT3, TNNI2 and IMOD 3.

[0281] In some embodiments, the disease is Parkinson’s disease and the gene is selected from SNCA, GDNF, and LRRK2. In some embodiments, the disease comprises Centronuclear myopathy and the gene is DNM2. In some embodiments, the disease is Huntington's disease and the gene is HTT. In some embodiments, the disease is Alpha- 1 antitrypsin deficiency (AATD) and the gene is SERPINA1. In some embodiments, the disease is amyotrophic lateral sclerosis (ALS) and the gene is selected fromS0D1, FUS, C9ORF72, ATXN2, TARDBP, and CHCHD10. In some embodiments, the disease comprises Alexander Disease and the gene is GFAP. In some embodiments, the disease comprises anaplastic large cell lymphoma and the gene is CD30. In some embodiments, the disease comprises Angelman Syndrome and the gene is UBE3A. In some embodiments, the disease comprises Calcific Aortic Stenosis and the gene is Apo(a). In some embodiments, the disease comprises CD3Z-associated primary T-cell immunodeficiency and the gene is CD3Z or CD247. In some embodiments, the disease comprises CD 18 deficiency and the gene is ITGB2. In some embodiments, the disease comprises CD40L deficiency and the gene is CD40L. In some embodiments, the disease comprises CNS trauma and the gene is VEGF. In some embodiments, the disease comprises coronary heart disease and the gene is selected from EGA. FGB. and FGG. In some embodiments, the disease comprises MECP2 Duplication syndrome and Rett syndrome and the gene is MECP2. In some embodiments, the disease comprises a bleeding disorder (coagulation) and the gene is FXI. In some embodiments, the disease comprises fragile X syndrome and the gene is FMRI. In some embodiments, the disease comprises Fuchs Comeal Dystrophy and the gene is selected from ZEB1. SLC4A11, and L0XHD1. In some embodiments, the disease comprises GM2-Gangliosidoses (e.g., Tay Sachs Disease, Sandhoff disease) and the gene is selected from HEXA and HEXB. In some embodiments, the disease comprises Hearing loss disorders and the gene is DFNA36. In some embodiments, the disease is Pompe disease, including infantile onset Pompe Disease (IOPD) and late onset Pompe Disease (LOPD) and the gene is GAA. In some embodiments, the disease is Retinitis pigmentosa and the gene is selected from PDE6B, RHO, RP1, RP2, RPGR, PRPH2, IMPDH1, PRPF31, CRB1, PRPF8, TULP1, CA4, HPRPF3, ABCA4, EYS, CERKL, FSCN2, TOPORS, SNRNP200, PRCD, NR2E3, MERTK, USH2A, PR0M1, KLHL7, CNGB1, TTC8, ARL6, DHDDS, BESTI, LRAT, SPARA7, CRX, CLRN1, RPE65. and WDR19. In some embodiments, the disease comprises Leber Congenital Amaurosis Type 10 and the gene is CEP 290. In some embodiments, the disease is cardiovascular disease and / or lipodystrophies and the gene is selected from ABCG5, ABCG8, AGP, ANGPTL3, APOCIII, APOA1, APOL1, ARH, CDKN2B, CFB, CXCL12, FXI, FXII, GATA-4, MIA3, MKL2, MTHFD1L, MYH7, NKX2-5. NOTCH1, PKK, PCSK9, PSRCI, SMAD3, and TTR. In some embodiments, the disease is cardiovascular disease and / or lipodystrophies and the gene is ANGPTL3. In some embodiments, the disease is cardiovascular disease and / or lipodystrophies and the gene is PCSK9. In some embodiments, the disease is cardiovascular disease and / or lipodystrophies and the gene is TTR. In some embodiments, the disease is severe hypertriglyceridemia (SHTG) and the gene is APOCIII or ANGPTL4. In some embodiments, the disease comprises acromegaly and the gene is GHR. In some embodiments, the disease comprises acute myeloid leukemia and the gene is CD22. In some embodiments, the disease is diabetes and the gene is GCGR. In some embodiments, the disease is NAFLD / NASH and the gene is selected from HSD17B13. PSD 3. GPAM. CIDEB. DGAT2 and PNPLA3. In some embodiments, the disease is NASH / cirrhosis and the gene is MARC1. In some embodiments, the disease is cancer and the gene is selected from STATS, YAP1, FOXP3, AR (Prostate cancer), anA IRI- 4 (multiple myeloma). In some embodiments, the diseaseis cystic fibrosis and the gene is CFTR. In some embodiments, the disease is Duchenne Muscular Dystrophy and the gene is DMD. In some embodiments, the disease is ornithine transcarbamylase deficiency (OTCD) and the gene is OTC. In some embodiments, the disease is congenital adrenal hyperplasia (CAH) and the gene is CYP21A2. In some embodiments, the disease is atherosclerotic cardiovascular disease (ASCVD) and the gene is LPA. In some embodiments, the disease is hepatitis B virus infection (CHB) and the gene is HBV covalently closed circular DNA (cccDNA). In some embodiments, the disease is citrullinemiatype I and the gene is ASS1. In some embodiments, the disease is citrullinemia type I and the gene is SLC25A13. In some embodiments, the disease is citrullinemia type I and the gene is ASS1. In some embodiments, the disease is arginase- 1 deficiency and the gene is ARG1. In some embodiments, the disease is carbamoyl phosphate synthetase I deficiency and the gene is CPS1. In some embodiments, the disease is argininosuccinic aciduria and the gene is ASL. In some embodiments, the disease comprises angioedema and the gene is PKK. In some embodiments, the disease comprises thalassemia and the gene is TMPRSS6. In some embodiments, the disease comprises achondroplasia and the gene is FGFR3. In some embodiments, the disease comprises Cri du chat syndrome and the gene is selected from CTNND2. In some embodiments, the disease comprises sickle cell anemia and the gene is Beta globin gene. In some embodiments, the disease comprises Alagille Syndrome and the gene is selected from JAG1 and N0TCH2. In some embodiments, the disease comprises Charcot Marie Tooth Disease and the gene is selected from PMP22 and MFN2. In some embodiments, the disease comprises Crouzon syndrome and the gene is selected from FGFR2, FGFR3, and FGFR3. In some embodiments, the disease comprises Dravet Syndrome and the gene is selected from SCN1A and SCN2A. In some embodiments, the disease comprises Emery-Dreifuss syndrome and the gene is selected from EMD, LMNA, SYNE1, SYNE2, FHL1 , and TMEM43. In some embodiments, the disease comprises