Genetically modified endonucleases

Abstract

The present disclosure relates to compositions and methods for modifying target nucleic acids, and methods for detecting nucleic acids. Various compositions are described herein, including compositions comprising an endonuclease, an endonuclease system, and a chimeric protein having the endonuclease and a nucleic acid modulating domain or a nucleic acid modifying domain.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 384,937 filed on November 23, 2022, U.S. Provisional Application No. 63 / 386,784 filed on December 9, 2022, U.S. Provisional Application No. 63 / 500,779 filed on May 8, 2023, and U.S. Provisional Application No. 63 / 504,721 filed on May 26, 2023, the entire contents of which are incorporated herein in their entirety. Technical Field

[0003] The present disclosure relates to compositions, systems, and methods for modifying target RNA, as well as methods for detecting nucleic acids.

[0004] Description of the text file submitted electronically

[0005] This application contains a sequence listing, which is submitted in XML format via EFS-Web. The contents of the XML copy, titled "AMR-006PC / 134241-5006_SequenceListing," were created on November 24, 2023, and are 150,000 bytes in size, the contents of which are incorporated herein by reference in their entirety. Background Art

[0006] The bacterial adaptive immune system utilizes CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins to perform RNA-guided nucleic acid cleavage. Thus, the CRISPR-Cas system confers adaptive immunity to bacteria and archaea through RNA-guided nucleic acid interference. To provide antiviral immunity, processed CRISPR array transcripts (crRNA) are assembled with a surveillance complex containing Cas proteins, which recognize nucleic acids with sequence complementarity to viral-derived segments of crRNA (called spacers).

[0007] CRISPR-Cas tools have been widely used in gene editing, gene activation, gene inactivation, protein imaging and other fields. For example, the RNA-guided nuclease of the CRISPR-Cas9 system can be used as a gene editing tool for certain organisms, including the most widely used Streptococcus pyogenes Cas9 (SpCas9). Although many current Cas9 polypeptides are capable of efficient gene modification, there are still limitations due to off-target activity, such as the generation of undesirable modifications at sites within the genome other than the desired target. In addition, the use of current nucleases may be limited due to the specificity of the protospacer adjacent motif (PAM) and packaging limitations of system component delivery.

[0008] Therefore, new genetic engineering techniques are needed, for example for use in therapy and / or diagnostics. Summary of the Invention

[0009] Thus, in various aspects, the present disclosure provides a composition comprising an endonuclease comprising a sequence, optionally comprising a higher eukaryotic and prokaryotic nucleotide binding domain (HEPN) domain, or a fragment or variant thereof, identical to SEQ ID NOs: 1-4 and / or SEQ ID NOs: any of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In an embodiment, the sequence comprises at least two HEPN domains or fragments or variants thereof.

[0010] Additionally, in various aspects, the present disclosure provides a composition comprising an endonuclease comprising a sequence optionally comprising one or more higher eukaryotic and prokaryotic nucleotide binding domain (HEPN) domains or fragments or variants thereof, and corresponding to SEQ ID NOs: 1-4 and / or SEQ ID NOs: any of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In an embodiment, the sequence comprises a fragment or variant of a HEPN domain. In an embodiment, the sequence comprises at least two HEPN domains or fragments or variants thereof.

[0011] In various aspects, the present disclosure provides a composition comprising a nucleic acid encoding an endonuclease comprising a sequence optionally comprising one or more HEPN domains or fragments or variants thereof and corresponding to SEQ ID NOs: 1-4 and / or SEQ ID NOs: one or more of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In an embodiment, the sequence comprises at least one HEPN domain, or a fragment or variant thereof. In an embodiment, the sequence comprises at least two HEPN domains, or fragments or variants thereof.

[0012] In various aspects, the present disclosure provides a composition comprising a nuclease system comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) similarity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., or about 17, or about 18, or about 19, or about 20 modifications); and (b) an RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule. In embodiments, the sequence comprises at least one HEPN domain or a fragment or variant thereof. In embodiments, the sequence comprises at least two HEPN domains or fragments or variants thereof.

[0013] In embodiments, the composition further comprises one or more donor polynucleotides and / or is suitable for introducing one or more donor polynucleotides into the target nucleic acid molecule.

[0014] In embodiments, the endonuclease is suitable for introducing one or more excisions into a target nucleic acid molecule.

[0015] In various aspects, the present disclosure provides a composition comprising a chimeric protein comprising: an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5 , or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications); and a nucleic acid regulatory domain or nucleic acid modifying domain, or a nucleic acid interaction / binding domain, the domain comprising a sequence comprising a catalytic domain or a fragment or variant thereof, wherein (a) and (b) do not naturally appear together in the same reading frame. In embodiments, the sequence comprises at least one HEPN domain or a fragment or variant thereof. In embodiments, the sequence comprises at least two HEPN domains or fragments or variants thereof. In embodiments, the nucleic acid regulatory domain or nucleic acid modifying domain is a nucleic acid interaction domain, for example selected from MCP, lambdaN, PP7, QBeta, SLBP, and TBP / TAR. In embodiments, the endonuclease reduces or enhances incidental activity for nucleic acid detection.

[0016] In various aspects, the present disclosure provides a composition comprising a complex comprising a chimeric protein and an RNA molecule, wherein the chimeric protein comprises an endonuclease comprising a sequence, the sequence optionally comprising one or more HEPN domains or fragments or variants thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7). or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications); and a nucleic acid regulatory domain or a nucleic acid modification domain that comprises a sequence that comprises a catalytic domain or a fragment or variant thereof, wherein (a) and (b) do not naturally appear together in the same reading frame and the RNA molecule comprises a sequence that is complementary to one strand of the target nucleic acid molecule.

[0017] In embodiments, the sequence comprises at least one HEPN domain or a fragment or variant thereof. In embodiments, the sequence comprises at least two HEPN domains or fragments or variants thereof. In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has one or more activities: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, debranching activity, transesterification activity, photolyase activity, and glycosylase activity. In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is a methyltransferase-like protein 3 (METTL3) methyltransferase domain, a METTL3:methyltransferase-like protein 1 (METTL1) fusion, or a fragment or variant thereof.

[0018] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is selected from deaminases, reverse transcriptases, transposases, integrases and recombinases. In embodiments, the deaminases are cytidine or cytosine deaminases or fragments or variants thereof. In embodiments, the cytidine or cytosine deaminases are selected from activation-induced cytidine deaminase (AID), cytidine deaminase 1 (CDA1) and apolipoprotein B mRNA editing complex (APOBEC) or fragments or variants thereof. In embodiments, the APOBEC is selected from A3A, AB3, APOBEC1, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H or fragments or variants thereof. In embodiments, the APOBEC has an amino acid sequence of one of SEQ ID NO:39 [A3A], SEQ ID NO:40 [AB3], SEQ ID NO:41 [APOBEC1], SEQ ID NO:42 [APOBEC3C], SEQ ID NO:43 [APOBEC3D], SEQ ID NO:44 [APOBEC3F], SEQ ID NO:45 [APOBEC3G], and SEQ ID NO:46 [APOBEC3H], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

[0019] In an embodiment, the deaminase is a DNA-specific adenine or adenosine deaminase or a fragment or variant thereof. In an embodiment, the DNA-specific adenine or adenosine deaminase is selected from tRNA-specific adenosine deaminase 7.10 (TadA7.10), tRNA-specific adenosine deaminase 6.3 (TadA6.3), tRNA-specific adenosine deaminase 7.8 (TadA7.8), tRNA-specific adenosine deaminase 7.9 (TadA7.9) and tRNA-specific adenosine deaminase 8e (TadA8e (TadA-8eV106W)) or a fragment or variant thereof. In an embodiment, the TadA has the amino acid sequence of one of SEQ ID NO:48 [TadA7.10], SEQ ID NO:49 [TadA6.3], SEQ ID NO:50 [TadA7.8], SEQ ID NO:51 [TadA7.9] and SEQ ID NO:52 [TadA8e], or a fragment or variant thereof, or an amino acid sequence that is at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

[0020] In embodiments, the deaminase is an RNA-specific adenine or adenosine deaminase or a fragment or variant thereof. In embodiments, the RNA-specific adenine or adenosine deaminase is an adenosine deaminase acting on an RNA (ADAR) enzyme or a fragment or variant thereof. In embodiments, the ADAR is selected from ADAR1, ADAR2 and ADAR3 or a fragment or variant thereof. In embodiments, the ADAR has an amino acid sequence of one of SEQ ID NO:53 [ADAR1] and SEQ ID NO:54 [ADAR2] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In an embodiment, and as a non-limiting example, the catalytic deaminase domain of ADAR1 comprises amino acids 833-1226 of SEQ ID NO: 53. As another non-limiting example, the catalytic deaminase domain of ADAR2 comprises amino acids 299-701 of SEQ ID NO:54.

[0021] In embodiments, the deaminase further comprises a nuclear localization signal. In embodiments, the endonuclease further comprises a uracil glycosylase inhibitor (UGI) or a fragment or variant thereof. In embodiments, the RNA molecule is a guide RNA (gRNA). In embodiments, the gRNA comprises a sequence that interacts with the endonuclease. In embodiments, the endonuclease forms a complex with the gRNA.

[0022] In embodiments, the composition is suitable for base editing. In embodiments, the composition is suitable for DNA base editing. In embodiments, the composition is suitable for RNA base editing. In embodiments, the composition is suitable for catalyzing a C>T nucleotide conversion or an A>G nucleotide conversion in a target nucleic acid.

[0023] In an embodiment, the composition comprises both adenosine deaminase and cytidine deaminase.

[0024] In an embodiment, the composition is suitable for double base editing.

[0025] In an embodiment, the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or M-MLV RT (D200N / L603W / T330P / T306K / W313F) or a fragment or variant thereof. In an embodiment, the M-MLV RT has the amino acid sequence of SEQ ID NO: 55 [M-MLV RT] or SEQ ID NO 56 [M-MLV RT (D200N / L603W / T330P / T306K / W313F)] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0026] In embodiments, the composition further comprises a dominant negative human MutL homolog (MLH1).In embodiments, the composition is suitable for use with dominant negative MLH1.

[0027] In embodiments, the RNA molecule is or comprises a lead editing guide RNA (pegRNA). In embodiments, the endonuclease forms a complex with the pegRNA. In embodiments, the pegRNA is used as a template for transcribing a new DNA sequence. In embodiments, the pegRNA is bound to a DNA chain opposite to a typical gRNA binding site. In embodiments, the pegRNA comprises a gRNA containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. In embodiments, the RNA molecule is or comprises a gRNA. In embodiments, the gRNA comprises a sequence that interacts with the endonuclease. In embodiments, the endonuclease forms a complex with the gRNA. In embodiments, the composition comprises both gRNA and pegRNA.

[0028] In embodiments, the composition is suitable for use in lead editing.

[0029] In embodiments, the transposase is selected from the group consisting of Tn1, Tn2, Tn3, Tn5, Tn7, Tn9, Tn10, Tn552, Tn903, Tn1000 / γ-δ, Tn / O, tnsA, tnsB, tnsC, tniQ, IS10, ISS, IS911, Minos, Sleeping Beauty, piggyBac, Tol2, Mos1, Himar1, Hermes, Tol2, Minos, Tel, P-element, MuA, Ty1, Chapaev, transib, Tc1 / mariner, and Tc3 donor DNA systems.

[0030] In embodiments, the transposase is a transposon 7-like (Tn7-like) transposon system or a fragment or variant thereof. In embodiments, the transposase is one or more of transposon 7 protein A (TnsA), transposon 7 protein B (Tns B), transposon 7 protein C (Tns C), and integron transposase protein Q (TniQ), or a fragment or variant thereof. In embodiments, the Tn7-like transposon system is derived from Vibrio cholerae Tn6677.

[0031] In embodiments, the transposase has the amino acid sequence of one or more of SEQ ID NO:57 [TnsA], SEQ ID NO:58 [TnsB], SEQ ID NO:59 [TnsC], and SEQ ID NO:60 [TniQ], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

[0032] In an embodiment, the integrase is a serine recombinase or a fragment or variant thereof.

[0033] In an embodiment, the serine recombinase is Bxb1 or a fragment or variant thereof. In an embodiment, the recombinase is a Gin convertase or a Tn3 resolvase or a fragment or variant thereof.

[0034] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain comprises one or more modifications (such as, but not limited to, mutations) to reduce activity relative to the unmutated form.

[0035] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain comprises one or more modifications (such as, but not limited to, mutations) to increase activity relative to the unmutated form.

[0036] In an embodiment, the sequence of (a) is positioned at the N-terminus of said chimeric protein, and the sequence of (b) is positioned at the C-terminus of said chimeric protein.

[0037] In an embodiment, the sequence of (a) is positioned at the C-terminus of said chimeric protein and the sequence of (b) is positioned at the N-terminus of said chimeric protein.

[0038] In an embodiment, the composition further comprises a linker connecting the sequence of (a) and the sequence of (b). In an embodiment, the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids. In an embodiment, the linker substantially comprises glycine and serine residues. In an embodiment, the linker is (GGS) n, wherein n is 1, or 2, or 3, or 4, or 5. In an embodiment, the linker is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS) n (n=1-4) (SEQ ID NO:65), (Gly)8 (SEQ ID NO:66), (Gly)6 (SEQ ID NO:67), (EAAAK) n (n=1-3)(SEQ ID NO:68), A(EAAAK) n A(n=2-5) (SEQ ID NO:69), AEAAAKEAAAKA (SEQ ID NO:70), A(EAAAK)4ALEA(EAAAK)4A (SEQID NO:71), PAPAP (SEQ ID NO:72), KESGSVSSEQLAQFRSLD (SEQID NO:73), EGKSSGSGSESKST (SEQ ID NO:74) and GSAGSAAGSGEF (SEQ ID NO:75) or variants thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations selected from substitutions or deletions.

[0039] In embodiments, the endonuclease is suitable for producing double-strand breaks in nucleic acids. In embodiments, the endonuclease is suitable for producing nicks in nucleic acids. In embodiments, the endonuclease is suitable for carrying out nucleic acid modification by homology-directed repair (HDR). In embodiments, the endonuclease is suitable for carrying out nucleic acid modification by non-homologous end joining (NHEJ). In embodiments, the endonuclease recognizes PAM. In embodiments, the endonuclease recognizes multiple PAMs. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to mutations) to reduce catalytic activity relative to unmutated forms. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to mutations) to render the endonuclease substantially catalytically inactive relative to unmutated forms. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to, modifications (such as but not limited to, mutations) to increase catalytic activity relative to the unmutated form. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to, mutations) to render the endonuclease substantially catalytically overactive relative to the unmutated form. In embodiments, the endonuclease has nickase activity. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to, mutations) to produce nickase activity. In embodiments, the endonuclease has incidental cleavage activity. In embodiments, the endonuclease comprises one or more modifications (such as but not limited to, mutations) to produce incidental cleavage activity. In embodiments, the endonuclease has at least about 75% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease has at least about 80% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease has at least about 90% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. Nos. 1-4 and / or one or more of SEQ ID NOs: 80-89 are at least about 85% identical.

[0040] In embodiments, the endonuclease is at least about 90% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 95% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 97% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 99% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89.

[0041] In embodiments, the endonuclease has about 1 to about 15 amino acid modifications. In embodiments, the endonuclease has about 1 to about 10 amino acid modifications. In embodiments, the endonuclease has about 1 to about 5 amino acid modifications. In embodiments, the endonuclease has about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications. In embodiments, the amino acid modifications are selected from substitution and deletion.

[0042] In embodiments, the endonuclease (or chimeric protein) comprises domains from different endonucleases. In embodiments, the different endonucleases are Cas endonucleases. In embodiments, the domain is a PAM interaction domain. In embodiments, the target nucleic acid is or comprises single-stranded RNA (ssRNA). In embodiments, the target nucleic acid is or comprises double-stranded RNA (dsRNA). In embodiments, the target nucleic acid is or comprises single-stranded DNA (ssDNA). In embodiments, the target nucleic acid is or comprises double-stranded DNA (dsDNA). In embodiments, the target nucleic acid is about 2 to about 6 nucleotides upstream of the PAM sequence. In embodiments, the RNA molecule is or comprises a guide ribonucleic acid structure, which is configured to form a complex with the endonuclease.

[0043] In embodiments, the guide RNA structure (i) comprises (a) a CRISPR RNA (crRNA) suitable for hybridizing to a target nucleic acid molecule and / or (b) a trans-activating CRISPR RNA (tracrRNA) suitable for interacting with the endonuclease, or (ii) lacks (a) a crRNA suitable for hybridizing to a target nucleic acid molecule and / or (b) a tracrRNA suitable for interacting with the endonuclease.

[0044] In embodiments, the RNA molecule is or comprises a gRNA. In embodiments, the gRNA comprises a sequence that interacts with the endonuclease. In embodiments, the endonuclease forms a complex with the gRNA.

[0045] In an embodiment, the RNA molecule is or comprises a nucleic acid sequence of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or a fragment or variant thereof, or a nucleic acid sequence that is at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

[0046] In an embodiment, the RNA molecule has complete sequence complementarity with one strand of the target nucleic acid molecule. In an embodiment, the RNA molecule has partial sequence complementarity with one strand of the target nucleic acid molecule.

[0047] In embodiments, the composition further comprises a viral vector. In embodiments, the viral vector is or comprises AAV. In embodiments, the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8 and AAV3 / 9. In embodiments, the composition further comprises a non-viral vector. In embodiments, the composition further comprises lipid nanoparticles (LNP) liposomes, lipid complexes or polymer nanoparticles. In embodiments, the LNP comprises one or more of ionizable lipids, amino lipids, anionic lipids, neutral lipids, amphipathic lipids, helper lipids, structural lipids, PEG lipids and lipids. In embodiments, the composition further comprises virus-like particles (VLPs).