Factor V Leiden Thrombophilia and the gene is F5. In some embodiments, the disease is fabry disease and the gene is GLA. In some embodiments, the disease is facioscapulohumeral muscular dystrophy (FSHD) and the gene is FSHD1. In some embodiments, the disease comprises Fanconi anemia and the gene is selected from FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCE FANCM, FANCN, FANCP, FANCS, RAD51C, and XPF. In some embodiments, the disease comprises Familial Creutzfeld-Jakob Disease and the gene is PRNP. In some embodiments, the disease comprises Familial Mediterranean Fever and the gene is MEFV. In some embodiments, the disease comprises Friedreich's ataxia and the gene is FXN. In some embodiments, the disease comprises Gaucher disease and the gene is GBA. In some embodiments, the disease comprises human papilloma virus (HPV) infection and the gene is HPV E7. In some embodiments, the disease comprises Hemochromatosis and the gene is HFE. optionally comprising a C282Y mutation. In some embodiments, the disease comprises Hemophilia A and the gene is FVIII. In some embodiments, the disease is hereditary angioedema and the gene is SERPING1 or KLKB1. In some embodiments, the disease comprises histiocytosis and the gene is CD1. In some embodiments, the disease comprises immunodeficiency 17 and the gene is CD3D. In some embodiments, the diseasecomprises immunodeficiency 13 and the gene is CD4. In some embodiments, the disease comprises Common Variable Immunodeficiency and the gene is selected from CD 19 and CD81. In some embodiments, the disease comprises Joubert syndrome and the gene is selected from INPP5E, TMEM216, AHI1, NPHP1, CEP 290, TMEM67, RPGRIP1L, ARL13B, CC2D2A, OFD1, TMEM138, TCTN3, ZNF423, and AMRC9. In some embodiments, the disease comprises leukocyte adhesion deficiency and the gene is CD18. In some embodiments, the disease comprises Li-Fraumeni syndrome and the gene is TP53. In some embodiments, the disease comprises lymphoproliferative syndrome and the gene is CD27. In some embodiments, the disease comprises Lynch syndrome and the gene is selected from MSH2, MLH1, MSH6, PMS2, PMS1, TGFBR2, wA MLH3. In some embodiments, the disease comprises mantle cell lymphoma and the gene is CD5. In some embodiments, the disease comprises Marfan syndrome and the gene is FBN1. In some embodiments, the disease comprises mastocytosis and the gene is CD2. In some embodiments, the disease comprises methylmalonic acidemia and the gene is selected from MMAA, MMAB, and MUT. In some embodiments, the disease is mycosis fiingoides and the gene is CD7. In some embodiments, the disease is myotonic dystrophy and the gene is selected from CNBP and DMPK. In some embodiments, the disease comprises neurofibromatosis and the gene is selected from NF1, and NF2. In some embodiments, the disease comprises osteogenesis imperfecta and the gene is selected from COL1A1, COL1A2, and IFITM5. In some embodiments, the disease is non-small cell lung cancer and the gene is selected from KRAS, EGFR, ALK, METexl4, BRAF V600E, ROS1, RET, and NTRK. In some embodiments, the disease comprises Peutz-Jeghers syndrome and the gene is STK11. In some embodiments, the disease comprises polycystic kidney disease and the gene is selected from PKD1 and PKD2. In some embodiments, the disease comprises Severe Combined Immune Deficiency and the gene is selected from II 77R. RAG1..JAK3, In some embodiments, the disease comprises PRKAG2 cardiac syndrome and the gene is PRKAG2. In some embodiments, the disease comprises Spinocerebellar ataxia and the gene is selected from ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, ATXN8OS, ATXN10, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND3. and FGF14. In some embodiments, the disease is thrombophilia due to antithrombin III deficiency and the gene is SERPINC1. In some embodiments the disease is spinal muscular atrophy and the gene is SMN1. In some embodiments, the disease comprises Usher Syndrome and the gene is selected from MY07A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, DFNB31, and CLRNI. In some embodiments, the disease comprises von Willebrand disease and the gene is VWF In some embodiments, the disease comprises Waardenburg syndrome and the gene is selected from PAX3, MITF, WS2B, WS2C, SNAI2, EDNRB, EDN3, and SOXIO. In some embodiments, the disease comprises Wiskott-Aldrich Syndrome and the gene is WAS. In some embodiments, the disease comprises von Hippel-Lindau disease and the gene is VHP. In some embodiments, the disease comprises Wilson disease and the gene is ATP7B. In some embodiments, the disease comprises Zellweger syndrome and the gene is selected from PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In someembodiments, the disease comprises infantile myofibromatosis and the gene is CD34. In some embodiments, the disease comprises platelet glycoprotein IV deficiency and the gene is CD 36. In some embodiments, the disease comprises immunodeficiency with hyper-IgM type 3 and the gene is CD40. In some embodiments, the disease comprises hemolytic uremic syndrome and the gene is CD46. In some embodiments, the disease comprises complement hyperactivation, angiopathic thrombosis, or protein-losing enteropathy and the gene is CD55. In some embodiments, the disease comprises hemolytic anemia and the gene is CD59. In some embodiments, the disease comprises calcification of joints and arteries and the gene is CD73. In some embodiments, the disease comprises immunoglobulin alpha deficiency and the gene is CD79A. In some embodiments, the disease comprises C syndrome and the gene is CD96. In some embodiments, the disease comprises hairy cell leukemia and the gene is CD 123. In some embodiments, the disease comprises histiocytic sarcoma and the gene is CD163. In some embodiments, the disease comprises autosomal dominant deafness and the gene is CD 164. In some embodiments, the disease comprises immunodeficiency 25 and the gene is CD247. In some embodiments, the disease comprises methymalonic acidemia due to transcobalamin receptor defect and the gene is CD320.

[0282] In some embodiments, treatment of a disease comprises administration of a gene therapy. In some embodiments, a gene therapy comprises use of a vector to introduce a functional gene or transgene. In some embodiments, vectors comprise nonviral vectors, including cationic polymers, cationic lipids, or bio-responsive polymers. In some embodiments, the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space. In some embodiments, vectors comprise viral vectors, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. In some embodiments, the vector comprises a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects. Methods of gene therapy are described in more detail in Ingusci et al., “Gene Therapy Tools for Brain Diseases". Front. Pharmacol. 10:724 (2019) which is hereby incorporated by reference in its entirety.

[0283] The target nucleic acid may be from any organism, including, but not limited to, a bacterium, a virus, a parasite, a protozoon, a fungus, a mammal, a plant, and an insect. As another non-limiting example, the target nucleic acid may be responsible for a disease, contain a mutation (e.g. , single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g., contain a unique sequence of nucleotides). In some embodiments, the target nucleic acid is from a bacteria. In some embodiments, the bacteria is Acholeplasma laidlawii, Brucella abortus, Chlamydia psittaci, Chlamydia trachomatis, Cryptococcus neoformans, Escherichia coli, Legionella pneumophila, Lyme disease spirochetes, methicillin-resistant Staphylococcus aureus, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma genitalium, Mycoplasma hyorhinis, Mycoplasma orale,Mycoplasma pneumoniae, Mycoplasma salivarium, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus pyogenes, Treponema pallidum, or any combination thereof.

[0284] In some embodiments, the target nucleic acid is from a virus. In some embodiments, the virus is adenovirus, blue tongue virus, chikungunya, coronavirus (e.g., SARS-CoV-2), cytomegalovirus, Dengue virus, Ebola, Epstein-Barr virus, feline leukemia virus, Hemophilus influenzae B, Hepatitis Virus A, Hepatitis Virus B, Hepatitis Virus C, herpes simplex virus I, herpes simplex virus II, human papillomavirus (HPV) including HPV16 and HPV18, human serum parvo-like virus, human T-cell leukemia viruses, immunodeficiency virus (e.g., HIV), influenza virus, lymphocytic choriomeningitis virus, measles virus, mouse mammary tumor virus, mumps virus, murine leukemia virus, polio virus, rabies virus, Reovirus, respiratory syncytial virus (RSV), rubella virus, Sendai virus, simian virus 40, Sindbis virus, varicella-zoster virus, vesicular stomatitis virus, wart virus, West Nile virus, yellow fever virus, or any combination thereof. In some embodiments, the target nucleic acid comprises a portion of a nucleic acid that is associated with a hemorrhagic fever.

[0285] In some embodiments, the target nucleic acid is from a parasite. In some embodiments, the parasite is a helminth, an annelid, a platyhelminth, a nematode, or a thorny-headed worms. In some embodiments, the parasite is Babesia bovis, Echinococcus granulosus, Eimeria tenella, Leishmania tropica, Mesocestoides corti, Onchocerca volvulus, Plasmodium falciparum, Plasmodium vivax, Schistosoma japonicum, Schistosoma mansoni, Schistosoma spp., Taenia hydatigena, Taenia ovis, Taenia saginata, Theileria parva, Toxoplasma gondii, Toxoplasma spp., Trichinella spiralis, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma rhodesiense, Balantidium coli, Entamoeba histolytica, Giardia spp. , Isospora spp. , Trichomonas spp. , or any combination thereof.

[0286] In some embodiments, the target nucleic acid comprises a nucleic acid sequence from a pathogen responsible for a disease. Non-limiting examples of pathogens are bacteria, a virus and a fungus. The target nucleic acid, in some embodiments, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease. In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any DNA amplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are notlimited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans. Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermaiiiidis. Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to coronavirus (e.g., SARS-CoV-2); immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae. In some embodiments, the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment.

[0287] In some embodiments, the target nucleic acid comprises a nucleic acid sequence of a virus, a bacterium, or other pathogen responsible for a disease in a plant (e.g., a crop). Methods and compositions of the disclosure may be used to treat or detect a disease in a plant. For example, the methods of the disclosure may be used to target a viral nucleic acid sequence in a plant. An effector protein of the disclosure may cleave the viral nucleic acid. In some embodiments, the target nucleic acid comprises a nucleic acid sequence of a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid comprises RNA. The target nucleic acid, in some embodiments, is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any NAamplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g, a crop). A virus infecting the plant may be an RNA virus. A virus infecting the plant may be a DNA virus. Non-limiting examples of viruses that may be targeted with the disclosure include Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PVX).

[0288] In some embodiments, the target nucleic acid is any one of: a naturally occurring eukaryotic sequence, a naturally occurring prokaryotic sequence, a naturally occurring viral sequence, a naturally occurring bacterial sequence, a naturally occurring fungal sequence, an engineered eukaryotic sequence, an engineered prokaryotic sequence, an engineered viral sequence, an engineered bacterial sequence, an engineered fungal sequence, a fragment of a naturally occurring sequence, a fragment of an engineered sequence, and combinations thereof.