[0048] In various aspects, the present disclosure provides a nucleic acid encoding an endonuclease or chimeric protein of any embodiment and / or aspect disclosed herein. In embodiments, the nucleic acid is or comprises a DNA molecule or an RNA molecule. In embodiments, the RNA is or comprises mRNA or modified mRNA (mmRNA). In embodiments, the DNA is or comprises a vector or plasmid. In embodiments, the nucleic acid comprises a codon-optimized sequence. In embodiments, the nucleic acid comprises one or more modifications. In embodiments, the modification is one or more of a base modification and a backbone modification.

[0049] In various aspects, the present disclosure provides a viral vector comprising a nucleic acid of any embodiment and / or aspect disclosed herein. In embodiments, the viral vector is or comprises AAV. In embodiments, the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

[0050] In various aspects, the present disclosure provides a viral vector comprising a nucleic acid according to any one of the embodiments and / or aspects disclosed herein. In an embodiment, the viral vector is or comprises a VLP.

[0051] In embodiments, the endonuclease mediates a trans-splicing event.

[0052] In embodiments, the endonuclease mediates an exon skipping or exon inclusion event.

[0053] In various aspects, the present disclosure provides a lipid nanoparticle comprising a nucleic acid according to any embodiment and / or aspect disclosed herein.

[0054] In various aspects, the present disclosure provides a cell comprising a nucleic acid according to any embodiment and / or aspect disclosed herein, a viral vector according to any embodiment and / or aspect disclosed herein, or a lipid nanoparticle according to any embodiment and / or aspect disclosed herein.

[0055] In embodiments, the cell is a prokaryotic cell. In embodiments, the cell is a eukaryotic cell. In embodiments, the cell is a mammalian cell. In embodiments, the cell is a human cell. In embodiments, the cell is an immortalized cell. In embodiments, the cell is harvested from a subject.

[0056] In various aspects, the present disclosure provides a pharmaceutical composition comprising a composition according to any embodiment and / or aspect disclosed herein, a nucleic acid according to any embodiment and / or aspect disclosed herein, a viral vector according to any embodiment and / or aspect disclosed herein, a lipid nanoparticle according to any embodiment and / or aspect disclosed herein, or a cell according to any embodiment and / or aspect disclosed herein, and a pharmaceutically acceptable carrier.

[0057] In various aspects, the present disclosure provides a composition comprising an RNA molecule comprising a nucleic acid sequence of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or a fragment or variant thereof, or a nucleic acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0058] In an embodiment, the RNA molecule interacts with an endonuclease comprising a sequence optionally comprising a HEPN domain or a fragment or variant thereof and interacting with SEQ ID NOs: 1-4 and / or SEQ ID NOs: one or more of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%), or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In an embodiment, the sequence comprises at least one HEPN domain, or a fragment or variant thereof. In an embodiment, the sequence comprises at least two HEPN domains, or fragments or variants thereof.

[0059] In embodiments, the RNA molecule comprises one or more modifications. In embodiments, the modifications are one or more of base modifications and backbone modifications. In embodiments, the RNA molecule comprises a sequence complementary to one strand of the target nucleic acid molecule. In embodiments, the RNA molecule has complete sequence complementarity with one strand of the target nucleic acid molecule.

[0060] In embodiments, the RNA molecule has partial sequence complementarity with one strand of the target nucleic acid molecule.

[0061] In various aspects, the present disclosure provides a composition comprising a nucleic acid encoding a nuclease comprising a sequence, optionally comprising a HEPN domain or a fragment or variant thereof, and an RNA comprising a repeat sequence having at least about 70% identity to one or more of SEQ ID NOs: 28-31 and / or SEQ ID NOs: 90-97. In embodiments, the composition has at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to SEQ ID NOs: 28-31 and / or SEQ ID NOs: 90-97, or has about 1 to about 20 nucleotide modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In an embodiment, the sequence comprises at least one HEPN domain, or a fragment or variant thereof. In an embodiment, the sequence comprises at least two HEPN domains, or fragments or variants thereof.

[0062] In various aspects, the present disclosure provides a kit comprising a container, wherein the kit comprises a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein, and instructions for regulating and / or modifying the nucleic acid.

[0063] In various aspects, the present disclosure provides a method of regulating and / or modifying a nucleic acid in a cell, the method comprising contacting the cell with a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein.

[0064] In various aspects, the present disclosure provides a method of regulating and / or modifying nucleic acids in a subject in need thereof, the method comprising administering to the subject an effective amount of cells and a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein.

[0065] In embodiments, the regulation and / or modification is selected from one or more of cleavage, cleavage, methylation, labeling of nucleic acids, and mutation of nucleic acids. In embodiments, the regulation and / or modification is selected from one or more of: cleavage of nucleic acids; insertion of nucleic acids; editing of nucleic acids; regulation of transcription of nucleic acids; isolation of nucleic acids; binding of nucleic acids; and imaging of nucleic acids.

[0066] In various aspects, the present disclosure provides a method for disrupting, correcting and / or replacing a gene in a cell, the method comprising contacting the cell with a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein.

[0067] In various aspects, the present disclosure provides a method for disrupting, correcting and / or replacing a gene in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein.

[0068] In various aspects, the present disclosure provides a method for treating, ameliorating or preventing a disease or condition in a subject, the method comprising (a) contacting a cell with a composition of any embodiment and / or aspect disclosed herein, a nucleic acid of any embodiment and / or aspect disclosed herein, a viral vector of any embodiment and / or aspect disclosed herein, a lipid nanoparticle of any embodiment and / or aspect disclosed herein, a cell of any embodiment and / or aspect disclosed herein, or a pharmaceutical composition of any embodiment and / or aspect disclosed herein, and (b) administering an effective amount of the cell to the subject.

[0069] In various aspects, the present disclosure provides a method for treating, ameliorating or preventing a disease or condition in a subject, the method comprising administering to the subject an effective amount of a composition according to any embodiment and / or aspect disclosed herein, a nucleic acid according to any embodiment and / or aspect disclosed herein, a viral vector according to any embodiment and / or aspect disclosed herein, a lipid nanoparticle according to any embodiment and / or aspect disclosed herein, a cell according to any embodiment and / or aspect disclosed herein, or a pharmaceutical composition according to any embodiment and / or aspect disclosed herein.

[0070] In an embodiment, the composition of any embodiment and / or aspect disclosed herein, the nucleic acid of any embodiment and / or aspect disclosed herein, the viral vector of any embodiment and / or aspect disclosed herein, the lipid nanoparticle of any embodiment and / or aspect disclosed herein, the cell of any embodiment and / or aspect disclosed herein, or the pharmaceutical composition of any embodiment and / or aspect disclosed herein is used to treat, improve or prevent a disease or condition in a patient.

[0071] In various aspects, the present disclosure provides use of a composition according to any embodiment and / or aspect disclosed herein, a nucleic acid according to any embodiment and / or aspect disclosed herein, a viral vector according to any embodiment and / or aspect disclosed herein, a lipid nanoparticle according to any embodiment and / or aspect disclosed herein, a cell according to any embodiment and / or aspect disclosed herein, or a pharmaceutical composition according to any embodiment and / or aspect disclosed herein in the manufacture of a medicament for treating, ameliorating or preventing a disease or condition.

[0072] In various aspects, the present disclosure provides a method of detecting and / or quantifying nucleic acids in a sample, the method comprising contacting the sample with a composition according to any embodiment and / or aspect disclosed herein.

[0073] In embodiments, the nucleic acid is a target and / or reporter nucleic acid. In embodiments, the method comprises detecting a reporter signal, wherein the reporter signal is generated upon endonuclease cleavage. In embodiments, the reporter signal is a fluorescent signal. In embodiments, the endonuclease has incidental cleavage activity.

[0074] In various embodiments, the compositions disclosed herein or the trans-splicing systems disclosed herein further comprise a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; (b) a splicing donor and / or a splicing acceptor, wherein the repRNA is suitable for trans-splicing. In embodiments, the trans-splicing system comprises a splicing donor, a splicing acceptor, and replaces an internal exon. In embodiments, the repRNA is operably linked to an RNA molecule or the gRNA comprising a sequence complementary to one strand of the target nucleic acid molecule.

[0075] In various aspects, the present disclosure provides a system for targeting a nucleic acid for trans-splicing, the system comprising: (a) an endonuclease of any embodiment disclosed herein, and optionally an RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule; (b) an RNA-binding polypeptide associated with the endonuclease; and (c) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; and (ii) a splice donor and / or a splice acceptor.

[0076] In an embodiment, the RNA molecule is a gRNA.

[0077] In embodiments, the endonuclease is not linked to, associated with, and / or fused to an RNA binding protein.

[0078] In embodiments, the repRNA is not operably linked to one or more gRNAs. In embodiments, the repRNA is provided to one or more gRNAs in trans.

[0079] In embodiments, the repRNA further comprises a ribozyme site. In embodiments, the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS) or glmS ribozyme site, or a variant thereof. In embodiments, the ribozyme site is an HDV ribozyme site. In embodiments, the ribozyme site is upstream of one or more exons and / or introns of the repRNA.

[0080] In various aspects, the present disclosure provides a system for targeting nucleic acids for trans-splicing, the system comprising: (a) a nuclease as described in any one of claims 1-108 and an RNA molecule, the RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule; and (b) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; (ii) a splice donor and / or a splice acceptor.

[0081] In embodiments, the RNA molecule is a gRNA. In embodiments, the endonuclease is not connected to, associated with, and / or fused to an RNA binding protein. In embodiments, the repRNA is operably linked to one or more gRNAs.

[0082] In embodiments, the composition comprises a gRNA, a repRNA, and a Cas endonuclease operably linked to a single promoter or a bidirectional promoter.

[0083] In an embodiment, the gRNA and repRNA are located on a first side of a bidirectional promoter, and the Cas endonuclease is located on a second side of the bidirectional promoter.

[0084] In embodiments, disclosed herein is a composition comprising an endonuclease and having an amino acid sequence that is at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 3. In embodiments, substitutions are made to SEQ ID NO: 3:

[0085] Met Asp Lys His Pro Ser Asn Arg Tyr Ala Leu Pro Lys Val Ile SerGlu Val Asp His Glu Arg Ile Leu Glu Phe Lys Val Lys Tyr Glu Lys Leu Ala ArgLeu Asp Arg Phe Glu Val Lys Ala Met His Tyr Asp Gly Ala Glu Ile Glu Val Gly Val Glu Val Asp Tyr Gln Asp Asn Asn Lys ThrIle Thr Ile Asn Leu Asn Gly Lys Tyr Thr Ile Asn Gly Arg Lys Val Gly GlyLys Arg Arg Leu Leu Glu Asp Arg Ile Ser Arg Gly Lys Val Cys Leu Glu Leu HisAsp Ser G Serg Lys Arg Lys Ar G Lys Pro Asp Arg Glu LeuIle Thr Phe Asp Ser Thr Lys Leu Tyr Ser Gln Ile Gly Arg Asp Val SerThr Lys Glu Ile Tyr Leu Ile Lys Arg Phe Leu Ala Tyr Arg Ser Asp Leu Leu PheTyr Tyr Gly Phe Ile Asp Ala Asn Phe Arg Gly As Val Phe Ser Gly Asp Lys Asn Gln Glu Leu Ile Lys Tyr Phe Asn Phe ThrIle Asn Asp Lys Leu Lys Asn Asp Lys Gly Tyr Leu Lys Glu Tyr Thr Ala Asn AspGlu Gln Ile Lys Lys Asp Leu Gln Asn Thr Me Thr Lys P He Ala P Glu Tyr AspPhe Phe Glu Lys Leu Phe Asn Asn Glu Glu IleGlu Thr Leu Ser Lys Ile His Asp Ile Glu Leu Leu Asn Thr Met Ile Asn Lys LeuAsp Lys Leu Asn Ile Asp Thr Arg Lys Glu Tyr Ile Asp Asp Glu Lys Ile Thr ValPhe Gly Glu Glu Ile Ser Leu Lys Thr Leu Tyr Gly Leu Tyr Ala His Thr Ala IleAsn Arg Val Ala Phe Asn Lys Leu Ile Asn Arg Phe Met Val Glu Asn Gly Thr GluAsn Glu Ala Leu Lys Lys Tyr Phe Asn Ser Lys Ala Glu Gly Gly Ile Ala Tyr GluIle Asp Ile His Gln Asn Ser Glu Tyr Lys Gln Leu Tyr Ile Gln His Lys Asp LeuVal Ser Lys Leu Ser Ala Leu Ser Asp Gly Asp Glu Ile Ala Asp Thr Asn Lys LysIle Ser Glu Leu Lys Val Lys Met Lys Ala Ile Thr Lys Ala Asn Ser Leu Lys ArgLeu Glu His Lys Leu Arg Leu Thr Phe Gly Phe Ile Tyr Thr Glu Tyr Gln Asp TyrAsn Ala Phe Lys Asn Asn Phe Asp Thr Asp Ile Lys Ser Gly Arg Phe Ile Pro LysAsp Ser Glu Gly Lys Arg Arg Gly Phe Asp His Arg Glu Leu Asp Gln Leu Lys ArgTyr Tyr Asp Ala Thr Phe Ala Asp Lys Lys Pro Gln Thr Lys Glu Thr Phe Asp GluIle Asp Lys Gln Ile Asp Gln Leu Ser Leu Lys Asn Leu IleGly Asp Asp Thr LeuLeu Lys Val Ile Leu Leu Ile Tyr Ile Phe Leu Pro Arg Glu Ile Lys Gly Glu PheLeu Gly Phe Val Lys Tyr Tyr His Asp Thr Lys His Ile Glu Glu Glu Asp Thr LysAsp Lys Asp Glu Val Asp Le Gly Thly Lys Phe Asp LeuAsp Lys Asn Ile Arg Ala Leu Ser Val Leu Lys Ser Leu Ser Tyr Gln Ala LysTyr Asn Lys Lys Glu Glu Lys Lys Glu Gln Phe Tyr Glu Ala Gly Asn Arg His GlyArg Phe Tyr Ser Lys Lys Leu Gly Ile Val Asyr Hip Ala Gs Glu Asn Arg Tyr His Ala Ala Leu Phe Lys Leu Leu Asn Asp Phe GluIle Tyr Ser Leu Ala Gln His Ile Glu Gly Lys Glu Thr Leu Ala Gln Gln Ile GluLys Pro Gln Phe Ser Gln Tyr Glu Le His Tyr Asn Phe Arg Lys Ser Glys Thr Ala T Leu Asp Asn Asp Ala Phe Asp Thr Val IleAsn Met Arg Asp Ile Ala His Leu Ser His Glu Pro Leu Phe Glu Cys Pro LeuAsp Gly Lys Ser Tyr Lys Leu Lys Gln Gly Lys Arg Thr Asn Thr Ile Asn ValLys Pro Arle Valle Met Glu Ser Pro Ile AspMetLys Lys Thr Leu Gly Tyr Asp Ala Val Asn Asp Leu Thr Met Lys Ile Ile GlnLeu Arg Thr Arg Leu Lys Val Tyr Ala Asp Lys Ser Glu Thr Ile Lys Thr Leu ValAsp Ala Lys Thr Pro Asn Asp Phe Tyr His Ile Tyr Lys Val Lys Gly Val GluAla Ile Asn Arg His Leu Leu Glu Val Ile Gly Glu Thr Lys Asp Glu Lys Arg IleArg Lys Arg Ile Glu Ser Gly Asn Ala Ile Ala Gly Arg Thr Pro Ala Asp Ser GlnGlu Asn

[0086] As described herein, substitutions can be made to this sequence to generate the endonucleases of the invention (including taking into account the degeneracy of the genetic code).

[0087] In some embodiments, the endonuclease has one or more substitutions at positions corresponding to D38X, A59X, G172X, T236X, T319X, H375X, H419X, T424X, E529X, T541X, G562X, K564X, D569X, A586X, N641X, D642X, S647X, D721X, R779X, K13X, K566X, G554X, A35X, E110X, G314X, K114X, D498X, I86X, V57X, H249X, R704X of SEQ ID NO: 3, wherein the substitution is defined by X and wherein X is any amino acid. In some embodiments, X is an essential or non-essential amino acid.

[0088] In some embodiments, X is a hydrophilic or hydrophobic amino acid.

[0089] In some embodiments, X is a hydrophilic amino acid.

[0090] In some embodiments, X is a polar and positively charged hydrophilic amino acid. In some embodiments, X is selected from arginine (R) or lysine (K).

[0091] In some embodiments, X is a polar and neutrally charged hydrophilic amino acid. In some embodiments, X is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C).

[0092] In some embodiments, X is a polar and negatively charged hydrophilic amino acid. In some embodiments, X is selected from aspartic acid (D) or glutamic acid (E).

[0093] In some embodiments, wherein X is an aromatic, polar, and positively charged hydrophilic amino acid. In some embodiments, wherein X is histidine (H).

[0094] In some embodiments, X is a hydrophobic amino acid.

[0095] In some embodiments, X is a hydrophobic, aliphatic amino acid. In some embodiments, X is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).