[0289] In some embodiments, the target nucleic acid is isolated from any one of: a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, and a non-naturally occurring cell.

[0290] Nucleic acids, such as DNA and pre-mRNA, described herein can contain at least one intron and at least one exon, wherein as read in the 5 ’ to the 3 ’ direction of a nucleic acid strand, the 3 ’ end of an intron can be adjacent to the 5’ end of an exon, and wherein said intron and exon correspond for transcription purposes. If a nucleic acid strand contains more than one intron and exon, the 5’ end of the second intron is adjacent to the 3’ end of the first exon, and 5’ end of the second exon is adjacent to the 3 ’ end of the second intron. The junction between an intron and an exon can be referred to herein as a splice junction, wherein a 5’ splice site (SS) can refer to the +1 / +2 position at the 5’ end of intron and a 3’SS can refer to the last two positions at the 3’ end of an intron. Alternatively, a 5’ SS can refer to the 5’ end of an exon and a 3’SS can refer to the 3’ end of an exon. In some embodiments, nucleic acids can contain one or more elements that act as a signal during transcription, splicing, and / or translation. In some embodiments, signaling elements include a 5’SS, a 3’SS, a premature stop codon, U1 and / or U2 binding sequences, and cis acting elements such as branch site (BS), polypyridine tract (PYT), exonic and intronic splicing enhancers (ESEs and ISEs) or silencers (ESSs and ISSs). In some embodiments, nucleic acids may also comprise a untranslated region (UTR), such as a 5 ’ UTR or a 3 ’ UTR. In some embodiments, the start of an exon or intron is referred to interchangeably herein as the 5’ end of an exon or intron, respectively. Likewise, in some embodiments, the end of an exon or intron is referred to interchangeably herein as the 3’ end of an exon or intron, respectively.

[0291] In some embodiments, at least a portion of at least one target sequence is within about 1, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 ormore, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 to about 300 nucleotides adj acent to : the 5 ’ end of an exon; the 3 ’ end of an exon; the 5 ’ end of an intron; the 3 ’ end of an intron; one or more signaling element comprising a 5’SS, a 3’SS, a premature stop codon, U1 binding sequence, U2 binding sequence, a BS, a PYT, ESE, an ISE, an ESS, an ISS; a 5’ UTR; a 3’ UTR; more than one of the foregoing, or any combination thereof. In some embodiments, the target nucleic acid comprises a target locus. In some embodiments, the target nucleic acid comprises more than one target loci. In some embodiments, the target nucleic acid comprises two target loci. Accordingly, in some embodiments, the target nucleic acid can comprise one or more target sequences.

[0292] In some embodiments, compositions, systems, and methods described herein comprise an edited target nucleic acid which can describe a target nucleic acid wherein the target nucleic acid has undergone a change, for example, after contact with an effector protein. In some embodiments, the editing is an alteration in the sequence of the target nucleic acid. In some embodiments, the edited target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unedited target nucleic acid. In some embodiments, the editing is a mutation.Mutations

[0293] In some embodiments, target nucleic acids described herein comprise a mutation. In some embodiments, a composition, system or method described herein can be used to edit a target nucleic acid comprising a mutation such that the mutation is edited to be the wild-type nucleotide or nucleotide sequence. In some embodiments, a composition, system or method described herein can be used to detect a target nucleic acid comprising a mutation. A mutation may result in the insertion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the deletion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid may result in misfolding of a protein encoded by the target nucleic acid. A mutation may result in a premature stop codon, thereby resulting in a truncation of the encoded protein.

[0294] Non-limiting examples of mutations are insertion-deletion (indel), a point mutation, single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation or variation, and frameshift mutations. In some embodiments, an indel mutation is an insertion or deletion of one or more nucleotides. In some embodiments, a point mutation comprises a substitution, insertion, or deletion. In some embodiments, a frameshift mutation occurs when the number of nucleotides in the insertion / deletion is not divisible by three, and it occurs in a protein coding region. In some embodiments, a chromosomal mutation can comprise an inversion, a deletion, a duplication, or atranslocation of one or more nucleotides. In some embodiments, a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. In some embodiments, an SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some embodiments, an SNP is associated with altered phenotype from wild type phenotype. In some embodiments, the SNP is a synonymous substitution or a nonsynonymous substitution. In some embodiments, the nonsynonymous substitution is a missense substitution or a nonsense point mutation. In some embodiments, the synonymous substitution is a silent substitution.

[0295] In some embodiments, a target nucleic acid described herein comprises a mutation of one or more nucleotides. In some embodiments, the one or more nucleotides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the mutation comprises a deletion, insertion, and / or substitution of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. In some embodiments, the mutation comprises a deletion, insertion, and / or substitution of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides. The mutation may be located in a non-coding region or a coding region of a gene, wherein the gene is a target nucleic acid. A mutation may be in an open reading frame of a target nucleic acid. In some embodiments, guide nucleic acids described herein hybridize to a portion of the target nucleic acid comprising or adjacent to the mutation.

[0296] In some embodiments, target nucleic acids comprise a mutation, wherein the mutation is a SNP. In some embodiments, the single nucleotide mutation or SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some embodiments, the SNP is associated with altered phenotype from wild type phenotype. In some embodiments, a single nucleotide mutation, SNP, or deletion described herein is associated with a disease, such as a genetic disease. In some embodiments, the SNP is a synonymous substitution or a nonsynonymous substitution. In some embodiments, the nonsynonymous substitution is a missense substitution or a nonsense point mutation. In some embodiments, the synonymous substitution is a silent substitution. In some embodiments, the mutation is a deletion of one or more nucleotides. In some embodiments, the single nucleotide mutation, SNP, or deletion is associated with a disease such as a genetic disorder. In some embodiments, the mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell.

[0297] In some embodiments, the mutation is associated with a disease, such as a genetic disorder. In some embodiments, the mutation may be encoded in the sequence of a target nucleic acid from thegermline of an organism or may be encoded in a target nucleic acid from a diseased cell. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to or suffers from, a disease, disorder, condition, or syndrome. In some examples, a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. A mutation associated with a disease may also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some embodiments, a mutation associated with a disease, comprises the co-occurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation. In some embodiments, a target nucleic acid described herein comprises a mutation associated with a disease described herein.Detection and Identification of Target Nucleic Acid

[0298] In some embodiments, a target nucleic acid is in a cell. In some embodiments, the cell is a single-cell eukaryotic organism; a plant cell an algal cell; a fungal cell; an animal cell; a cell of an invertebrate animal; a cell of a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a cell of a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell, a human cell, or a plant cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a: muscle cell, liver cell, lung cell, cardiac cell, visceral cell, cardiac muscle cell, smooth muscle cell, cardiomyocyte, nodal cardiac muscle cell, smooth muscle cell, visceral muscle cell, skeletal muscle cell, myocyte, red (or slow) skeletal muscle cell, white (fast) skeletal muscle cell, intermediate skeletal muscle, muscle satellite cell, muscle stem cell, myoblast, muscle progenitor cell, induced pluripotent stem cell (iPS), or a cell derived from an iPS cell, modified to have its gene edited and differentiated into myoblasts, muscle progenitor cells, muscle satellite cells, muscle stem cells, skeletal muscle cells, cardiac muscle cells or smooth muscle cells.

[0299] In some embodiments, an effector protein-guide nucleic acid complex may comprise high selectivity for a target sequence. In some embodiments, an RNP comprise a selectivity of at least 200: 1, 100: 1, 50: 1, 20: 1, 10: 1, or 5: 1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. In some embodiments, an RNP may comprise a selectivity of at least 5: 1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid.

[0300] By leveraging such effector protein selectivity, some methods described herein may detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some embodiments, the method detects at least 2 target nucleic acid populations. In someembodiments, the method detects at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some embodiments, the method detects 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations. In some embodiments, the method detects at least 2 individual target nucleic acids. In some embodiments, the method detects at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 individual target nucleic acids. In some embodiments, the method detects 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 individual target nucleic acids. In some embodiments, the method detects target nucleic acid present at least at one copy per 10 non-target nucleic acids, 102non-target nucleic acids, 103non-target nucleic acids, 104non-target nucleic acids, 105non-target nucleic acids, 106non- target nucleic acids, 107non-target nucleic acids, 108non-target nucleic acids, 109non-target nucleic acids, or 1010non-target nucleic acids.

[0301] In some embodiments, compositions described herein exhibit indiscriminate trans-cleavage of a nucleic acid (e.g., ssRNA or ssDNA), enabling their use for detection of a nucleic acid (e.g, RNA or DNA, respectively) in samples. In some embodiments, target nucleic acids are generated from many nucleic acid templates (e.g., RNA) in order to achieve cleavage of a reporter (e.g., a FQ reporter) in a device (e.g., a DETECTR platform). Certain effector proteins may be activated by a nucleic acid (e.g., ssDNA or ssRNA), upon which they may exhibit trans-cleavage of the nucleic acid (e.g., ssDNA or ssRNA) and may, thereby, be used to cleave reporter molecules (e.g., ssDNA or ssRNA FQ reporter molecules) in a device (e.g., a DETECTR). These effector proteins may target nucleic acids present in the sample or nucleic acids generated and / or amplified from any number of nucleic acid templates (e.g., RNA). Described herein are reagents comprising a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid (e.g., a ssDNA-FQ reporter described herein) is capable of being cleaved by the effector protein, upon generation (e.g., cDNA) and amplification of nucleic acids from a nucleic acid template (e.g., ssRNA) using the methods disclosed herein, thereby generating a first detectable signal. While DNA and RNA are used as an exemplary reporter in the foregoing, any suitable reporter may be used.

[0302] In some embodiments, a target nucleic acid is an amplified nucleic acid of interest. In some embodiments, the nucleic acid of interest is any nucleic acid disclosed herein or from any sample as disclosed herein. In some embodiments, the nucleic acid of interest is an RNA that is reverse transcribed before amplification. In some embodiments, the nucleic acid of interest is amplified then the amplicons is transcribed into RNA.

[0303] In some embodiments, target nucleic acids may activate an effector protein to initiate sequenceindependent cleavage of a nucleic acid-based reporter (e.g., a reporter comprising an RNA sequence, or a reporter comprising DNA and RNA). For example, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA (also referred to herein as an “RNA reporter”). Alternatively, an effector protein of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA. Alternatively, an effector protein of the present disclosure isactivated by a target RNA to cleave reporters having an RNA (also referred to herein as a “RNA reporter”). The RNA reporter may comprise a single-stranded RNA labelled with a detection moiety or may be any RNA reporter as disclosed herein.

[0304] Further description of editing or detecting a target nucleic acid in a gene of interest can be found in more detail in Kim et al. , “Enhancement of target specificity of CRISPR-Cas 12a by using a chimeric DNA-RNA guide”, Nucleic Acids Res. 2020 Sep 4;48(15):8601-8616; Wang et al., “Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing”, Scientific Reports volume 10, Article number: 2269 (2020); Tuladhar et al. , “CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation”, Nature Communications volume 10, Article number: 4056 (2019); Dong et al., “Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring”, G3, Volume 9, Issue 11, 1 November 2019, Pages 3645-3651; Winter et al., “Genomewide CRISPR screen reveals novel host factors required for Staphylococcus aureus a-hemolysin- mediated toxicity”, Scientific Reports volume 6, Article number: 24242 (2016); and Ma et al., “A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death”, Cell Rep. 2015 Jul 28;12(4):673-83, which are hereby incorporated by reference in their entirety.Pharmaceutical Compositions and Modes of Administration

[0305] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, or fusion proteins described herein. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, or fusion proteins described herein. In some embodiments, pharmaceutical compositions comprise a guide nucleic acid. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.