[0096] In some embodiments, X is a hydrophobic, aromatic amino acid. In some embodiments, wherein X is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

[0097] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises one or more of the following substitutions:

[0098] a hydrophilic residue other than aspartic acid (D) at position corresponding to 38;

[0099] a hydrophobic residue other than alanine (A) at position corresponding to 59;

[0100] a hydrophobic residue other than glycine (G) at the position corresponding to 172;

[0101] a hydrophilic residue other than threonine (T) at position corresponding to 236;

[0102] a hydrophilic residue other than threonine (T) at the position corresponding to 319;

[0103] a hydrophilic residue other than histidine (H) at the position corresponding to 375;

[0104] a hydrophilic residue other than histidine (H) at the position corresponding to 419;

[0105] a hydrophilic residue other than threonine (T) at the position corresponding to 424;

[0106] a hydrophilic residue other than glutamic acid (E) at the position corresponding to 529;

[0107] a hydrophilic residue other than threonine (T) at the position corresponding to 541;

[0108] a hydrophobic residue other than glycine (G) at the position corresponding to 562;

[0109] a hydrophilic residue other than lysine (K) at the position corresponding to 564;

[0110] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 569;

[0111] a hydrophobic residue other than alanine (A) at the position corresponding to 586;

[0112] a hydrophilic residue other than asparagine (N) at position corresponding to 641;

[0113] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 642;

[0114] a hydrophilic residue other than serine (S) at position corresponding to 647;

[0115] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 721;

[0116] a hydrophilic residue other than arginine (R) at the position corresponding to 779;

[0117] a hydrophilic residue other than lysine (K) at position corresponding to 13;

[0118] a hydrophilic residue other than lysine (K) at the position corresponding to 566;

[0119] a hydrophobic residue other than glycine (G) at the position corresponding to 554;

[0120] a hydrophobic residue other than alanine (A) at position corresponding to 35;

[0121] a hydrophilic residue other than glutamic acid (E) at the position corresponding to 110;

[0122] a hydrophobic residue other than glycine (G) at the position corresponding to 314;

[0123] a hydrophilic residue other than lysine (K) at the position corresponding to 114;

[0124] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 498;

[0125] a hydrophobic residue other than isoleucine (I) at the position corresponding to 86;

[0126] a hydrophobic residue other than valine (V) at position corresponding to 57;

[0127] a hydrophilic residue other than histidine (H) at the position corresponding to 249; and

[0128] A hydrophilic residue other than arginine (R) at the position corresponding to 704.

[0129] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A59V. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G172L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T236L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T319I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H375L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H419Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T424F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises E529L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T541L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G562Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K564M. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D569L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A586I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises N641F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D642L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises S647L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D721L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises R779I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K13R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K566R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G554H. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises E110T. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G314Q. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K114P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D498P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises I86P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises V57E.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H249W. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises R704F.

[0130] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F and A59V. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, and G172L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, and T236L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, and T319I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, and H375L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, and H419Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, and T424F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, and E529L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, and T541L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, and G562X. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, and K564M. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, and D569L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, and A586I.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, and N641F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, and D642L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, and S647L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, and D721L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, and R779I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, and K13R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, and K566R.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, and G554H. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, and E110T. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, and G314Q.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, and K114P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, and D498P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, and I86P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, and V57E. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, and H249W.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, H249W, and R704F.

[0131] In some embodiments, disclosed herein are sequences that are at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identical to SEQ ID NO: 3 or to SEQ ID NO: 3. NO:3 has about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity) to one or more (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30) substitutions. In various embodiments, SEQ ID One or more amino acids in NO:3 are substituted by naturally occurring amino acids, such as hydrophilic amino acids (e.g., polar and positively charged hydrophilic amino acids, such as arginine (R) or lysine (K); polar and neutrally charged hydrophilic amino acids, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C); polar and negatively charged hydrophilic amino acids, such as aspartic acid (D) or glutamic acid (E); or aromatic, polar and positively charged hydrophilic amino acids, such as histidine (H)); or hydrophobic amino acids (e.g., hydrophobic aliphatic amino acids, such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V); hydrophobic aromatic amino acids, such as phenylalanine (F), tryptophan (C), threonine (T), proline (P) and cysteine ​​(C); (W) or tyrosine (Y); or non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids (such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general)).

[0132] In illustrative embodiments, substitutions of the invention include, but are not limited to, one or more (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or more) of SEQ ID NO: 3 or a sequence having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity. or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30) substitutions: D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, H249W, and R704F.

[0133] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following figures, the detailed description of several embodiments, and the appended claims. The details of the present disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the present invention will be apparent from the description and claims. In the description and the appended claims, unless the context clearly indicates otherwise, the singular also includes the plural. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1DFigure 1 is an image showing the protein sizes of the Cas13K2F system for SEQ ID NO: 1 (Cas13K2F1), SEQ ID NO: 2 (Cas13K2F2), SEQ ID NO: 3 (Cas13K2F3), SEQ ID NO: 4 (Cas13K2F5), SEQ ID NO: 80 (Cas13K2F7), SEQ ID NO: 81 (Cas13K2F8), SEQ ID NO: 82 (Cas13K2F9), SEQ ID NO: 83 (Cas13K2F10), SEQ ID NO: 84 (Cas13K2F11), SEQ ID NO: 85 (Cas13K2F12), SEQ ID NO: 86 (Cas13K2F13), and SEQ ID NO: 87 (Cas13K2F14). The red or black arrows in Figure 1 indicate the placement of the higher eukaryotic and prokaryotic nucleotide binding (HEPN) domains in each protein.

[0135] Figure 2 is a non-limiting image showing a representative guide RNA structure from the Cas13K2F system.

[0136] Figure 3 is an image showing the design of guide RNAs (gRNAs) targeting multiple sites in the eGFP coding sequence in HEK293T cells.

[0137] Figure 4 is an image showing the gRNA structure used for the Cas13K2F system (SEQ ID NO: 5).

[0138] Figure 5is a percent identity matrix for the following sequences: SEQ ID NO: 6 (Cas13X.1), SEQ ID NO: 7 (Cas13bt3), SEQ ID NO: 8 (Cas13bt2), SEQ ID NO: 9 (Cas13bt1), SEQ ID NO: 10 (Cas13bt8), SEQ ID NO: 11 (Cas13X.2), SEQ ID NO: 12 (Cas13bt9), SEQ ID NO: 13 (Cas13bt11), SEQ ID NO: 14 (Cas13bt5), SEQ ID NO: 15 (Cas13bt10), SEQ ID NO: 16 (Cas13bt15), SEQ ID NO: 17 (Cas13bt7), SEQ ID NO: 18 (Cas13bt6), SEQ ID NO: 19 (Cas13bt14), SEQ ID NO: 20 (Cas13Y.3), SEQ ID NO: 21 (Cas13bt12), SEQ ID NO: NO:22 (Cas13Y.1), SEQ ID NO:23 (Cas13bt4), SEQ ID NO:24 (Cas13bt16), SEQ ID NO:25 (Cas13Y.5) and SEQ ID NO:26 (Cas13Y.4).

[0139] Figure 6is an image showing a maximum likelihood phylogenetic tree of the following sequences: SEQ ID NO: 6 (Cas13X.1), SEQ ID NO: 7 (Cas13bt3), SEQ ID NO: 8 (Cas13bt2), SEQ ID NO: 9 (Cas13bt1), SEQ ID NO: 10 (Cas13bt8), SEQ ID NO: 11 (Cas13X.2), SEQ ID NO: 12 (Cas13bt9), SEQ ID NO: 13 (Cas13bt11), SEQ ID NO: 14 (Cas13bt5), SEQ ID NO: 15 (Cas13bt10), SEQ ID NO: 16 (Cas13bt15), SEQ ID NO: 17 (Cas13bt7), SEQ ID NO: 18 (Cas13bt6), SEQ ID NO: 19 (Cas13bt14), SEQ ID NO: 20 (Cas13Y.3), SEQ ID NO: 21 (Cas13bt12), SEQ ID NO:22 (Cas13Y.1), SEQ ID NO:23 (Cas13bt4), SEQ ID NO:24 (Cas13bt16), SEQ ID NO:25 (Cas13Y.5) and SEQ ID NO:26 (Cas13Y.4).

[0140] Figures 7A-7C Each shows SEQ ID NO: 2 ( Figure 7A )、SEQ ID NO:3( Figure 7B ) and SEQ ID NO: 27 ( Figure 7C ) results of RNA fragmentation experiments.

[0141] Figure 8A is an image showing a maximum likelihood phylogenetic tree of Cas13, where Cas13 is highly divergent and is a clade that includes the Cas13K2F system, a marginal CRISPR-Cas13 family with <7% identity to previously characterized RNA-targeting systems. Figure 8BGraph showing the percentage of GFP-positive cells after transfection with: an SD reporter gene encoding GFP 5', an SA reporter gene with an MS2 stem-loop and encoding GFP 3', and / or (i) a catalytically inactive Cas13K2F fused to the MS2 coat protein ("dCas13K2F-MS2") and a gRNA targeting the SD reporter gene (Cas13K2FgRNA1 or 2), or (ii) a catalytically inactive PspCas13 fused to the MS2 coat protein (dPspCas13b-MS2) and a gRNA targeting the SD reporter gene (PspCas13b gRNA). Control cells were transfected with non-targeting (NT) gRNA.

[0142] Figure 9 Graph showing the percentage of GFP-positive cells after transfection with: (i) a target containing sequences encoding the 5' end of GFP, a splice donor, a gene A intron, a splice acceptor, and a gene A exon, (ii) a template with two MS2 stem loops and a sequence encoding the 3' end of GFP, (iii) dCas13K2F fused to the MS2 coat protein (dCas13K2F-MS2) and a gRNA for the target (gRNA12, gRNA2, gRNA18, or gRNA19). Control cells were transfected with non-targeting (NT) gRNA or the target alone ("no RepRNA").

[0143] Figure 10A Figure 2 is an image showing the protein structure and domain organization of Cas13e (used interchangeably herein with “Cas13K2F”) and Cas13c representatives relative to EsiCas13d (PDB: 6E9F) based on ColabFold predictions. Figure 10B is an image illustrating the RNA knockdown strategy to test the trans-splicing activity of Cas13e in mammalian cells. Figure 10C Figure 2 is a graph showing relative Cas13eGFP fluorescence (=MFI targeted crRNA / MFI non-targeted crRNA) of HEK293T-GFP cells transfected with plasmids expressing Cas13e or RfxCas13d and GFP targeting crRNA, measured by flow cytometry to show trans-splicing. Percentage of GFP detected in mammalian cells relative to a non-targeted negative control. Figure 10C, plasmids expressing Cas13e1 (used interchangeably herein with “Cas13K2F1”), Cas13e2 (used interchangeably herein with “Cas13K2F2”), Cas13e3 (used interchangeably herein with “Cas13K2F3”), Cas13e4 (used interchangeably herein with “Cas13K2F4”), and Cas13e5 (used interchangeably herein with “Cas13K2F5”) are shown on the x-axis. Figure 10D Figure 2 is a graph showing validation of dCas13e activity for trans-splicing in mammalian cells.

[0144] Figure 11A is an image showing v1 SE3 AAV and the 3' end RNA replacement strategy. Figure 11B Figure 1 is an image of the experimental workflow used to evaluate the performance of SE3 as an AAV plasmid in alternative cell types. Figure 11C are images showing the editing performance of SE3 using targeted (T) and non-targeted (NT) guides in the HepG2 cell line. Figure 11D is a graph showing the number of pathogenic mutations plotted in maroon (dark) at each position in the USH2A gene, with exons shown in blue (grey) and introns shown in light cream (white). There are over 700 pathogenic variants across the entire length. Figure 11E is an image showing a non-limiting strategy for correcting the 5' end of a target RNA. Figure 11F is a graphic showing 5' editing applied to a USH2A reporter gene, where a gRNA targeting intron 12 is compared to a non-targeting guide whose activity is driven by the presence of repair RNA.

[0145] Figure 12 is an image showing the protein structure and domain organization of different Cas13 family members based on ColabFold predictions. Cas13e is used interchangeably with "Cas13K2F" in this article.

[0146] Figure 13A and Figure 13B Figure 2 shows the effects of Cas13K2F on two different RNA targets (MMP9 ( Figure 13A ) and USH2A( Figure 13B ))Graphic representation of trans-splicing.

[0147] Figure 14Figure 2 is a graph showing the percentage of GFP-positive cells after transfection with: an SD reporter gene encoding the 5' end of GFP and (i) an SA reporter gene encoding the 3' end of GFP with the sequence motif of the indicated RNA binding protein (RBP) ("SA reporter gene only" bar); or (ii) an SA reporter gene and a splicing editor ("SE"), which is dPspCas13b fused to the indicated RBP and a PspCas13b gRNA targeting the SD reporter gene ("SE+SA reporter gene" bar). Control cells were transfected with only the SD reporter gene or only the SD reporter gene and the SA reporter gene.

[0148] Figure 15A is an image and Figure 15B is a graph showing AAV delivery of dCas13K2F3 with targeting (T) or non-targeting (NT) gRNA and repRNA to promote trans-splicing in HEK293T cells.

[0149] Figure 16 is a graphic showing amino acid substitutions in dCas13K2F3 that improve or reduce trans-splicing efficacy in human cells compared to the native (WT) sequence.

[0150] Figure 17 are images showing, without wishing to be bound by theory, internal exon replacement using two nucleases and two separate gRNAs and RBPs, where the repRNA is shown in green (just to the left of the splice donor site in the image).

[0151] Figure 18 is an image showing, without wishing to be bound by theory, how internal exon replacement can be achieved with a single gRNA, nuclease, and RBP in combination with a binding motif (BM) in the repRNA, shown in orange (and labeled BM2 in the image).

[0152] Figure 19 is a diagram showing a combination of a gRNA, a nuclease and an RBP (PCP) with a binding motif (BM) (see Figure 18 Targeted (T) (first four bars on the left) or non-targeted (NT) (second four bars on the right) constructs in ) to promote internal exon replacement.

[0153] Figure 20 is an image showing, without wishing to be bound by theory, how internal exon replacement can be achieved with a combination of two gRNAs, two nucleases, and an RBP with or without binding to a binding motif (BM) in the repRNA, shown in orange (and labeled BM2 in the image).

[0154] Figure 21A and Figure 21B is a graph showing the combination of two gRNAs, two nucleases and RBP with or without binding to a binding motif (BM) in repRNA (see Figure 20 ) to promote internal exon replacement. Figure 21A , the first bar in each group is 3'SE(T) (far left), the next bar in each group is 3'SE(NT) (second from the left), the next bar in each group is 5'SE(T) (middle), the next bar in each group is 5'SE(NT) (second from the right), and the last bar in each group is 3' and 5'SE(T) (far right). Figure 21B , the first bar in each group is 3'SE(T) (far left), the next bar in each group is 5'SE(NT) (second from the left), the next bar in each group is 3'SE(T) (middle), the next bar in each group is 3'SE(NT) (second from the right), and the last bar in each group is 3' and 5'SE(T) (far right).

[0155] Figure 22A is an image and Figure 22B is a diagram illustrating, without wishing to be bound by theory, the design of a guide repair RNA ("grepRNA") lacking an RBP. Figure 22B The expression of grepRNA alone or grepRNA and dCas13 on two different RNA targets (USH2A, Figure 22B Left; and MMP9, Figure 22B Internal exon substitution (right). Figure 22A In the top image, from 3' to 5' are repRNA, gRNA and dCas13-RBP. Figure 22B In the bottom image, from 3' to 5' are grepRNA and dCas13.

[0156] Figure 23A is an image showing the integrated USH2A target for 5' substitution. Figure 23B is a diagram showing targeted (T) or non-targeted (NT) integration of the 5' USH2A target to promote internal exon replacement. DETAILED DESCRIPTION

[0157] The present disclosure provides, inter alia, compositions and methods relating to a novel family of CRISPR-Cas effector proteins, comprising nucleic acids encoding the CRISPR-Cas effector proteins and RNA components that induce DNA targeting; and methods of use thereof.

[0158] The present disclosure is based in part on the discovery of compositions and methods related to Type VI CRISPR-Cas effector proteins, optionally complexed with guide nucleic acids, capable of modifying target nucleic acids. The present disclosure also provides methods for modifying target nucleic acids using the disclosed endonucleases or chimeric proteins and, optionally, guide RNAs.

[0159] The Cas13 enzyme belongs to the type VI CRISPR-Cas enzyme and is identified as an RNA-guided RNA-targeting protein. While Cas9 cleaves DNA to interrupt DNA replication, Cas13 digests RNA to attenuate transcription. The CRISPR-Cas13 system can be divided into six subtypes (a, b1, b2, c, d, X, Y). Each subtype carries Cas13, a single effector protein. All Cas13 proteins exhibit two different RNase activities. One is RNA-targeted degradation and the other is precursor crRNA processing. To date, about six total Cas13 variants have been identified.

[0160] Without wishing to be bound by theory, the nucleases of the present invention (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, or fragments or variants thereof) belong to the Cas13K2F system.

[0161] Endonucleases (Proteins, Nucleic Acids, and Systems)

[0162] In various aspects, the present disclosure provides a composition comprising an endonuclease comprising a sequence or a fragment or variant thereof corresponding to SEQ ID NOs: 1-4 and / or SEQ ID any of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).

[0163] In various aspects, the present disclosure provides a composition comprising an endonuclease comprising a sequence optionally comprising a HEPN domain or a fragment or variant thereof and corresponding to SEQ ID NOs: 1-4 and / or SEQ ID any of NOs: 80-89 has at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identity, or has about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications).

[0164] In an embodiment, the sequence comprises at least one HEPN domain, or a fragment or variant thereof. In an embodiment, the sequence comprises at least two HEPN domains, or fragments or variants thereof.

[0165] In an embodiment, the sequence comprises one or more truncated HEPN domains.

[0166] In an embodiment, one or more HEPN domains are according to Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D The position of the arrows in and / or by referring to Table 1 or Table 2.

[0167] In an embodiment, a polypeptide (e.g., an endonuclease or chimeric protein) of the present disclosure is provided in the form of a nucleic acid encoding the endonuclease or chimeric protein (e.g., mRNA, DNA, plasmid, expression vector, viral vector, etc.). In an embodiment, an endonuclease or chimeric protein of the present disclosure is provided directly in protein form (e.g., without an associated guide RNA, or with an associated guide RNA, i.e., in the form of a ribonucleoprotein complex). An endonuclease or chimeric protein or nucleic acid of the present disclosure can be introduced into a cell (provided to a cell) by any convenient method; such methods are known to those of ordinary skill in the art.

[0168] In embodiments, the endonuclease is suitable for producing a double-strand break in a nucleic acid. In embodiments, the endonuclease is suitable for producing a nick in a nucleic acid. In embodiments, the endonuclease is suitable for nucleic acid modification by HDR. In embodiments, the endonuclease is suitable for nucleic acid modification by NHEJ.

[0169] In embodiments, the endonuclease recognizes a PAM. In embodiments, the endonuclease recognizes multiple PAMs (e.g., about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 PAMs). In embodiments, the PAM sequence is about 1 to about 20, or about 2 to about 12, or about 2 to about 6, or about 2, or about 3, or about 4, or about 5, or about 6, or about 8, or about 10 nucleotides long.

[0170] In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations that reduce catalytic activity relative to the unmutated form. In an embodiment, the one or more mutations that reduce catalytic activity relative to the unmutated form are in one or more HEPN domains of the endonuclease of the invention. Those skilled in the art can refer to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D , and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc., to select one or more mutations to reduce catalytic activity relative to the unmutated form.

[0171] In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations that render the endonuclease substantially catalytically inactive relative to the unmutated form. In an embodiment, the one or more mutations that render the endonuclease substantially catalytically inactive relative to the unmutated form are in one or more HEPN domains of the endonuclease of the invention. One skilled in the art can refer to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D , and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc., to select one or more mutations so that the nuclease is substantially catalytically inactive relative to the unmutated form.

[0172] In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations to increase catalytic activity relative to an unmutated form. In an embodiment, the one or more mutations that increase catalytic activity relative to an unmutated form are in one or more HEPN domains of an endonuclease of the present invention. Those skilled in the art can select one or more mutations to increase catalytic activity relative to an unmutated form by reference to, for example, Figure 1, and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other endonucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175 (1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc.

[0173] In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations that render the endonuclease substantially catalytically overactive relative to the unmutated form. In an embodiment, the one or more mutations that render the endonuclease substantially catalytically overactive relative to the unmutated form are in one or more HEPN domains of an endonuclease of the invention. One skilled in the art can refer to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D , and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc., to select one or more mutations so that the nuclease is substantially catalytically overactive relative to the unmutated form.

[0174] In an embodiment, the endonuclease has nickase activity. In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations to produce nickase activity. In an embodiment, the one or more mutations that produce nickase activity relative to the unmutated form are in one or more HEPN domains of the endonuclease of the invention. Those skilled in the art can refer to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D, and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc., to select one or more mutations to produce nickase activity relative to the unmutated form.

[0175] In an embodiment, the endonuclease has incidental cleavage activity. In an embodiment, the endonuclease (or chimeric protein) comprises one or more mutations to produce, increase, remove or reduce incidental cleavage activity. In an embodiment, the one or more mutations that produce, increase, remove or reduce incidental cleavage activity relative to the unmutated form are in one or more HEPN domains of the endonuclease of the invention. One skilled in the art can refer to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D , and / or by reference to Table 1 or Table 2, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety), etc., to select one or more mutations to produce, increase, remove or reduce incidental cleavage activity relative to the unmutated form.

[0176] In an embodiment, one skilled in the art can select the residues to be changed based on the desired percentage of sequence identity, and / or by reference to, for example, Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1D and / or by reference to Table 1 or Table 2, and / or by reference to Figure 5 Comparison information and / or Figure 6 Amino acid modifications are selected based on phylogenetic information, and / or by reference to structural information about other nucleases known in the art, such as Slaymaker et al., "High-Resolution Structure of Cas13 band Biochemical Characterization of RNA Targeting and Cleavage" 2019, Cell Reports 26, 3741-3751; Zhang et al., "Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d" Cell 175(1): 212-22, 2018 (each of which is hereby incorporated by reference in its entirety).

[0177] In embodiments, the amino acid modification is amino acid mutation or amino acid replacement.In embodiments, the amino acid replacement is conservative and / or non-conservative replacement.In embodiments, the amino acid modification is two or more amino acid whose amino acid truncation (for example, about to about 100 or about 2 to about 90 or about 2 to about 80 or about 2 to about 70 or about 2 to about 60 or about 2 to about 50 or about 2 to about 40 or about 2 to about 30 or about 2 to about 20 or about 2 to about 10 or about 20 to about 100 or about 50 to about 100 or about 70 to about 100 amino acids).

[0178] "Conservative substitutions" can be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0179] As used herein, a "conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, exchanging Asp with Glu retains a negative charge in the modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices.

[0180] As used herein, a "non-conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in a different group among the six standard amino acid groups (1) to (6) shown above.

[0181] In embodiments, the substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs in general).

[0182] In an embodiment, the percentage of sequence identity between a specific nucleic acid or amino acid sequence and a sequence cited by a specific sequence identification number is determined as follows. The nucleic acid or amino acid sequence is compared with the sequence shown in the specific sequence identification number using, for example, the BLAST 2 sequences (Bl2seq) program from a BLASTZ standalone version containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This BLASTZ standalone version is available online at or ncbi.nlm.nih.gov. Explanations for how to use the Bl2seq program can be found in the accompanying readme file for BLASTZ. Bl2seq uses the BLASTN or BLASTP algorithm to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options can be set as follows: -i is set to the file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default settings. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -pblastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options for Bl2seq are set as follows: -i is set to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, the specified output file will present those homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present an aligned sequence. Once the alignment is performed, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue occurs in the two sequences.The percent sequence identity is determined by dividing the number of matches by the length of the sequence shown in the identified sequence (e.g., any one of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89), or by the hinge length (e.g., 100 consecutive nucleotides or amino acid residues of the sequence shown in the identified sequence), and then multiplying the resulting value by 100. It should be noted that the percent sequence identity values ​​are rounded to one decimal place. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. It should also be noted that length values ​​should always be integers.

[0183] In embodiments, the endonuclease is at least about 75% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 80% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 85% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 90% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 95% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 97% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease is at least about 99% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89.

[0184] In embodiments, the endonuclease has about 1 to about 15 amino acid modifications. In embodiments, the endonuclease has about 1 to about 10 amino acid modifications. In embodiments, the endonuclease has about 1 to about 5 amino acid modifications. In embodiments, the endonuclease has about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications. In embodiments, the amino acid modifications are selected from substitution and deletion.

[0185] In an embodiment, the endonuclease is selected from Table 1 below.

[0186] In embodiments, the sequence comprises at least one HEPN domain, or a fragment or variant thereof. In embodiments, the sequence comprises at least two HEPN domains, or fragments or variants thereof. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 3. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 4. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:80.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 81. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 82. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 83. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 84. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 85. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 86. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO:87.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 88. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 89. In embodiments, the endonuclease comprises about 1 to about 20 amino acid modifications relative to SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 1. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 2. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 3. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 4.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 80. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 81. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 82. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 83. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 84. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 85. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:86.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 87. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 88. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:89.

[0187] In various aspects, the present disclosure provides a composition comprising a nucleic acid encoding a nuclease comprising a sequence, optionally comprising a HEPN domain or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In embodiments, the sequence comprises at least two HEPN domains or fragments or variants thereof. In embodiments, the HEPN domain is according to Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1DIn embodiments, the endonuclease is selected from Table 1 below. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 3. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 4. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:80.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 81. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 82. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 83. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 84. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 85. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 86. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO:87.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 88. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 89. In embodiments, the endonuclease comprises about 1 to about 20 amino acid modifications relative to SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 1. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 2. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 3. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 4.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 80. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 81. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 82. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 83. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 84. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 85. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:86.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 87. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 88. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:89.

[0188] In various aspects, the present disclosure provides a composition comprising a nuclease system comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications); and (b) an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule. In embodiments, the HEPN domain is according to Figure 1A 、 Figure 1B 、 Figure 1C and Figure 1DIn one embodiment, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 3. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 4. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:80.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 81. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 82. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 83. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 84. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 85. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 86. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO:87.In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 88. In embodiments, the endonuclease comprises a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 89. In embodiments, the endonuclease comprises about 1 to about 20 amino acid modifications relative to SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications). In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 1. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 2. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 3. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 4.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 80. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 81. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 82. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 83. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 84. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 85. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:86.In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 87. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO: 88. In embodiments, the endonuclease comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid modifications relative to SEQ ID NO:89.

[0189] Table 1: Amino acid sequences of SEQ ID NOs: 1-4 and 80-89

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197] In any aspect or embodiment herein, an endonuclease of the invention or a fragment or variant thereof having at least about 70% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 has the N-terminal M residue removed.

[0198] In any aspect or embodiment herein, an endonuclease of the invention or a fragment or variant thereof having at least about 70% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 has the N-terminal M residue removed and the SV40NLS SV40 sequence (MSPKKKRKVEAS (SEQ ID NO: 78)) added to the N-terminus.

[0199] In any aspect or embodiment herein, the endonucleases of the invention or fragments or variants thereof having at least about 70% identity to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89 have the N-terminal M residue removed and the SV40NLS SV40 sequence (MSPKKKRKVEAS (SEQ ID NO: 78)) added to the N-terminus and the HA tag (GSGPKKKRKVAAAYPYDVPDYA (SEQ ID NO: 77)) added to the C-terminus.

[0200] In an embodiment, the catalytic domain is selected from Table 2. In an embodiment, the mutation of any endonuclease of the invention involves one or more residues of Table 2.

[0201] Table 2: Illustrative Positioning of Catalytic Residues

[0202]

[0203]

[0204]

[0205]

[0206] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and has an amino acid modification at one or more positions with an R or H residue in the wild-type sequence.

[0207] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and has an amino acid modification at one or more positions in the HEPN domain.

[0208] In embodiments, the endonuclease is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, one of NO:89 having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity and having an amino acid modification at one or more positions in the endonuclease in a region from about 100 to about 300 amino acids, or about 150 to about 300 amino acids, or about 150 to about 250 amino acids, or about 100 to about 300 amino acids, or about 200 to about 250 amino acids, or about 240 to about 250 amino acids of the N-terminus of the endonuclease.

[0209] In embodiments, the endonuclease is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, one of NO:89 having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity and having an amino acid modification at one or more positions in the endonuclease in a region that is about 100 to about 300 amino acids, or about 150 to about 300 amino acids, or about 150 to about 250 amino acids, or about 100 to about 300 amino acids, or about 200 to about 250 amino acids, or about 240 to about 250 amino acids from the C-terminus of the endonuclease.

[0210] In embodiments, the amino acid modification is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification is a hydrophilic amino acid. In embodiments, the amino acid modification is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification is an arginine (R) or a lysine (K). In embodiments, the amino acid modification is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification is histidine (H). In embodiments, the amino acid modification is a hydrophobic amino acid. In embodiments, the amino acid modification is a hydrophobic aliphatic amino acid. In embodiments, the amino acid modification is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In embodiments, the amino acid modification is a hydrophobic aromatic amino acid. In embodiments, the amino acid modification is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

[0211] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 1 and has an amino acid modification at position R244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R244 relative to SEQ ID NO: 1 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic amino acid. In embodiments, the amino acid modification at R244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is lysine (K). In embodiments, the amino acid modification at R244 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R244 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R244 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is histidine (H). In embodiments, the amino acid modification at R244 is a hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0212] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 1 and has an amino acid modification at position H249. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H249 relative to SEQ ID NO: 1 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic amino acid. In embodiments, the amino acid modification at H249 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H249 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H249 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H249 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H249 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H249 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H249 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0213] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 1 and has an amino acid modification at position H669. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H669 relative to SEQ ID NO: 1 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H669 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H669 is a hydrophilic amino acid. In embodiments, the amino acid modification at H669 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H669 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H669 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H669 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H669 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H669 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H669 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H669 is hydrophobic amino acid. In embodiments, the amino acid modification at H669 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H669 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H669 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H669 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0214] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 1 and has an amino acid modification at position R664. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R664 relative to SEQ ID NO: 1 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R664 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R664 is a hydrophilic amino acid. In embodiments, the amino acid modification at R664 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R664 is lysine (K). In embodiments, the amino acid modification at R664 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R664 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R664 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R664 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R664 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R664 is histidine (H). In embodiments, the amino acid modification at R664 is a hydrophobic amino acid. In embodiments, the amino acid modification at R664 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R664 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R664 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R664 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0215] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 2, and has an amino acid modification at position R244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R244 relative to SEQ ID NO: 2 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic amino acid. In embodiments, the amino acid modification at R244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is lysine (K). In embodiments, the amino acid modification at R244 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R244 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R244 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is histidine (H). In embodiments, the amino acid modification at R244 is a hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0216] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 2, and has an amino acid modification at position H249. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H249 relative to SEQ ID NO: 2 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic amino acid. In embodiments, the amino acid modification at H249 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H249 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H249 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H249 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H249 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H249 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H249 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0217] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 2, and has an amino acid modification at position H687. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H687 relative to SEQ ID NO: 2 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H687 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H687 is a hydrophilic amino acid. In embodiments, the amino acid modification at H687 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H687 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H687 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H687 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is hydrophobic amino acid. In embodiments, the amino acid modification at H687 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H687 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H687 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H687 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0218] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 2, and has an amino acid modification at position R682. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R682 relative to SEQ ID NO: 2 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R682 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R682 is a hydrophilic amino acid. In embodiments, the amino acid modification at R682 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R682 is lysine (K). In embodiments, the amino acid modified at R682 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modified at R682 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modified at R682 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is histidine (H). In embodiments, the amino acid modified at R682 is hydrophobic amino acid. In embodiments, the amino acid modified at R682 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R682 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R682 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R682 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0219] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 3, and has an amino acid modification at position R244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R244 relative to SEQ ID NO: 3 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic amino acid. In embodiments, the amino acid modification at R244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is lysine (K). In embodiments, the amino acid modification at R244 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R244 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R244 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is histidine (H). In embodiments, the amino acid modification at R244 is a hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0220] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 3 and has an amino acid modification at position H249. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H249 relative to SEQ ID NO: 3 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic amino acid. In embodiments, the amino acid modification at H249 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H249 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H249 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H249 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H249 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H249 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H249 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0221] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 3 and has an amino acid modification at position R704. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R704 relative to SEQ ID NO: 3 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R704 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R704 is a hydrophilic amino acid. In embodiments, the amino acid modification at R704 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R704 is lysine (K). In embodiments, the amino acid modification at R704 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R704 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R704 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R704 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R704 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R704 is histidine (H). In embodiments, the amino acid modification at R704 is a hydrophobic amino acid. In embodiments, the amino acid modification at R704 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R704 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V). In an embodiment, the amino acid modification at R704 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R704 is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