[0306] In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent allows the active ingredient to retain biological activity. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is non-reactive with the subject's immune system. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent provides for long-term stabilization of the composition. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is provided as a bulking agent in solid formulations that contain potent active ingredients in small amounts. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent confers a therapeutic enhancement on the active ingredient in the final dosage form. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent facilitates absorption, reduces viscosity, or enhances solubility. In some embodiments, the pharmaceutically acceptable excipient, carrier or diluent is selected based upon the route of administration, dosage form, active ingredient, other factors, or any combination thereof. In some embodiments, the pharmaceutically acceptable excipient, carrier ordiluent can be formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0307] In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, effector partner, fusion protein, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, effector partner, fusion protein, or combination thereof may be any one of those described herein. The one or more nucleic acids may comprise a plasmid. The one or more nucleic acids may comprise a nucleic acid expression vector. The one or more nucleic acids may comprise a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, compositions, including pharmaceutical compositions, comprise a viral vector encoding a fusion protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion protein. In some embodiments, pharmaceutical compositions comprise a virus comprising a viral vector encoding a fusion protein, an effector protein, an effector partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.

[0308] Pharmaceutical compositions described herein may comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNOs. In some embodiments, the salt is Mg2+ SO42-.

[0309] Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives. In some embodiments, the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method. In some embodiments, the pharmaceutical compositions comprise a virus vector or a non-viral vector.

[0310] In some embodiments, pharmaceutical compositions are in the form of a solution (e.g. , a liquid). The solution may be formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH of the solution is less than 7. In some embodiments, the pH is greater than 7.Systems for Detection[3H] Disclosed herein, in some aspects, are systems for detecting a target nucleic acid, comprising any one of the effector proteins described herein. In some embodiments, systems comprise a guide nucleic acid. Systems may be used to detect a target nucleic acid. In some embodiments, systems comprise an effector protein described herein, a reagent, support medium, or a combination thereof. In some embodiments, systems comprise a fusion protein described herein.

[0312] Systems may be used for detecting the presence of a target nucleic acid associated with or causative of a disease, such as cancer, a genetic disorder, or an infection. In some embodiments, systems are useful for phenotyping, genotyping, or determining ancestry. Unless specified otherwise, systems include kits and may be referred to as kits. Unless specified otherwise, systems include devices and may also be referred to as devices. Systems described herein may be provided in the form of a companion diagnostic assay or device, a point-of-care assay or device, or an over-the-counter diagnostic assay / device.

[0313] In some embodiments, in vitro can be used to describe an event that takes places in a container for holding laboratory reagents such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. In some embodiments, in vivo can be used to describe an event that takes place in a subject’s body. In some embodiments, ex vivo can be used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an in vitro assay.

[0314] Reagents and effector proteins of various systems may be provided in a reagent chamber or on a support medium. Alternatively, the reagent and / or effector protein may be contacted with the reagent chamber or the support medium by the individual using the system. An exemplary reagent chamber is a test well or container. The opening of the reagent chamber may be large enough to accommodate the support medium. Optionally, the system comprises a buffer and a dropper. The buffer may be provided in a dropper bottle for ease of dispensing. The dropper may be disposable and transfer a fixed volume. The dropper may be used to place a sample into the reagent chamber or on the support medium.System solutions

[0315] In general, systems comprise a solution in which the activity of an effector protein occurs. Often, the solution comprises or consists essentially of a buffer. The solution or buffer may comprise a buffering agent, a salt, a crowding agent, a detergent, a reducing agent, a competitor, or a combination thereof. Often the buffer is the primary component or the basis for the solution in which the activity occurs. Thus, concentrations for components of buffers described herein (e.g., buffering agents, salts, crowding agents, detergents, reducing agents, and competitors) are the same or essentially the same asthe concentration of these components in the solution in which the activity occurs. In some embodiments, a buffer is required for cell lysis activity or viral lysis activity.

[0316] In some embodiments, systems comprise a buffer, wherein the buffer comprise at least one buffering agent. Exemplary buffering agents include HEPES, TRIS, MES, ADA, PIPES, ACES, MOPSO, BIS-TRIS propane, BES, MOPS, TES, DISO, Trizma, TRICINE, GLY-GLY, HEPPS, BICINE, TAPS, A MPD, A MPSO, CHES, CAPSO, AMP, CAPS, phosphate, citrate, acetate, imidazole, or any combination thereof. In some embodiments, the concentration of the buffering agent in the buffer is 1 mM to 200 mM. A buffer compatible with an effector protein may comprise a buffering agent at a concentration of 10 mM to 30 mM. A buffer compatible with an effector protein may comprise a buffering agent at a concentration of about 20 mM. A buffering agent may provide a pH for the buffer or the solution in which the activity of the effector protein occurs. The pH may be 3 to 4, 3.5 to 4.5, 4 to 5, 4.5 to 5.5, 5 to 6, 5.5 to 6.5, 6 to 7, 6.5 to 7.5, 7 to 8, 7.5 to 8.5, 8 to 9, 8.5 to 9.5, 9 to 10, or 9.5 to 10.5.