[0222] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 3, and has an amino acid modification at position H709. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H709 relative to SEQ ID NO: 3 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H709 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H709 is a hydrophilic amino acid. In embodiments, the amino acid modification at H709 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H709 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H709 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H709 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H709 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H709 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H709 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H709 is hydrophobic amino acid. In embodiments, the amino acid modification at H709 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H709 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H709 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H709 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0223] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 4 and has an amino acid modification at position R244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R244 relative to SEQ ID NO: 4 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic amino acid. In embodiments, the amino acid modification at R244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is lysine (K). In embodiments, the amino acid modification at R244 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R244 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R244 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is histidine (H). In embodiments, the amino acid modification at R244 is a hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0224] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 4 and has an amino acid modification at position H249. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H249 relative to SEQ ID NO: 4 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic amino acid. In embodiments, the amino acid modification at H249 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H249 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H249 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H249 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H249 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H249 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H249 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0225] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 4 and has an amino acid modification at position H687. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H687 relative to SEQ ID NO: 4 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H687 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H687 is a hydrophilic amino acid. In embodiments, the amino acid modification at H687 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H687 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H687 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H687 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H687 is hydrophobic amino acid. In embodiments, the amino acid modification at H687 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H687 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H687 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H687 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0226] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 4, and has an amino acid modification at position R682. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R682 relative to SEQ ID NO: 4 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R682 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R682 is a hydrophilic amino acid. In embodiments, the amino acid modification at R682 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R682 is lysine (K). In embodiments, the amino acid modified at R682 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modified at R682 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modified at R682 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modified at R682 is histidine (H). In embodiments, the amino acid modified at R682 is hydrophobic amino acid. In embodiments, the amino acid modified at R682 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R682 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R682 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R682 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0227] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO:80 and has an amino acid modification at position R234. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R234 relative to SEQ ID NO:80 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic amino acid. In embodiments, the amino acid modification at R234 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is lysine (K). In embodiments, the amino acid modification at R234 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R234 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R234 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is histidine (H). In embodiments, the amino acid modification at R234 is a hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R234 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R234 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R234 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0228] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 80 and has an amino acid modification at position H239. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H239 relative to SEQ ID NO: 80 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic amino acid. In embodiments, the amino acid modification at H239 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H239 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H239 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H239 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H239 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H239 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H239 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0229] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 80, and has an amino acid modification at position R659. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R659 relative to SEQ ID NO: 80 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R659 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R659 is a hydrophilic amino acid. In embodiments, the amino acid modification at R659 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is lysine (K). In embodiments, the amino acid modification at R659 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R659 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R659 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is histidine (H). In embodiments, the amino acid modification at R659 is a hydrophobic amino acid. In embodiments, the amino acid modification at R659 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R659 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R659 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R659 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0230] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 80, and has an amino acid modification at position H664. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H664 relative to SEQ ID NO: 80 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic amino acid. In embodiments, the amino acid modification at H664 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H664 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H664 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H664 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H664 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H664 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H664 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0231] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 81 and has an amino acid modification at position R234. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R234 relative to SEQ ID NO: 81 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic amino acid. In embodiments, the amino acid modification at R234 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is lysine (K). In embodiments, the amino acid modification at R234 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R234 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R234 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is histidine (H). In embodiments, the amino acid modification at R234 is a hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R234 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R234 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R234 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0232] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 81 and has an amino acid modification at position H239. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H239 relative to SEQ ID NO: 81 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic amino acid. In embodiments, the amino acid modification at H239 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H239 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H239 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H239 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H239 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H239 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H239 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0233] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 81 and has an amino acid modification at position R659. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R659 relative to SEQ ID NO: 81 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R659 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R659 is a hydrophilic amino acid. In embodiments, the amino acid modification at R659 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is lysine (K). In embodiments, the amino acid modification at R659 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R659 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R659 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is histidine (H). In embodiments, the amino acid modification at R659 is a hydrophobic amino acid. In embodiments, the amino acid modification at R659 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R659 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R659 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R659 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0234] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 81 and has an amino acid modification at position H664. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H664 relative to SEQ ID NO: 81 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic amino acid. In embodiments, the amino acid modification at H664 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H664 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H664 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H664 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H664 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H664 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H664 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0235] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 82, and has an amino acid modification at position R234. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R234 relative to SEQ ID NO: 82 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophilic amino acid. In embodiments, the amino acid modification at R234 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is lysine (K). In embodiments, the amino acid modification at R234 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R234 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R234 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R234 is histidine (H). In embodiments, the amino acid modification at R234 is a hydrophobic amino acid. In embodiments, the amino acid modification at R234 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R234 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R234 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R234 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0236] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 82 and has an amino acid modification at position H239. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H239 relative to SEQ ID NO: 82 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H239 is a hydrophilic amino acid. In embodiments, the amino acid modification at H239 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H239 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H239 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H239 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic amino acid. In embodiments, the amino acid modification at H239 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H239 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H239 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H239 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0237] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 82, and has an amino acid modification at position R659. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R659 is essential or non-essential amino acid relative to SEQ ID NO: 82. In embodiments, the amino acid modification at R659 is hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R659 is hydrophilic amino acid. In embodiments, the amino acid modification at R659 is polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is lysine (K). In embodiments, the amino acid modification at R659 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R659 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R659 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R659 is histidine (H). In embodiments, the amino acid modification at R659 is a hydrophobic amino acid. In embodiments, the amino acid modification at R659 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R659 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R659 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R659 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0238] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 82 and has an amino acid modification at position H664. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H664 relative to SEQ ID NO: 82 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H664 is a hydrophilic amino acid. In embodiments, the amino acid modification at H664 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H664 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H664 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H664 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic amino acid. In embodiments, the amino acid modification at H664 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H664 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H664 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H664 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0239] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 83, and has an amino acid modification at position R239. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R239 relative to SEQ ID NO: 83 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R239 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R239 is a hydrophilic amino acid. In embodiments, the amino acid modification at R239 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R239 is lysine (K). In embodiments, the amino acid modification at R239 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R239 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R239 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R239 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R239 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R239 is histidine (H). In embodiments, the amino acid modification at R239 is a hydrophobic amino acid. In embodiments, the amino acid modification at R239 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R239 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R239 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R239 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0240] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 83 and has an amino acid modification at position H244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H244 relative to SEQ ID NO: 83 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H244 is a hydrophilic amino acid. In embodiments, the amino acid modification at H244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H244 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H244 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H244 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H244 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H244 is hydrophobic amino acid. In embodiments, the amino acid modification at H244 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0241] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 83, and has an amino acid modification at position R677. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R677 relative to SEQ ID NO: 83 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R677 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R677 is a hydrophilic amino acid. In embodiments, the amino acid modification at R677 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R677 is lysine (K). In embodiments, the amino acid modification at R677 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R677 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R677 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R677 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R677 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R677 is histidine (H). In embodiments, the amino acid modification at R677 is hydrophobic amino acid. In embodiments, the amino acid modification at R677 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R677 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R677 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R677 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0242] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 83 and has an amino acid modification at position H682. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H682 relative to SEQ ID NO: 83 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H682 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H682 is a hydrophilic amino acid. In embodiments, the amino acid modification at H682 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H682 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H682 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H682 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H682 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H682 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H682 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H682 is hydrophobic amino acid. In embodiments, the amino acid modification at H682 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H682 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H682 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H682 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0243] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 84 and has an amino acid modification at position R241. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R241 relative to SEQ ID NO: 84 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R241 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R241 is a hydrophilic amino acid. In embodiments, the amino acid modification at R241 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R241 is lysine (K). In embodiments, the amino acid modification at R241 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R241 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R241 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R241 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R241 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R241 is histidine (H). In embodiments, the amino acid modification at R241 is a hydrophobic amino acid. In embodiments, the amino acid modification at R241 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R241 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R241 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R241 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0244] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 84 and has an amino acid modification at position H246. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H246 relative to SEQ ID NO: 84 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H246 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H246 is a hydrophilic amino acid. In embodiments, the amino acid modification at H246 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H246 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H246 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H246 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H246 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H246 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H246 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H246 is hydrophobic amino acid. In embodiments, the amino acid modification at H246 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H246 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H246 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H246 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0245] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 84 and has an amino acid modification at position R681. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R681 relative to SEQ ID NO: 84 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R681 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R681 is a hydrophilic amino acid. In embodiments, the amino acid modification at R681 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R681 is lysine (K). In embodiments, the amino acid modification at R681 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R681 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R681 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R681 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R681 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R681 is histidine (H). In embodiments, the amino acid modification at R681 is hydrophobic amino acid. In embodiments, the amino acid modification at R681 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R681 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R681 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R681 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0246] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 84 and has an amino acid modification at position H686. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H686 relative to SEQ ID NO: 84 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H686 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H686 is a hydrophilic amino acid. In embodiments, the amino acid modification at H686 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H686 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H686 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H686 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H686 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H686 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H686 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H686 is hydrophobic amino acid. In embodiments, the amino acid modification at H686 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H686 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H686 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H686 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0247] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 85 and has an amino acid modification at position R244. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R244 relative to SEQ ID NO: 85 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophilic amino acid. In embodiments, the amino acid modification at R244 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is lysine (K). In embodiments, the amino acid modification at R244 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R244 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R244 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R244 is histidine (H). In embodiments, the amino acid modification at R244 is a hydrophobic amino acid. In embodiments, the amino acid modification at R244 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R244 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R244 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R244 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0248] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 85 and has an amino acid modification at position H249. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H249 relative to SEQ ID NO: 85 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H249 is a hydrophilic amino acid. In embodiments, the amino acid modification at H249 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H249 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H249 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H249 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic amino acid. In embodiments, the amino acid modification at H249 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H249 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H249 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H249 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0249] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 85 and has an amino acid modification at position R701. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R701 relative to SEQ ID NO: 85 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R701 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R701 is a hydrophilic amino acid. In embodiments, the amino acid modification at R701 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R701 is lysine (K). In embodiments, the amino acid modification at R701 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R701 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R701 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R701 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R701 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R701 is histidine (H). In embodiments, the amino acid modification at R701 is a hydrophobic amino acid. In embodiments, the amino acid modification at R701 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R701 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R701 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R701 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0250] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 85, and has an amino acid modification at position H706. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H706 relative to SEQ ID NO: 85 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H706 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H706 is a hydrophilic amino acid. In embodiments, the amino acid modification at H706 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H706 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H706 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H706 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H706 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H706 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H706 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H706 is hydrophobic amino acid. In embodiments, the amino acid modification at H706 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H706 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H706 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H706 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0251] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 86, and has an amino acid modification at position R247. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R247 relative to SEQ ID NO: 86 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R247 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R247 is a hydrophilic amino acid. In embodiments, the amino acid modification at R247 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R247 is lysine (K). In embodiments, the amino acid modification at R247 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R247 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R247 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R247 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R247 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R247 is histidine (H). In embodiments, the amino acid modification at R247 is a hydrophobic amino acid. In embodiments, the amino acid modification at R247 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R247 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R247 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R247 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0252] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 86 and has an amino acid modification at position H252. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H252 relative to SEQ ID NO: 86 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H252 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H252 is a hydrophilic amino acid. In embodiments, the amino acid modification at H252 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H252 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H252 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H252 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H252 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H252 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H252 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H252 is hydrophobic amino acid. In embodiments, the amino acid modification at H252 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H252 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H252 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H252 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0253] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 86, and has an amino acid modification at position R711. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R711 relative to SEQ ID NO: 86 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R711 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R711 is a hydrophilic amino acid. In embodiments, the amino acid modification at R711 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R711 is lysine (K). In embodiments, the amino acid modification at R711 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R711 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R711 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R711 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R711 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R711 is histidine (H). In embodiments, the amino acid modification at R711 is hydrophobic amino acid. In embodiments, the amino acid modification at R711 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R711 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R711 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R711 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0254] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 86, and has an amino acid modification at position H716. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H716 relative to SEQ ID NO: 86 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H716 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H716 is a hydrophilic amino acid. In embodiments, the amino acid modification at H716 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H716 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H716 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H716 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H716 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H716 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H716 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H716 is hydrophobic amino acid. In embodiments, the amino acid modification at H716 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H716 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H716 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H716 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0255] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 87 and has an amino acid modification at position R316. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R316 relative to SEQ ID NO: 87 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R316 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R316 is a hydrophilic amino acid. In embodiments, the amino acid modification at R316 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R316 is lysine (K). In embodiments, the amino acid modification at R316 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R316 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R316 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R316 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R316 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R316 is histidine (H). In embodiments, the amino acid modification at R316 is a hydrophobic amino acid. In embodiments, the amino acid modification at R316 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R316 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R316 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R316 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0256] In embodiments, the endonuclease is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 87 and has an amino acid modification at position H321. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H321 relative to SEQ ID NO: 87 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H321 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H321 is a hydrophilic amino acid. In embodiments, the amino acid modification at H321 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H321 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H321 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H321 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H321 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H321 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H321 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H321 is a hydrophobic amino acid. In embodiments, the amino acid modification at H321 is a hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H321 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H321 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H321 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0257] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 87, and has an amino acid modification at position R781. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R781 relative to SEQ ID NO: 87 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R781 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R781 is a hydrophilic amino acid. In embodiments, the amino acid modification at R781 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R781 is lysine (K). In embodiments, the amino acid modification at R781 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R781 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R781 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R781 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R781 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R781 is histidine (H). In embodiments, the amino acid modification at R781 is hydrophobic amino acid. In embodiments, the amino acid modification at R781 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R781 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R781 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R781 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0258] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 87, and has an amino acid modification at position H786. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H786 relative to SEQ ID NO: 87 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H786 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H786 is a hydrophilic amino acid. In embodiments, the amino acid modification at H786 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H786 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H786 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H786 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H786 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H786 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H786 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H786 is hydrophobic amino acid. In embodiments, the amino acid modification at H786 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H786 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H786 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H786 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0259] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 88, and has an amino acid modification at position R274. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R274 relative to SEQ ID NO: 88 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R274 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R274 is a hydrophilic amino acid. In embodiments, the amino acid modification at R274 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R274 is lysine (K). In embodiments, the amino acid modification at R274 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R274 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R274 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R274 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R274 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R274 is histidine (H). In embodiments, the amino acid modification at R274 is a hydrophobic amino acid. In embodiments, the amino acid modification at R274 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R274 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R274 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R274 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0260] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 88 and has an amino acid modification at position H279. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H279 relative to SEQ ID NO: 88 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H279 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H279 is a hydrophilic amino acid. In embodiments, the amino acid modification at H279 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H279 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H279 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H279 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H279 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H279 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H279 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H279 is hydrophobic amino acid. In embodiments, the amino acid modification at H279 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H279 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H279 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H279 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0261] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 88, and has an amino acid modification at position R723. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R723 relative to SEQ ID NO: 88 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R723 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R723 is a hydrophilic amino acid. In embodiments, the amino acid modification at R723 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R723 is lysine (K). In embodiments, the amino acid modified at R723 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modified at R723 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modified at R723 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modified at R723 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modified at R723 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modified at R723 is histidine (H). In embodiments, the amino acid modified at R723 is hydrophobic amino acid. In embodiments, the amino acid modified at R723 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R723 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R723 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R723 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0262] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 88, and has an amino acid modification at position H728. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H728 relative to SEQ ID NO: 88 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H728 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H728 is a hydrophilic amino acid. In embodiments, the amino acid modification at H728 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H728 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H728 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H728 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H728 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H728 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H728 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H728 is hydrophobic amino acid. In embodiments, the amino acid modification at H728 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H728 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H728 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H728 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0263] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position R237. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R237 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R237 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R237 is a hydrophilic amino acid. In embodiments, the amino acid modification at R237 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is lysine (K). In embodiments, the amino acid modification at R237 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R237 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R237 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is histidine (H). In embodiments, the amino acid modification at R237 is a hydrophobic amino acid. In embodiments, the amino acid modification at R237 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R237 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R237 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R237 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0264] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position H242. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H242 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H242 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H242 is a hydrophilic amino acid. In embodiments, the amino acid modification at H242 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H242 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H242 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H242 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is hydrophobic amino acid. In embodiments, the amino acid modification at H242 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H242 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H242 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H242 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0265] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position R694. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R694 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R694 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R694 is a hydrophilic amino acid. In embodiments, the amino acid modification at R694 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is lysine (K). In embodiments, the amino acid modification at R694 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R694 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R694 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is histidine (H). In embodiments, the amino acid modification at R694 is hydrophobic amino acid. In embodiments, the amino acid modification at R694 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R694 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R694 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R694 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0266] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position H699. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H699 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H699 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H699 is a hydrophilic amino acid. In embodiments, the amino acid modification at H699 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H699 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H699 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H699 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is hydrophobic amino acid. In embodiments, the amino acid modification at H699 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H699 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H699 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H699 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0267] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position R237. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R237 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R237 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R237 is a hydrophilic amino acid. In embodiments, the amino acid modification at R237 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is lysine (K). In embodiments, the amino acid modification at R237 is a polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R237 is a polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R237 is an aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R237 is histidine (H). In embodiments, the amino acid modification at R237 is a hydrophobic amino acid. In embodiments, the amino acid modification at R237 is a hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R237 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R237 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R237 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0268] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position H242. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H242 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H242 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H242 is a hydrophilic amino acid. In embodiments, the amino acid modification at H242 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H242 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H242 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H242 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H242 is hydrophobic amino acid. In embodiments, the amino acid modification at H242 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H242 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H242 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H242 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0269] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position R694. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at R694 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at R694 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at R694 is a hydrophilic amino acid. In embodiments, the amino acid modification at R694 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is lysine (K). In embodiments, the amino acid modification at R694 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at R694 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at R694 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at R694 is histidine (H). In embodiments, the amino acid modification at R694 is hydrophobic amino acid. In embodiments, the amino acid modification at R694 is hydrophobic aliphatic amino acid. In an embodiment, the amino acid modification at R694 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at R694 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at R694 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0270] In embodiments, the endonuclease has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to SEQ ID NO: 89 and has an amino acid modification at position H699. In embodiments, the amino acid modification is selected from substitution and deletion. In embodiments, the amino acid modification at H699 relative to SEQ ID NO: 89 is an essential or non-essential amino acid. In embodiments, the amino acid modification at H699 is a hydrophilic or hydrophobic amino acid. In embodiments, the amino acid modification at H699 is a hydrophilic amino acid. In embodiments, the amino acid modification at H699 is a polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from arginine (R) or lysine (K). In embodiments, the amino acid modification at H699 is polar and neutrally charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C). In embodiments, the amino acid modification at H699 is polar and negatively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is selected from aspartic acid (D) or glutamic acid (E). In embodiments, the amino acid modification at H699 is aromatic, polar and positively charged hydrophilic amino acid. In embodiments, the amino acid modification at H699 is hydrophobic amino acid. In embodiments, the amino acid modification at H699 is hydrophobic aliphatic amino acid. In embodiments, the amino acid modification at H699 is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V). In an embodiment, the amino acid modification at H699 is a hydrophobic aromatic amino acid. In an embodiment, the amino acid modification at H699 is selected from phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0271] In embodiments, disclosed herein is a composition comprising an endonuclease and having an amino acid sequence that is at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 3. In embodiments, substitutions are made to SEQ ID NO: 3:

[0272] Met Asp Lys His Pro Ser Asn Arg Tyr Ala Leu Pro Lys Val Ile SerGlu Val Asp His Glu Arg Ile Leu Glu Phe Lys Val Lys Tyr Glu Lys Leu Ala ArgLeu Asp Arg Phe Glu Val Lys Ala Met His Tyr Asp Gly Ala Glu Ile Glu Val Gly Val Glu Val Asp Tyr Gln Asp Asn Asn Lys ThrIle Thr Ile Asn Leu Asn Gly Lys Tyr Thr Ile Asn Gly Arg Lys Val Gly GlyLys Arg Arg Leu Leu Glu Asp Arg Ile Ser Arg Gly Lys Val Cys Leu Glu Leu HisAsp Ser G Serg Lys Arg Lys Ar G Lys Pro Asp Arg Glu LeuIle Thr Phe Asp Ser Thr Lys Leu Tyr Ser Gln Ile Gly Arg Asp Val SerThr Lys Glu Ile Tyr Leu Ile Lys Arg Phe Leu Ala Tyr Arg Ser Asp Leu Leu PheTyr Tyr Gly Phe Ile Asp Ala Asn Phe Arg Gly As Val Phe Ser Gly Asp Lys Asn Gln Glu Leu Ile Lys Tyr Phe Asn Phe ThrIle Asn Asp Lys Leu Lys Asn Asp Lys Gly Tyr Leu Lys Glu Tyr Thr Ala Asn AspGlu Gln Ile Lys Lys Asp Leu Gln Asn Thr Me Thr Lys P He Ala P Glu Tyr AspPhe Phe Glu Lys Leu Phe Asn Asn Glu Glu IleGlu Thr Leu Ser Lys Ile His Asp Ile Glu Leu Leu Asn Thr Met Ile Asn Lys LeuAsp Lys Leu Asn Ile Asp Thr Arg Lys Glu Tyr Ile Asp Asp Glu Lys Ile Thr ValPhe Gly Glu Glu Ile Ser Leu Lys Thr Leu Tyr Gly Leu Tyr Ala His Thr Ala IleAsn Arg Val Ala Phe Asn Lys Leu Ile Asn Arg Phe Met Val Glu Asn Gly Thr GluAsn Glu Ala Leu Lys Lys Tyr Phe Asn Ser Lys Ala Glu Gly Gly Ile Ala Tyr GluIle Asp Ile His Gln Asn Ser Glu Tyr Lys Gln Leu Tyr Ile Gln His Lys Asp LeuVal Ser Lys Leu Ser Ala Leu Ser Asp Gly Asp Glu Ile Ala Asp Thr Asn Lys LysIle Ser Glu Leu Lys Val Lys Met Lys Ala Ile Thr Lys Ala Asn Ser Leu Lys ArgLeu Glu His Lys Leu Arg Leu Thr Phe Gly Phe Ile Tyr Thr Glu Tyr Gln Asp TyrAsn Ala Phe Lys Asn Asn Phe Asp Thr Asp Ile Lys Ser Gly Arg Phe Ile Pro LysAsp Ser Glu Gly Lys Arg Arg Gly Phe Asp His Arg Glu Leu Asp Gln Leu Lys ArgTyr Tyr Asp Ala Thr Phe Ala Asp Lys Lys Pro Gln Thr Lys Glu Thr Phe Asp GluIle Asp Lys Gln Ile Asp Gln Leu Ser Leu Lys Asn Leu IleGly Asp Asp Thr LeuLeu Lys Val Ile Leu Leu Ile Tyr Ile Phe Leu Pro Arg Glu Ile Lys Gly Glu PheLeu Gly Phe Val Lys Tyr Tyr His Asp Thr Lys His Ile Glu Glu Glu Asp Thr LysAsp Lys Asp Glu Val Asp Le Gly Thly Lys Phe Asp LeuAsp Lys Asn Ile Arg Ala Leu Ser Val Leu Lys Ser Leu Ser Tyr Gln Ala LysTyr Asn Lys Lys Glu Glu Lys Lys Glu Gln Phe Tyr Glu Ala Gly Asn Arg His GlyArg Phe Tyr Ser Lys Lys Leu Gly Ile Val Asyr Hip Ala Gs Glu Asn Arg Tyr His Ala Ala Leu Phe Lys Leu Leu Asn Asp Phe GluIle Tyr Ser Leu Ala Gln His Ile Glu Gly Lys Glu Thr Leu Ala Gln Gln Ile GluLys Pro Gln Phe Ser Gln Tyr Glu Le His Tyr Asn Phe Arg Lys Ser Glys Thr Ala T Leu Asp Asn Asp Ala Phe Asp Thr Val IleAsn Met Arg Asp Ile Ala His Leu Ser His Glu Pro Leu Phe Glu Cys Pro LeuAsp Gly Lys Ser Tyr Lys Leu Lys Gln Gly Lys Arg Thr Asn Thr Ile Asn ValLys Pro Arle Valle Met Glu Ser Pro Ile AspMetLys Lys Thr Leu Gly Tyr Asp Ala Val Asn Asp Leu Thr Met Lys Ile Ile GlnLeu Arg Thr Arg Leu Lys Val Tyr Ala Asp Lys Ser Glu Thr Ile Lys Thr Leu ValAsp Ala Lys Thr Pro Asn Asp Phe Tyr His Ile Tyr Lys Val Lys Gly Val GluAla Ile Asn Arg His Leu Leu Glu Val Ile Gly Glu Thr Lys Asp Glu Lys Arg IleArg Lys Arg Ile Glu Ser Gly Asn Ala Ile Ala Gly Arg Thr Pro Ala Asp Ser GlnGlu Asn

[0273] As described herein, substitutions can be made to this sequence to generate the endonucleases of the invention (including taking into account the degeneracy of the genetic code).

[0274] In some embodiments, the endonuclease has one or more substitutions at positions corresponding to D38X, A59X, G172X, T236X, T319X, H375X, H419X, T424X, E529X, T541X, G562X, K564X, D569X, A586X, N641X, D642X, S647X, D721X, R779X, K13X, K566X, G554X, A35X, E110X, G314X, K114X, D498X, I86X, V57X, H249X, R704X of SEQ ID NO: 3, wherein the substitution is defined by X and wherein X is any amino acid. In some embodiments, X is an essential or non-essential amino acid.

[0275] In some embodiments, X is a hydrophilic or hydrophobic amino acid.

[0276] In some embodiments, X is a hydrophilic amino acid.

[0277] In some embodiments, X is a polar and positively charged hydrophilic amino acid. In some embodiments, X is selected from arginine I or lysine (K).

[0278] In some embodiments, X is a polar and neutrally charged hydrophilic amino acid. In some embodiments, X is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C).

[0279] In some embodiments, X is a polar and negatively charged hydrophilic amino acid. In some embodiments, X is selected from aspartic acid (D) or glutamic acid (E).

[0280] In some embodiments, wherein X is an aromatic, polar, and positively charged hydrophilic amino acid. In some embodiments, wherein X is histidine (H).

[0281] In some embodiments, X is a hydrophobic amino acid.

[0282] In some embodiments, X is a hydrophobic, aliphatic amino acid. In some embodiments, X is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).

[0283] In some embodiments, X is a hydrophobic, aromatic amino acid. In some embodiments, wherein X is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

[0284] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises one or more of the following substitutions:

[0285] a hydrophilic residue other than aspartic acid (D) at position corresponding to 38;

[0286] a hydrophobic residue other than alanine (A) at position corresponding to 59;

[0287] a hydrophobic residue other than glycine (G) at the position corresponding to 172;

[0288] a hydrophilic residue other than threonine (T) at position corresponding to 236;

[0289] a hydrophilic residue other than threonine (T) at the position corresponding to 319;

[0290] a hydrophilic residue other than histidine (H) at the position corresponding to 375;

[0291] a hydrophilic residue other than histidine (H) at the position corresponding to 419;

[0292] a hydrophilic residue other than threonine (T) at the position corresponding to 424;

[0293] a hydrophilic residue other than glutamic acid (gIamate) (E) at the position corresponding to 529;

[0294] a hydrophilic residue other than threonine (T) at the position corresponding to 541;

[0295] a hydrophobic residue other than glycine (G) at the position corresponding to 562;

[0296] a hydrophilic residue other than lysine (K) at the position corresponding to 564;

[0297] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 569;

[0298] a hydrophobic residue other than alanine (A) at the position corresponding to 586;

[0299] a hydrophilic residue other than asparagine (N) at position corresponding to 641;

[0300] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 642;

[0301] a hydrophilic residue other than serine (S) at position corresponding to 647;

[0302] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 721;

[0303] a hydrophilic residue other than arginine (R) at the position corresponding to 779;

[0304] a hydrophilic residue other than lysine (K) at position corresponding to 13;

[0305] a hydrophilic residue other than lysine (K) at the position corresponding to 566;

[0306] a hydrophobic residue other than glycine (G) at the position corresponding to 554;

[0307] a hydrophobic residue other than alanine (A) at position corresponding to 35;

[0308] a hydrophilic residue other than glutamic acid (E) at the position corresponding to 110;

[0309] a hydrophobic residue other than glycine (G) at the position corresponding to 314;

[0310] a hydrophilic residue other than lysine (K) at the position corresponding to 114;

[0311] a hydrophilic residue other than aspartic acid (D) at the position corresponding to 498;

[0312] a hydrophobic residue other than isoleucine (I) at the position corresponding to 86;

[0313] a hydrophobic residue other than valine (V) at position corresponding to 57;

[0314] a hydrophilic residue other than histidine (H) at the position corresponding to 249; and

[0315] A hydrophilic residue other than arginine (R) at the position corresponding to 704.

[0316] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A59V. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G172L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T236L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T319I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H375L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H419Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T424F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises E529L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises T541L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G562Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K564M. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D569L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A586I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises N641F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D642L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises S647L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D721L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises R779I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K13R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K566R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G554H. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises E110T. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises G314Q. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises K114P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D498P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises I86P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises V57E.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises H249W. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises R704F.

[0317] In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F and A59V. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, and G172L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, and T236L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, and T319I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, and H375L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, and H419Y. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, and T424F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, and E529L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, and T541L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, and G562X. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541I, G562X, and K564M. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, and D569L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, and A586I.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, and N641F. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, and D642L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, and S647L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, and D721L. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, and R779I. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, and K13R. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, and K566R.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, and G554H. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, and E110T. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, and G314Q.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, and K114P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, and D498P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, and I86P. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, and V57E. In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, and H249W.In some embodiments, the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, H249W, and R704F.

[0318] In some embodiments, disclosed herein are sequences that are at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identical to SEQ ID NO: 3 or to SEQ ID NO: 3. NO:3 has about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% identity) to one or more (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30) substitutions. In various embodiments, SEQ ID One or more amino acids in NO:3 are substituted by naturally occurring amino acids, such as hydrophilic amino acids (e.g., polar and positively charged hydrophilic amino acids, such as arginine (R) or lysine (K); polar and neutrally charged hydrophilic amino acids, such as asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P) and cysteine ​​(C); polar and negatively charged hydrophilic amino acids, such as aspartic acid (D) or glutamic acid (E); or aromatic, polar and positively charged hydrophilic amino acids, such as histidine (H)); or hydrophobic amino acids (e.g., hydrophobic aliphatic amino acids, such as glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) or valine (V); hydrophobic aromatic amino acids, such as phenylalanine (F), tryptophan (C), threonine (T), proline (P) and cysteine ​​(C); (W) or tyrosine (Y); or non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids (such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general)).

[0319] In illustrative embodiments, substitutions of the invention include, but are not limited to, one or more (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or more) of SEQ ID NO: 3 or a sequence having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity. or about 7, or about 8, or about 9, or about 10, or about 15, or about 20, or about 30) substitutions: D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, H249W, and R704F.

[0320] In embodiments, the endonucleases disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, or fragments or variants thereof) comprise a bilobed structure. In embodiments, the endonuclease comprises two HEPN domains in a nuclease (NUC) lobe for cleaving RNA targets; and a recognition (REC) lobe comprising an α-helical domain and an N-terminal domain (NTD). In embodiments, the REC lobe binds to the CRISPR repeat sequence of the gRNA. In embodiments, the NTD comprises one or more β-sheets.

[0321] In embodiments, the endonuclease comprises a C-terminal domain (CTD) in the NUC lobe.

[0322] In embodiments, the endonuclease and / or system for targeting nucleic acid for trans-splicing comprises an inactive endonuclease (dCas). In embodiments, the dCas is a variant of Cas13e3. In embodiments, the Cas13e3 comprises R244A / H249A / R704A / H709A mutations in the HEPN domain or mutations corresponding thereto relative to SEQ ID NO:3.

[0323] In an embodiment, a miniature splicing editor is disclosed herein. In an embodiment, the miniature splicing editor is based on dCas13e3. In an embodiment, the sequence length of the miniature splicing editor is at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% shorter than SE1 (SE1 fused, linked, or associated with dPspCas13b-MCP).

[0324] In embodiments, the endonuclease (or chimeric protein) comprises domains from different endonucleases. In embodiments, the different endonucleases are Cas endonucleases. In embodiments, the domains are one or more PAM interaction domains. In embodiments, the domain is derived from one or more of Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), Cas12b (C2c1), Cas13a (C2c2), Cas13b, Cas13c, Cas13d, Cas13X / Cas13bt, Cas13Y, Cas12c (C2c3), GeoCas9, CjCas9, NmeCas9, Cas12J (CasPhi), Cas12L (CasLambda), Cas12f (Cas14), Cas12g, Cas12h, Cas12i, Cas12k, NmeCas9, Nme2Cas9, CjCas9, GeoCas9, BlatCas9, PpCas9, and Cas14. In embodiments, the domain is derived from Cas of one or more of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus, or Treponema denticola.

[0325] In embodiments, the compositions also includes one or more donor polynucleotides. In embodiments, the endonuclease is applicable to one or more donor polynucleotides being introduced into the target nucleic acid molecule. In embodiments, the donor polynucleotides include transgenic. In embodiments, the donor polynucleotides include the sequence of correction mutation. In embodiments, the donor polynucleotides have any length, such as a length between about 2 and about 10000 nucleotides (or any integer value therebetween), such as a length between about 100 and about 1000 nucleotides (or any integer therebetween), or a length between about 200 and about 500 nucleotides.

[0326] In embodiments, the endonuclease (or chimeric protein) comprises a nuclear localization signal (NLS). Examples of NLSs are provided in Kosugi et al. (J. Biol. Chem. (2009) 284:478-485; hereby incorporated by reference herein). In embodiments, the NLS comprises the consensus sequence K(K / R)X(K / R) (SEQ ID NO: 32). In embodiments, the NLS comprises the consensus sequence (K / R)(K / R)X 10-12 (K / R) 3 / 5 (SEQ ID NO:33), wherein (K / R) 3 / 5 Indicates that at least three of the five amino acids are lysine or arginine. In embodiments, the NLS comprises a c-myc NLS. In embodiments, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 34). In embodiments, the NLS is a nucleoplasmin NLS. In embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 35). In embodiments, the NLS comprises an SV40 large T antigen NLS. In embodiments, the SV40 large T antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 36). In a specific embodiment, the NLS comprises three SV40 large T antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 37)). In embodiments, the NLS is or comprises SEQ ID NO: 72. In embodiments, the NLS comprises mutations / variations in the above sequences such that they contain one or more substitutions, additions or deletions (e.g., about one, or about two, or about three, or about four, or about five, or about ten substitutions, additions or deletions).

[0327] In embodiments, the endonuclease (or chimeric protein) comprises a polypeptide penetration domain to promote cellular uptake. In embodiments, the penetration domain is a peptide, a peptide mimetic or a non-peptide carrier. In embodiments, the penetrating peptide is derived from the third alpha helix of the Drosophila melanogaster transcription factor antennapedia gene (antennapaedia) (called a membrane-penetrating peptide), and the third alpha helix comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO:38). In embodiments, the penetrating peptide comprises an HIV-1tat basic region amino acid sequence, and the amino acid sequence may include, for example, amino acids 49-57 of naturally occurring tat protein. In embodiments, the penetrating peptide is a polyarginine motif, such as the region of amino acids 34-56 of HIV-1rev protein, nine arginines, eight arginines, etc. (See, e.g., Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr;4(2):87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. USA 2000 Nov 21;97(24):13003-8; U.S. Patent Publications 2003 / 0220334; 2003 / 0083256; 2003 / 0032593; and 2003 / 0022831, herein incorporated by reference in their entireties).

[0328] In embodiments, the endonuclease (or chimeric protein) comprises a polypeptide that facilitates or is suitable for VLP delivery, including but not limited to a retroviral gag polyprotein comprising a matrix polypeptide, a capsid polypeptide, and a nucleocapsid polypeptide (optionally with one or more heterologous protease cleavage sites (e.g., a TEV cleavage site, a PreScission (a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease) cleavage site, a human rhinovirus 3C protease cleavage site, an enterokinase cleavage site, an Epstein-Barr virus (Epstein-Barr virus) cleavage site, a cleavage site of Epstein-Barr virus ... virus) protease cleavage site, cathepsin D cleavage site and / or thrombin cleavage site): matrix polypeptide and capsid polypeptide; and capsid polypeptide and nucleocapsid polypeptide, such as lentiviral gag polyprotein, such as bovine immunodeficiency virus gag polyprotein, murine leukemia virus (MLV) gag protein, simian immunodeficiency virus gag polyprotein, feline immunodeficiency virus gag polyprotein, human immunodeficiency virus gag polyprotein, equine infectious anemia virus gag polyprotein and caprine arthritis encephalitis virus gag polyprotein or gag polyprotein of alpha retrovirus, beta retrovirus, gamma retrovirus, delta retrovirus, epsilon retrovirus or foamy virus. In an embodiment, the polypeptide that promotes or is suitable for VLP delivery is co-delivered with a protease to promote the cleavage of the chimeric protein. In an embodiment, the cleavage of the chimeric protein occurs between the nuclease and the polypeptide that promotes or is suitable for VLP delivery. In an embodiment, the protease is fused to the polypeptide that promotes or is suitable for VLP delivery.

[0329] In embodiments, the endonuclease (or chimeric protein) or a delivery vehicle associated therewith (e.g., one or more lipids) comprises a polypeptide or other portion that interacts with a targeting moiety, such as an antibody or antibody-like molecule, a ligand that binds a receptor or a receptor that binds a ligand (or a fragment thereof), an aptamer, etc., for targeted delivery. In embodiments, the endonuclease (or chimeric protein) or a delivery vehicle associated therewith (e.g., one or more lipids) comprises a targeting moiety, such as an antibody or antibody-like molecule, a ligand that binds a receptor or a receptor that binds a ligand (or a fragment thereof), an aptamer, etc., for targeted delivery.

[0330] In embodiments, the endonuclease is suitable for introducing one or more excisions into the target nucleic acid molecule. In embodiments, the excision is a double-stranded DNA break in two chains of the target nucleic acid molecule. In embodiments, the excision is a nick in one or more chains of the target nucleic acid molecule.

[0331] Chimeric constructs / nucleic acid regulatory / modifying domains

[0332] In various aspects, the present disclosure provides a composition comprising a chimeric protein comprising: an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% (or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) identical to one or more of SEQ ID NOs: 1-4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications (e.g., about 1, or about 2, or about 3, or about 4). or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 modifications); and a nucleic acid regulatory domain or a nucleic acid modifying domain that comprises a sequence that comprises a catalytic domain or a fragment or variant thereof, wherein (a) and (b) do not naturally appear together in the same reading frame.

[0333] In embodiments, the endonuclease reduces or enhances non-specific degradation of transcripts. In embodiments, the endonuclease reduces or enhances incidental activities, e.g., for nucleic acid detection. In embodiments, the endonuclease reduces or enhances incidental activities, e.g., for nucleic acid detection using electrochemical methods.

[0334] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has one or more activities: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, debranching activity, transesterification activity, photolyase activity and glycosylase activity. In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is a METTL3 methyltransferase domain, a METTL3:METTL1 fusion domain or a fragment or variant thereof.

[0335] In an embodiment, the nucleic acid regulatory domain or the nucleic acid modifying domain is a nucleic acid interaction / binding domain. Non-limiting examples of nucleic acid interaction / binding domains are MCP, lambdaN, PP7, QBeta, SLBP, and TBP / TAR.

[0336] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is a splicing regulatory domain. In embodiments, the splicing regulatory domain is the RS-rich domain of SRSF1, the Gly-rich domain of hnRNP A1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1. In embodiments, the endonuclease (or chimeric protein) induces exon skipping. In embodiments, the endonuclease (or chimeric protein) induces exon inclusion.