[0317] In some embodiments, systems comprise a solution, wherein the solution comprises at least one salt. In some embodiments, the at least one salt is selected from potassium acetate, magnesium acetate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and any combination thereof. In some embodiments, the concentration of the at least one salt in the solution is 5 mM to 100 mM, 5 mM to 10 mM, 1 mM to 60 mM, or 1 mM to 10 mM. In some embodiments, the concentration of the at least one salt is about 105 mM. In some embodiments, the concentration of the at least one salt is about 55 mM. In some embodiments, the concentration of the at least one salt is about 7 mM. In some embodiments, the solution comprises potassium acetate and magnesium acetate. In some embodiments, the solution comprises sodium chloride and magnesium chloride. In some embodiments, the solution comprises potassium chloride and magnesium chloride. In some embodiments, the salt is a magnesium salt and the concentration of magnesium in the solution is at least 5 mM, 7 mM, at least 9 mM, at least 11 mM, at least 13 mM, or at least 15 mM. In some embodiments, the concentration of magnesium is less than 20 mM, less than 18 mM, or less than 16 mM.

[0318] In some embodiments, systems comprise a solution, wherein the solution comprises at least one crowding agent. A crowding agent may reduce the volume of solvent available for other molecules in the solution, thereby increasing the effective concentrations of said molecules. Exemplary crowding agents include glycerol and bovine serum albumin. In some embodiments, the crowding agent is glycerol. In some embodiments, the concentration of the crowding agent in the solution is 0.01% (v / v) to 10% (v / v). In some embodiments, the concentration of the crowding agent in the solution is 0.5% (v / v) to 10% (v / v).

[0319] In some embodiments, systems comprise a solution, wherein the solution comprises at least one detergent. Exemplary detergents include Tween, Triton-X, and IGEPAL. A solution may comprise Tween, Triton-X, or any combination thereof. A solution may comprise Triton-X. A solution may comprise IGEPAL CA-630. In some embodiments, the concentration of the detergent in the solution is2% (v / v) or less. In some embodiments, the concentration of the detergent in the solution is 1% (v / v) or less. In some embodiments, the concentration of the detergent in the solution is 0.00001% (v / v) to 0.01% (v / v). In some embodiments, the concentration of the detergent in the solution is about 0.01% (v / v).

[0320] In some embodiments, systems comprise a solution, wherein the solution comprises at least one reducing agent. Exemplary reducing agents comprise dithiothreitol (DTT), B-mercaptoethanol (BME), or tris(2 -carboxyethyl) phosphine (TCEP). In some embodiments, the reducing agent is DTT. In some embodiments, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.5 mM to 2 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.01 mM to 100 mM. In some embodiments, the concentration of the reducing agent in the solution is 0.1 mM to 10 mM. In some embodiments, the concentration of the reducing agent in the solution is about 1 mM.

[0321] In some embodiments, systems comprise a solution, wherein the solution comprises a competitor. In general, competitors compete with the target nucleic acid or the reporter nucleic acid for cleavage by the effector protein or a dimer thereof. Exemplary competitors include heparin, and imidazole, and salmon sperm DNA. In some embodiments, the concentration of the competitor in the solution is 1 pg / mL to 100 pg / mL. In some embodiments, the concentration of the competitor in the solution is 40 pg / mL to 60 pg / mL.

[0322] In some embodiments, systems comprise a solution, wherein the solution comprises a co-factor. In some embodiments, the co-factor allows an effector protein or a multimeric complex thereof to perform a function, including pre-crRNA processing and / or target nucleic acid cleavage . The suitability of a cofactor for an effector protein or a multimeric complex thereof may be assessed, such as by methods based on those described by Sundaresan et al. (Cell Rep. 2017 Dec 26; 21(13): 3728-3739). In some embodiments, an effector or a multimeric complex thereof forms a complex with a co-factor. In some embodiments, the co-factor is a divalent metal ion. In some embodiments, the divalent metal ion is selected from Mg2+, Mn2+, Zn2+, Ca2+, Cu2+. In some embodiments, the divalent metal ion is Mg2+. In some embodiments, the co-factor is Mg2+.Reporters

[0323] In some embodiments, systems disclosed herein comprise a reporter. By way of non-limiting and illustrative example, a reporter may comprise a single stranded nucleic acid and a detection moiety (e.g., a labeled single stranded RNA reporter), wherein the nucleic acid is capable of being cleaved by an effector protein or a multimeric complex thereof, releasing the detection moiety, and generating a detectable signal. In some embodiments, a detectable signal comprises a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical and other detection methods known in the art. The effector proteins disclosed herein, activated upon hybridization of a guide nucleic acid toa target nucleic acid, may cleave the reporter. Cleaving the “reporter” may be referred to herein as cleaving the “reporter nucleic acid,” the “reporter molecule,” or the “nucleic acid of the reporter.” Reporters may comprise RNA. Reporters may comprise DNA. Reporters may be double-stranded. Reporters may be single-stranded.

[0324] In some embodiments, reporters comprise a protein capable of generating a signal. A signal may be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some embodiments, the reporter comprises a detection moiety. Suitable detectable labels and / or moieties that may provide a signal include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like.

[0325] In some embodiments, the reporter comprises a detection moiety and a quenching moiety. In some embodiments, the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter and the quenching moiety is located at a second site on the reporter, wherein the first site and the second site are separated by the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some embodiments, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some embodiments, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the nucleic acid of a reporter. Sometimes the detection moiety is at the 3’ terminus of the nucleic acid of a reporter. In some embodiments, the detection moiety is at the 5’ terminus of the nucleic acid of a reporter. In some embodiments, the quenching moiety is at the 3 ’ terminus of the nucleic acid of a reporter.

[0326] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B -Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, [3-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).

[0327] In some embodiments, the detection moiety comprises an invertase. The substrate of the invertase may be sucrose. A DNS reagent may be included in the system to produce a colorimetric change when the invertase converts sucrose to glucose. In some embodiments, the reporter nucleic acid and invertase are conjugated using a heterobifimctional linker via sulfo-SMCC chemistry.

[0328] Suitable fluorophores may provide a detectable fluorescence signal in the same range as 6- Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA T...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: a) an effector protein, or a nucleic acid encoding the effector protein, comprising an amino acid sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-161, and 391-847; and b) an engineered guide nucleic acid, or a nucleic acid encoding the engineered guide nucleic acid, comprising a protein binding sequence and a spacer sequence.