[0337] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is a degradation domain. In embodiments, the degradation domain is an E2 ubiquitin or ubiquitin-like domain. In embodiments, the degradation domain comprises a ubiquitin core catalytic (UBC) domain. In embodiments, the degradation domain is SUMO, NEDD8, ATG8, ATG12, ISG15, UFM1, FAT10, URM1 or FUBI or a fragment or variant thereof. In embodiments, the degradation domain is UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UBE2G), UBE2G2 (UBC7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (U In one embodiment, the degradation domain is a cereblon (CRBN) E3 ligase. In one embodiment, the degradation domain is regulated by a proteolysis targeting chimera (PROTAC).

[0338] In embodiments, the degradation domain is a protease, such as a protease conditionally regulated by another molecule (eg, a protease inhibitor), such as a matrix metalloproteinase (MMP) and TIMP-1, TIMP-2, TIMP-3, or TIMP-4.

[0339] In embodiments, the degradation domain is regulated by a small molecule. In embodiments, the degradation domain is active in the presence of a small molecule. In embodiments, the degradation domain is inactive in the presence of a small molecule. In embodiments, the small molecule is an antiviral drug. In embodiments, the small molecule is one or more of the following: abscisic acid (ABA), rapamycin (or rapamycin analogs), FK506, cyclosporine A, FK1012, gibberellin 3-AM, FKCsA, AP1903 / AP20187, and auxin.

[0340] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has nuclease activity, such as the activity provided by a restriction enzyme (eg, FokI nuclease).

[0341] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has methyltransferase activity, such as the activity 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 (e.g., plants), ZMET2, CMT1, CMT2 (e.g., plants), etc.).

[0342] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has demethylase activity, such as the activity provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.).

[0343] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has deamination activity, eg, activity provided by a deaminase (eg, a cytosine deaminase, such as rat APOBEC1).

[0344] In an embodiment, the nucleic acid regulatory domain or the nucleic acid modifying domain has integrase and / or resolvase activity (e.g., Gin convertase, such as an overactive mutant of Gin convertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; etc.).

[0345] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain has recombinase activity, such as the activity provided by a recombinase (eg, the catalytic domain of Gin recombinase).

[0346] In embodiments, the nucleic acid regulatory domain or the nucleic acid modifying domain is selected from a deaminase, a reverse transcriptase, a transposase, an integrase, and a recombinase. In embodiments, the deaminase is a cytidine or cytosine deaminase or a fragment or variant thereof. In embodiments, the cytidine or cytosine deaminase is selected from AID, CDA1, and APOBEC or a fragment or variant thereof. In embodiments, the APOBEC is selected from A3A, AB3, APOBEC1, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H or a fragment or variant thereof. In embodiments, the APOBEC has an amino acid sequence of one of SEQ ID NO:39 [A3A], SEQ ID NO:40 [AB3], SEQ ID NO:41 [APOBEC1], SEQ ID NO:42 [APOBEC3C], SEQ ID NO:43 [APOBEC3D], SEQ ID NO:44 [APOBEC3F], SEQ ID NO:45 [APOBEC3G], and SEQ ID NO:46 [APOBEC3H], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto. In an embodiment, the APOBEC has an amino acid sequence selected from one of SEQ ID NOs: 32-39 as shown in Table 5 below.

[0347] Table 5: APOBEC amino acid sequences

[0348]

[0349]

[0350] In an embodiment, the deaminase is a DNA-specific adenine or adenosine deaminase or an adenosine base editor (ABE) or a fragment or variant thereof.

[0351] In embodiments, the ABE is generated by replacing the APOBEC1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In embodiments, the ABE comprises an evolved TadA variant. In embodiments, the ABE is ABE 1.2 (TadA*-XTEN-nCas9-NLS). In some embodiments, TadA* comprises A106V and D108N mutations. In embodiments, the ABE is a second-generation ABE. In embodiments, the ABE is ABE2.1, which comprises the additional mutations D147Y and E155V in TadA* (TadA*2.1). In embodiments, the ABE is ABE2.2, which is a fusion of ABE2.1 with a catalytically inactive form of human alkyladenine DNA glycosylase (AAG with an E125Q mutation). In embodiments, the ABE is ABE2.3, which is a fusion of ABE2.1 with a catalytically inactivated form of E. coli Endo V (inactivated with a D35A mutation). In embodiments, the ABE is ABE2.6, which has a linker twice as long as the linker in ABE2.1 (32 amino acids, (SGGS)2-XTEN-(SGGS)2 ("(SGGS)2," as disclosed in SEQ ID NO:47). In embodiments, the ABE is ABE2.7, which is ABE2.1 tethered to an additional wild-type TadA monomer. In embodiments, the ABE is ABE2.8, which is ABE2.1 tethered to an additional TadA*2.1 monomer. In embodiments, the ABE is ABE2.9, which is a direct fusion of an evolved TadA (TadA*2.1) to the N-terminus of ABE2.1. In embodiments, the ABE is ABE2.10, which is a direct fusion of wil...

Claims

1. A composition comprising an endonuclease comprising a sequence, optionally comprising a higher eukaryotic and prokaryotic nucleotide binding domain (HEPN) domain, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications.

2. A composition comprising a nucleic acid encoding a nuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications.

3. A composition comprising a nuclease system, the nuclease system comprising (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications; and (b) An RNA molecule comprising a sequence complementary to one strand of the target nucleic acid molecule.

4. The composition of any one of claims 1-3, further comprising one or more donor polynucleotides.

5. The composition of any one of claims 1 to 4, wherein the endonuclease is suitable for introducing one or more donor polynucleotides into a target nucleic acid molecule.

6. The composition of any one of claims 1 to 5, wherein the endonuclease is suitable for introducing one or more excisions into a target nucleic acid molecule.

7. A composition comprising a chimeric protein comprising: (a) an endonuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications; and (b) a nucleic acid regulatory domain or a nucleic acid modifying domain comprising a sequence comprising a catalytic domain or a fragment or variant thereof, Where (a) and (b) do not naturally appear together in the same reading frame.

8. The composition of claim 7, wherein the nucleic acid regulatory domain or the nucleic acid modifying domain is selected from the group consisting of MCP, lambdaN, PP7, QBeta, SLBP, and TBP / TAR.

9. The composition of any one of claims 1-8, wherein the endonuclease reduces or enhances incidental activities for nucleic acid detection.

10. A composition comprising a complex comprising a chimeric protein and an RNA molecule, wherein the chimeric protein comprises (a) an endonuclease, optionally comprising a sequence comprising a HEPN domain, or a fragment or variant thereof, and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications; and (b) a nucleic acid regulatory domain or a nucleic acid modifying domain comprising a sequence comprising a catalytic domain or a fragment or variant thereof, where (a) and (b) do not naturally occur together in the same reading frame and The RNA molecule comprises a sequence that is complementary to one strand of the target nucleic acid molecule.

11. The composition of any one of claims 7 to 10, wherein the nucleic acid regulatory domain or the nucleic acid modification domain has one or more activities: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, debranching activity, transesterification activity, photolyase activity, and glycosylase activity.

12. The composition of any one of claims 7-11, wherein the nucleic acid regulatory domain or the nucleic acid modifying domain is a methyltransferase-like protein 3 (METTL3) methyltransferase domain, a METTL3:methyltransferase-like protein 1 (METTL1) fusion, or a fragment or variant thereof.

13. The composition of any one of claims 7 to 12, wherein the nucleic acid regulatory domain or the nucleic acid modifying domain is selected from the group consisting of a deaminase, a reverse transcriptase, a transposase, an integrase, and a recombinase.

14. The composition of claims 7-13, wherein the deaminase is a cytidine or cytosine deaminase or a fragment or variant thereof.

15. The composition of claim 14, wherein the cytidine or cytosine deaminase is selected from activation-induced cytidine deaminase (AID), cytidine deaminase 1 (CDA1) and apolipoprotein B mRNA editing complex (APOBEC) or a fragment or variant thereof.

16. The composition of claim 15, wherein the APOBEC is selected from A3A, AB3, APOBEC1, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H, or fragments or variants thereof.

17. The composition of claim 15 or 16, wherein the APOBEC has an amino acid sequence of one of SEQ ID NO:39 [A3A], SEQ ID NO:40 [AB3], SEQ ID NO:41 [APOBEC1], SEQ ID NO:42 [APOBEC3C], SEQ ID NO:43 [APOBEC3D], SEQ ID NO:44 [APOBEC3F], SEQ ID NO:45 [APOBEC3G], and SEQ ID NO:46 [APOBEC3H], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

18. The composition of any one of claims 7-17, wherein the deaminase is a DNA-specific adenine or adenosine deaminase or a fragment or variant thereof.

19. The composition of claim 18, wherein the DNA-specific adenine or adenosine deaminase is selected from tRNA-specific adenosine deaminase 7.10 (TadA7.10), tRNA-specific adenosine deaminase 6.3 (TadA 6.3), tRNA-specific adenosine deaminase 7.8 (TadA7.8), tRNA-specific adenosine deaminase 7.9 (TadA 7.9) and tRNA-specific adenosine deaminase 8e (TadA8e (TadA-8e V106W)) or a fragment or variant thereof.

20. The composition of claim 19, wherein the tRNA-specific adenosine deaminase has an amino acid sequence of one of SEQ ID NO: 48 [TadA 7.10], SEQ ID NO: 49 [TadA 6.3], SEQ ID NO: 50 [TadA 7.8], SEQ ID NO: 51 [TadA 7.9], and SEQ ID NO: 52 [TadA 8e], or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

21. The composition of any one of claims 7-17, wherein the deaminase is an RNA-specific adenine or adenosine deaminase or a fragment or variant thereof.

22. The composition of claim 21, wherein the RNA-specific adenine or adenosine deaminase is an adenosine deaminase acting on RNA (ADAR) enzyme or a fragment or variant thereof.

23. The composition of claim 22, wherein the ADAR is selected from ADAR1, ADAR2, and ADAR3, or fragments or variants thereof.

24. A composition as described in claim 23, wherein the ADAR has the amino acid sequence of one of SEQ ID NO:53 [ADAR1] and SEQ ID NO:54 [ADAR2] or a fragment or variant thereof, or an amino acid sequence having at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

25. The composition of any one of claims 11-24, wherein the deaminase further comprises a nuclear localization signal.

26. The composition of any one of claims 1-25, wherein the endonuclease further comprises a uracil glycosylase inhibitor (UGI) or a fragment or variant thereof.

27. The composition of any one of claims 1-26, wherein the RNA molecule is a guide RNA (gRNA).

28. The composition of claim 27, wherein the gRNA comprises a sequence that interacts with the endonuclease.

29. The composition of any one of claims 1-28, wherein the endonuclease forms a complex with the gRNA.

30. The composition of any one of claims 1-29, wherein the composition is suitable for base editing.

31. The composition of any one of claims 1-30, wherein the composition is suitable for DNA base editing.

32. The composition of any one of claims 1-30, wherein the composition is suitable for RNA base editing.

33. The composition of any one of claims 1-32, wherein the composition is suitable for catalyzing a C>T nucleotide conversion or an A>G nucleotide conversion in a target nucleic acid.

34. The composition of any one of claims 1-33, wherein the composition comprises both adenosine deaminase and cytidine deaminase.

35. The composition of any one of claims 1-34, wherein the composition is suitable for dual base editing.

36. The composition of any one of claims 13-35, wherein the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase (M-MLV RT) or M-MLV RT (D200N / L603W / T330P / T306K / W313F) or a fragment or variant thereof.

37. The composition of claim 36, wherein the M-MLV RT has the amino acid sequence of SEQ ID NO: 55 [M-MLV RT] or SEQ ID NO: 56 [M-MLV RT (D200N / L603W / T330P / T306K / W313F)], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

38. The composition of any one of claims 1-37, wherein the composition further comprises a dominant negative human MutL homolog (MLH1).

39. The composition of any one of claims 1-38, wherein the composition is suitable for use with dominant-negative MLH1.

40. The composition of any one of claims 1-39, wherein the RNA molecule is or comprises a prime editing guide RNA (pegRNA).

41. The composition of any one of claims 1-40, wherein the endonuclease forms a complex with the pegRNA.

42. The composition of claim 40 or 41, wherein the pegRNA is used as a template for transcribing a new DNA sequence.

43. The composition of any one of claims 40-42, wherein the pegRNA binds to the DNA strand opposite to the typical gRNA binding site.

44. The composition of any one of claims 40-43, wherein the pegRNA comprises a gRNA containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence.

45. The composition of any one of claims 1-44, wherein the RNA molecule is or comprises a gRNA.

46. ​​The composition of claim 45, wherein the gRNA comprises a sequence that interacts with the endonuclease.

47. The composition of any one of claims 1-46, wherein the endonuclease forms a complex with the gRNA.

48. The composition of any one of claims 1-47, wherein the composition comprises both gRNA and pegRNA.

49. The composition of any one of claims 1-48, wherein the composition is suitable for lead editing.

50. The composition of any one of claims 11-49, wherein the transposase is selected from the group consisting of Tnl, Tn2, Tn3, Tn5, Tn7, Tn9, TnlO, Tn552, Tn903, TnlOO / γ-δ, Tn / O, tnsA, tnsB, tnsC, tniQ, ISlO, ISS, IS911, Minos, Sleeping Beauty, piggyBac, Tol2, Mosl, Himarl, Hermes, Tol2, Minos, Tel, P-element, MuA, Tyl, Chapaev, transib, Tc1 / mariner, and Tc3 donor DNA systems.

51. The composition of claim 50, wherein the transposase is a transposon 7-like (Tn7-like) transposon system or a fragment or variant thereof.

52. The composition of claim 50, wherein the transposase is one or more of transposon 7 protein A (TnsA), transposon 7 protein B (Tns B), transposon 7 protein C (Tns C), and integron transposase protein Q (TniQ), or a fragment or variant thereof.

53. The composition of claim 51, wherein the Tn7-like transposon system is derived from Vibrio cholerae Tn6677.

54. The composition of claim 52, wherein the transposase has the amino acid sequence of one or more of SEQ ID NO: 57 [TnsA], SEQ ID NO: 58 [TnsB], SEQ ID NO: 59 [TnsC], and SEQ ID NO: 60 [TniQ], or a fragment or variant thereof, or an amino acid sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical thereto.

55. The composition of claim 13, wherein the integrase is a serine recombinase or a fragment or variant thereof.

56. The composition of claim 55, wherein the serine recombinase is Bxb1 or a fragment or variant thereof.

57. The composition of claim 13, wherein the recombinase is a Gln convertase or a Tn3 resolvase or a fragment or variant thereof.

58. The composition of any one of claims 1-57, wherein the nucleic acid regulatory domain or the nucleic acid modifying domain comprises one or more mutations to reduce activity relative to an unmutated form.

59. The composition of any one of claims 1-58, wherein the nucleic acid regulatory domain or the nucleic acid modifying domain comprises one or more mutations to increase activity relative to an unmutated form.

60. The composition of any one of claims 1-59, wherein the sequence of (a) is positioned at the N-terminus of the chimeric protein and the sequence of (b) is positioned at the C-terminus of the chimeric protein.

61. The composition of any one of claims 1-60, wherein the sequence of (a) is positioned at the C-terminus of the chimeric protein and the sequence of (b) is positioned at the N-terminus of the chimeric protein.

62. The composition of any one of claims 1-61, further comprising a linker connecting the sequence of (a) and the sequence of (b).

63. The composition of claim 62, wherein the linker is between about 4 and about 40 amino acids, or about 10 and about 40 amino acids, or about 20 and about 40 amino acids, or about 30 and about 40 amino acids, or about 4 and about 30 amino acids, or about 4 and about 20 amino acids, or about 4 and about 10 amino acids, or about 5 amino acids, or about 10 amino acids, or about 15 amino acids, or about 20 amino acids, or about 25 amino acids, or about 30 amino acids, or about 35 amino acids, or about 40 amino acids.

64. The composition of claim 62 or 63, wherein the linker comprises substantially glycine and serine residues.

65. The composition of claim 64, wherein the linker is (GGS) n , where n is 1, or 2, or 3, or 4, or 5.

66. The composition of claim 64, wherein the linker is GGSGGSGGSG (SEQ ID NO: 61), GGSGGSGGGGSGGGGS (SEQ ID NO: 62), GGGGS (SEQ ID NO: 63), GGS (SEQ ID NO: 64), (GGGGS) n (n=1-4) (SEQ ID NO:65), (Gly)8 (SEQ ID NO:66), (Gly)6 (SEQ ID NO:67), (EAAAK) n (n=1-3)(SEQ ID NO:68), A(EAAAK) n A(n=2-5) (SEQ ID NO:69), AEAAAKEAAAKA (SEQ ID NO:70), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:71), PAPAP (SEQ ID NO:72), KESGSVSSEQLAQFRSLD (SEQ ID NO:73), EGKSSGSGSESKST (SEQ ID NO:74) and GSAGSAAGSGEF (SEQ ID NO:75) or variants thereof, wherein the variant comprises about 1, or about 2, or about 3, or about 4, or about 5 mutations selected from substitutions or deletions.

67. The composition of any one of claims 1-66, wherein the endonuclease is suitable for producing double-strand breaks in nucleic acids.

68. The composition of any one of claims 1-67, wherein the endonuclease is suitable for producing a nick in a nucleic acid.

69. The composition of any one of claims 1-68, wherein the endonuclease is suitable for nucleic acid modification by homology directed repair (HDR).

70. The composition of any one of claims 1-69, wherein the endonuclease is suitable for nucleic acid modification by non-homologous end joining (NHEJ).

71. The composition of any one of claims 1-70, wherein the endonuclease recognizes a protospacer adjacent motif (PAM).

72. The composition of any one of claims 1-71, wherein the endonuclease recognizes multiple PAMs.

73. The composition of any one of claims 1-72, wherein the endonuclease comprises one or more mutations to reduce catalytic activity relative to the unmutated form.

74. The composition of any one of claims 1-73, wherein the endonuclease comprises one or more mutations rendering the endonuclease substantially catalytically inactive relative to the unmutated form.