2. The composition of claim 1, wherein the protein binding sequence comprises a nucleotide sequence that is 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% identical to SEQ ID NO: 178-373, 375-390, and 848-1168, listed in TABLE 2.

3. The composition of claim 1 or 2, wherein the composition comprises a combination of the effector protein and the engineered guide nucleic acid comprising a protein binding sequence as described in TABLE 24. The composition of any one of claims 1-3, wherein the spacer sequence is 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% identical, complementary, or reverse complementary to a eukaryotic sequence.

5. The composition of any one of claims 1-4, wherein the spacer sequence is linked to a 5’ end of the protein binding sequence.

6. The composition of any one of claims 1-5, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, or about 540 contiguous amino acids of the sequence selected from SEQ ID NO: 1-161, and 391-847.

7. The composition of any one of claims 1-6, wherein a length of the effector protein is less than 600, less than 580, less than 560, less than 540, less than 520, less than 500, less than 480, less than 460, less than 440, less than 420, or less than 400 amino acids; and at least 300, at least 320, at least 340, at least 360, or at least 380 amino acids.

8. The composition of any one of claims 1-7, wherein the amino acid sequence is less than 100% identical to SEQ ID NO: 1-161 and 391-847, and wherein not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the amino acid sequence are non-conservative amino acid substitutions relative to a respective sequence selected from SEQ ID NO: 1-161 and 391-847.

9. The composition of claim 8, wherein the effector protein comprises at least one amino acid substitution in a RuvC domain, an HNH domain, or a combination thereof.

10. The composition of claim 9, wherein the at least one amino acid substitution is a non-conservative amino acid substitution.

11. The composition of claim 9 or 10, wherein the at least one amino acid substitution replaces a catalytic residue in the RuvC domain, the HNH domain, or a combination thereof.

12. The composition of any one of claims 1-11, wherein the effector protein has nickase activity.

13. The composition of any one of claims 1-12, wherein the effector protein is linked to a nuclear localization signal.

14. The composition of any one of claims 1-13, further comprising a donor nucleic acid.

15. The composition of any one of claims 1-14, further comprising an effector partner linked to the effector protein.

16. The composition of claim 15, wherein the effector partner comprises a polypeptide selected from a deaminase, a reverse transcriptase, a transcriptional activator, a transcriptional repressor, or a functional domain thereof.

17. The composition of any one of claims 1-16, wherein the effector protein comprises at least one mutation that reduces its nuclease activity relative to the effector protein without the at least one mutation as measured in a cleavage assay, optionally wherein the effector protein is a catalytically inactive nuclease.

18. The composition of any one of claims 1-17, further comprising a lipid nanoparticle containing the effector protein or the nucleic acid encoding the effector protein, the engineered guide nucleic acid, or a combination thereof.

19. The composition of any one of claims 1-18, wherein the effector protein or the nucleic acid encoding the effector protein, and the engineered guide nucleic acid or the nucleic acid encoding the engineered guide nucleic acid are provided in separate compositions.

20. The composition of any one of claims 1-19, wherein the nucleic acid encoding the effector protein, the nucleic acid encoding the engineered guide nucleic acid, or a combination thereof is an expression vector.

21. The composition of claim 20, wherein the expression vector is a viral vector, optionally wherein the viral vector is an adeno-associated viral (AAV) vector.

22. A pharmaceutical composition comprising the composition of any one of claims 1-21, and a pharmaceutically acceptable excipient.

23. A system comprising: a) an effector protein, or a nucleic acid encoding the effector protein, comprising an amino acid sequence that is 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% identical to a sequence selected from SEQ ID NOs: 1-161, and 391-847; andb) an engineered guide nucleic acid, or a nucleic acid encoding the engineered guide nucleic acid, comprising a protein binding sequence and a spacer sequence.

24. The system of claim 23, wherein the protein binding sequence comprises a nucleotide sequence that is 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% identical to SEQ ID NOs: 178-373, 375-390, and 848-1168, listed in TABLE 2.

25. The system of claim 23 or 24 , wherein the system comprises a combination of the effector protein and the engineered guide nucleic acid comprising a protein binding sequence as described in TABLE 226. The system of any one of claims 23-25, wherein the spacer sequence is 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% identical, complementary, or reverse complementary to a eukaryotic sequence.

27. The system of any one of claims 23-26, wherein the spacer sequence is linked to a 5’ end of the protein binding sequence.

28. The system of any one of claims 23-27, wherein the effector protein comprises about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, about 420, about 440, about 460, about 480, about 500, about 520, about 540 contiguous amino acids of the sequence selected from SEQ ID NOs: 1-161, and 391-847.

29. A method of detecting a target nucleic acid in a sample, comprising the steps of: a. contacting the sample with: i. the composition of any one of claims 1-21, the pharmaceutical composition of claim 22, or the system of any one of claims 23-28; and ii. a reporter nucleic acid comprising a detectable moiety that produces a detectable signal in the presence of the target nucleic acid and the composition, pharmaceutical composition, or system, and b. detecting the detectable signal.

30. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the composition of any one of claims 1-21, the pharmaceutical composition of claim 22, or the system of any one of claims 23-28, thereby modifying the target nucleic acid.

31. A cell comprising the composition of any one of claims 1-21.

32. A cell modified by the composition of any one of claims 1-21, the pharmaceutical composition of claim 22, or the system of any one of claims 23-28.

33. A cell comprising a modified target nucleic acid, wherein the modified target nucleic acid is a target nucleic acid modified according to claim 32.

34. A method of treating a disease comprising administering to a subject in need thereof any one of: a composition according to any one of claims 1-21; a pharmaceutical composition according to claim 22; the components of a system according to any one of claims 23-28; and a cell according to any one of claims 31-33.