75. The composition of any one of claims 1-74, wherein the endonuclease comprises one or more mutations to increase catalytic activity relative to the unmutated form.

76. The composition of any one of claims 1-75, wherein the endonuclease comprises one or more mutations such that the endonuclease is substantially catalytically hyperactive relative to an unmutated form.

77. The composition of any one of claims 1-76, wherein the endonuclease has nickase activity.

78. The composition of any one of claims 1-77, wherein the endonuclease comprises one or more mutations to confer nickase activity.

79. The composition of any one of claims 1-78, wherein the endonuclease has incidental cleavage activity.

80. The composition of any one of claims 1-79, wherein the endonuclease comprises one or more mutations to confer incidental cleavage activity.

81. The composition of any one of claims 1-80, wherein the endonuclease is at least about 75% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

82. The composition of any one of claims 1-81, wherein the endonuclease is at least about 80% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

83. The composition of any one of claims 1-82, wherein the endonuclease is at least about 85% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

84. The composition of any one of claims 1-83, wherein the endonuclease is at least about 90% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

85. The composition of any one of claims 1-84, wherein the endonuclease is at least about 95% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

86. The composition of any one of claims 1-85, wherein the endonuclease is at least about 97% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

87. The composition of any one of claims 1-86, wherein the endonuclease is at least about 99% identical to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89.

88. The composition of any one of claims 1-87, wherein the endonuclease has about 1 to about 15 amino acid modifications.

89. The composition of any one of claims 1-88, wherein the endonuclease has about 1 to about 10 amino acid modifications.

90. The composition of any one of claims 1-89, wherein the endonuclease has about 1 to about 5 amino acid modifications.

91. The composition of any one of claims 1-90, wherein the endonuclease has about 1, or about 2, or about 3, or about 4, or about 5, or about 10, or about 15, or about 20 amino acid modifications.

92. The composition of any one of claims 1-91, wherein the amino acid modification is selected from the group consisting of substitution and deletion.

93. The composition of any one of claims 1 to 92, wherein the endonuclease comprises a domain from a different endonuclease, optionally from a Cas endonuclease.

94. The composition of any one of claims 1-93, wherein the endonuclease comprises one or two HEPN domains or a truncated form thereof.

95. The composition of any one of claims 1-94, wherein the domain is a PAM interaction domain.

96. The composition of any one of claims 1-95, wherein the target nucleic acid is or comprises single-stranded RNA (ssRNA).

97. The composition of any one of claims 1-95, wherein the target nucleic acid is or comprises double-stranded RNA (dsRNA).

98. The composition of any one of claims 1-95, wherein the target nucleic acid is or comprises single-stranded DNA (ssDNA).

99. The composition of any one of claims 1-95, wherein the target nucleic acid is or comprises double-stranded DNA (dsDNA).

100. The composition of any one of claims 1-99, wherein the target nucleic acid is about 2 to about 6 nucleotides upstream of a PAM sequence.

101. The composition of any one of claims 1-100, wherein the RNA molecule is or comprises a guide RNA structure configured to form a complex with the endonuclease.

102. The composition of claim 101, wherein the guide RNA structure: (i) comprising (a) a CRISPR RNA (crRNA) suitable for hybridizing to a target nucleic acid molecule and / or (b) a transactivating CRISPR RNA (tracrRNA) suitable for interacting with said endonuclease, or (ii) lacking (a) a crRNA suitable for hybridizing to a target nucleic acid molecule and / or (b) a tracrRNA suitable for interacting with the endonuclease.

103. The composition of any one of claims 1 to 102, wherein the RNA molecule is or comprises a single gRNA.

104. The composition of any one of claims 1-103, wherein the gRNA comprises a sequence that interacts with the endonuclease.

105. The composition of any one of claims 1-104, wherein the endonuclease forms a complex with the gRNA.

106. The composition of any one of claims 1-105, wherein the RNA molecule is or comprises a nucleic acid sequence of SEQ ID NOs: 28-31 and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, or a nucleic acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identical thereto.

107. The composition of any one of claims 1-106, wherein the RNA molecule has perfect sequence complementarity with one strand of a target nucleic acid molecule.

108. The composition of any one of claims 1-106, wherein the RNA molecule has partial sequence complementarity with a strand of a target nucleic acid molecule.

109. The composition of any one of claims 1-108, further comprising a viral vector or a non-viral vector.

110. The composition of claim 109, wherein the viral vector is or comprises AAV, optionally wherein the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

111. The composition of any one of claims 1-108, wherein the endonuclease mediates a trans-splicing event.

112. The composition of any one of claims 1-108, wherein the endonuclease mediates exon skipping or exon inclusion events.

113. The composition of any one of claims 1-108, further comprising lipid nanoparticle (LNP) liposomes, lipid complexes, or polymer nanoparticles.

114. The composition of claim 113, wherein the LNP comprises one or more of an ionizable lipid, an amino lipid, an anionic lipid, a neutral lipid, an amphiphilic lipid, a helper lipid, a structural lipid, a PEG lipid, and a lipid.

115. A nucleic acid encoding the endonuclease or chimeric protein of any one of claims 1-114.

116. The nucleic acid of claim 115, wherein the nucleic acid is or comprises a DNA molecule or an RNA molecule.

117. The nucleic acid of claim 116, wherein the RNA is or comprises mRNA or modified mRNA (mmRNA).

118. The nucleic acid of claim 116, wherein the DNA is or comprises a vector or a plasmid.

119. The nucleic acid of any one of claims 115 to 118, wherein the nucleic acid comprises a codon-optimized sequence.

120. The nucleic acid of any one of claims 115-119, wherein the nucleic acid comprises one or more modifications.

121. The nucleic acid of claim 120, wherein the modification is one or more of a base modification and a backbone modification.

122. A viral vector comprising the nucleic acid of any one of claims 115-121.

123. The viral vector of claim 122, wherein the viral vector is or comprises AAV or virus-like particles (VLPs).

124. The viral vector of claim 123, wherein the AAV is or comprises one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2 / 1, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV3 / 1, AAV3 / 5, AAV3 / 8, and AAV3 / 9.

125. A lipid nanoparticle comprising the nucleic acid of any one of claims 115-121.

126. A cell comprising the nucleic acid of claim 115, the viral vector of claim 122, or the lipid nanoparticle of claim 125.

127. The cell of claim 126, wherein the cell is a prokaryotic cell.

128. The cell of claim 126, wherein the cell is a eukaryotic cell.

129. The cell of claim 126, wherein the cell is a mammalian cell.

130. The cell of claim 126, wherein the cell is a human cell.

131. The cell of claim 126, wherein the cell is an immortalized cell.

132. The cell of claim 126, wherein the cell is harvested from a subject.

133. A pharmaceutical composition comprising the composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, or the cell of any one of claims 126-132, and a pharmaceutically acceptable carrier.

134. A composition comprising an RNA molecule comprising a nucleic acid sequence of SEQ ID NOs: 28-31 and / or SEQ ID NOs: 90-97, or a fragment or variant thereof, or a nucleic acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identical thereto.

135. The composition of claim 134, wherein the RNA molecule interacts with an endonuclease comprising a sequence optionally comprising at least one HEPN domain or a fragment or variant thereof and having at least about 70% identity to one or more of SEQ ID NOs: 3 or 1, 2 or 4 and / or SEQ ID NOs: 80-89, or having about 1 to about 20 amino acid modifications.

136. The composition of claim 134 or 135, wherein the RNA molecule comprises one or more modifications.

137. The composition of any one of claims 134-136, wherein the modification is one or more of a base modification and a backbone modification.

138. The composition of any one of claims 134-137, wherein the RNA molecule comprises a sequence complementary to one strand of a target nucleic acid molecule.

139. The composition of any one of claims 134-138, wherein the RNA molecule has perfect sequence complementarity with one strand of a target nucleic acid molecule.

140. The composition of any one of claims 134-137, wherein the RNA molecule has partial sequence complementarity with a strand of a target nucleic acid molecule.

141. A composition comprising a nucleic acid encoding a nuclease comprising a sequence, optionally comprising a HEPN domain, or a fragment or variant thereof, and an RNA comprising a repeat sequence at least about 70% identical to one or more of SEQ ID NOs: 28-31 and / or SEQ ID NOs: 90-97.

142. A kit comprising a container, the kit comprising a composition of any one of claims 1-114, a nucleic acid of any one of claims 115-121, a viral vector of any one of claims 122-124, a lipid nanoparticle of claim 125, a cell of any one of claims 126-132, or a pharmaceutical composition of claim 133, and instructions for modulating and / or modifying the nucleic acid.

143. A method of regulating and / or modifying a nucleic acid in a cell, the method comprising contacting the cell with a composition as described in any one of claims 1-114, a nucleic acid as described in any one of claims 115-121, a viral vector as described in any one of claims 122-124, a lipid nanoparticle as described in claim 125, a cell as described in any one of claims 126-132, or a pharmaceutical composition as described in claim 133.

144. A method of regulating and / or modifying a nucleic acid in a subject in need thereof, the method comprising administering to the subject an effective amount of a cell and a composition as described in any one of claims 1-114, a nucleic acid as described in any one of claims 115-121, a viral vector as described in any one of claims 122-124, a lipid nanoparticle as described in claim 125, a cell as described in any one of claims 126-132, or a pharmaceutical composition as described in claim 133.

145. The method of claim 144, wherein the modulation and / or modification is selected from one or more of cleaving, cleaving, methylating, marking and mutating the nucleic acid.

146. The method of claim 144 or 145, wherein the regulation and / or modification is selected from one or more of: cleaving the nucleic acid; inserting the nucleic acid; editing the nucleic acid; regulating the transcription of the nucleic acid; isolating the nucleic acid; binding the nucleic acid; and imaging the nucleic acid.

147. A method of disrupting, correcting and / or replacing a gene in a cell, the method comprising contacting the cell with a composition as described in any one of claims 1-114, a nucleic acid as described in any one of claims 115-121, a viral vector as described in any one of claims 122-124, a lipid nanoparticle as described in claim 125, a cell as described in any one of claims 126-132, or a pharmaceutical composition as described in claim 133.

148. A method of disrupting, correcting and / or replacing a gene in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, the cell of any one of claims 126-132, or the pharmaceutical composition of claim 133.

149. A method for treating, ameliorating or preventing a disease or condition in a subject, the method comprising (a) contacting a cell with a composition according to any one of claims 1 to 114, a nucleic acid according to any one of claims 115 to 121, a viral vector according to any one of claims 122 to 124, a lipid nanoparticle according to claim 125, a cell according to any one of claims 126 to 132, or a pharmaceutical composition according to claim 133, and (b) administering an effective amount of the cells to the subject.

150. A method for treating, ameliorating or preventing a disease or condition in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, the cell of any one of claims 126-132, or the pharmaceutical composition of claim 133.

151. The composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, the cell of any one of claims 126-132, or the pharmaceutical composition of claim 133, for treating, ameliorating, or preventing a disease or condition in a patient.

152. Use of the composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, the cell of any one of claims 126-132, or the pharmaceutical composition of claim 133 in the manufacture of a medicament for treating, ameliorating or preventing a disease or disorder.

153. A method of detecting and / or quantifying nucleic acids in a sample, the method comprising contacting the sample with a composition as described in any one of claims 1-114.

154. The method of claim 153, wherein the nucleic acid is a target and / or reporter nucleic acid.

155. The method of claim 153 or 154, wherein the method comprises detecting a reporter signal generated upon endonuclease cleavage.

156. The method of claim 155, wherein the reporter signal is a fluorescent signal.

157. The method of claim 153, wherein the endonuclease has incidental cleavage activity.

158. A system for targeting a nucleic acid for trans-splicing, the system comprising: (a) the endonuclease of any one of claims 1 to 108 and, optionally, an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide associated with the endonuclease; and (c) a trans-splicing nucleic acid template, the template comprising: (i) a splice donor and / or a splice acceptor, and (ii) an RNA sequence that binds to the RNA-binding polypeptide.

159. The system of claim 158, wherein the RNA molecule is a gRNA.

160. The system of claim 158 or 159, wherein the endonuclease is linked to, associated with, and / or fused to the RNA binding protein.

161. The system of any one of claims 158-160, wherein the RNA binding protein is a viral protein.

162. The system of any one of claims 158-161, wherein the RNA binding protein is a MS binding protein.

163. The system of any one of claims 158-161, wherein the RNA binding protein is PP7 coat protein.

164. The system of any one of claims 158-161, wherein the RNA binding protein is PRR1.

165. The system of any one of claims 158-161, wherein the RNA binding protein is HgaII.

166. The system of any one of claims 158-161, wherein the RNA binding protein is Qbeta coat protein.

167. The system of any one of claims 158-161, wherein the RNA binding protein is IN protein.

168. The system of any one of claims 158-161, wherein the RNA binding protein is M protein.

169. A method for targeted trans-splicing of pre-mRNA in a cell, the method comprising contacting the cell with the system of any one of claims 158-167.

170. A method of treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, the method comprising: An effective amount of the composition of any one of claims 1-114, the nucleic acid of any one of claims 115-121, the viral vector of any one of claims 122-124, the lipid nanoparticle of claim 125, the cell of any one of claims 126-132, the pharmaceutical composition of claim 133, or the trans-splicing system of any one of claims 158-167 is administered to the subject.

171. A method for treating, ameliorating or preventing Usher syndrome or a symptom thereof in a subject in need thereof, the method comprising: (a) contacting a cell with a composition according to any one of claims 1 to 114, a nucleic acid according to any one of claims 115 to 121, a viral vector according to any one of claims 122 to 124, a lipid nanoparticle according to claim 125, a cell according to any one of claims 126 to 132, a pharmaceutical composition according to claim 133, or a trans-splicing system according to any one of claims 158 to 169, and (b) administering an effective amount of the cells to the subject.

172. The method of any one of claims 170-171, wherein the cell is derived from the subject.

173. The method of any one of claims 170-172, wherein the Usher syndrome is selected from Usher syndrome type I, Usher syndrome type II, or Usher syndrome type III.

174. The method of any one of claims 170-172, wherein the Usher syndrome is Usher syndrome type I.

175. The method of any one of claims 170-172, wherein the Usher syndrome is Usher syndrome type II.

176. The method of any one of claims 170-172, wherein the Usher syndrome is Usher syndrome type III.

177. The method of any one of claims 171-176, wherein the method targets one or more Usher syndrome-associated genes.

178. The method of claim 177, wherein the method targets one or more genes selected from the group consisting of CDH23, MY07A, PCDH15, USH1C, USH1G, USH2A, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

179. The method of claim 178, wherein the method targets one or more of USH2A, GPR98, and DFNB31.

180. The method of claim 179, wherein the method targets USH2A.

181. The method of any one of claims 171-180, wherein the method corrects a mutation or defect in one or more Usher syndrome-associated genes.

182. The method of claim 181, wherein the method corrects a mutation or defect in one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

183. The method of claim 182, wherein the method corrects a mutation or deficiency in one or more of USH2A, GPR98, and DFNB31.

184. The method of claim 183, wherein the method corrects a mutation or defect in USH2A.

185. The method of any one of claims 171-184, wherein the method results in trans-splicing of one or more genes selected from the group consisting of: USH2A, CDH23, MY07A, PCDH15, USH1C, USH1G, ADGRV1, WHRN, GPR98, DFNB31, and CLRN1.

186. The method of any one of claims 171-185, wherein the method causes trans-splicing of one or more of USH2A, GPR98, and DFNB31.

187. The method of any one of claims 171-186, wherein the method results in trans-splicing of USH2A.

188. The method of any one of claims 170-187, wherein the method treats, ameliorates, or prevents one or more symptoms of retinitis pigmentosa.

189. The method of any one of claims 170-188, wherein the method treats, ameliorates, or prevents hearing loss or impairment.

190. The method of any one of claims 170-189, wherein the method treats, ameliorates, or prevents vision loss or impairment.

191. The method of any one of claims 170-190, wherein the method treats, ameliorates, or prevents one or more of night blindness and loss or reduction of peripheral vision.

192. A system for targeting a nucleic acid for trans-splicing, the system comprising: (a) dPspCas13b and an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide associated with an endonuclease, the RNA-binding polypeptide selected from one or more of the following: PP7, M, PRR1, HgaII, or Qbeta coat protein; and (c) a trans-splicing template, comprising: (i) a splice donor and / or a splice acceptor, and (ii) an RNA sequence that binds to the RNA-binding polypeptide.

193. The system of claim 192, wherein the RNA molecule is a gRNA.

194. The system of claim 192 or 193, wherein the endonuclease is linked to, associated with, and / or fused to the RNA binding protein.

195. A method for targeted trans-splicing of pre-mRNA in a cell, the method comprising contacting the cell with the system of any one of claims 192-194.

196. The composition of claim 111, or the trans-splicing system of any one of claims 158-167, further comprising a repair RNA (repRNA) sequence comprising: (a) one or more exons and / or introns; (b) a splice donor and / or a splice acceptor, The repRNA is suitable for trans-splicing.

197. The composition of claim 196, wherein the trans-splicing system comprises a splice donor, a splice acceptor, and replaces an internal exon.

198. The composition of claim 197, wherein the repRNA is operably linked to the RNA molecule comprising a sequence complementary to a strand of a target nucleic acid molecule or the gRNA.

199. A system for targeting a nucleic acid for trans-splicing, the system comprising: (a) the endonuclease of any one of claims 1 to 108 and, optionally, an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; (b) an RNA-binding polypeptide associated with the endonuclease; and (c) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; (ii) splice donor and / or splice acceptor.

200. The system of claim 199, wherein the RNA molecule is a gRNA.

201. The system of claim 199 or 200, wherein the endonuclease is not linked to, associated with, and / or fused to an RNA binding protein.

202. The composition or system of any one of claims 196-201, wherein the repRNA is not operably linked to one or more gRNAs.

203. The composition or system of any one of claims 196-201, wherein the repRNA is provided to one or more gRNAs in trans.

204. The composition or system of any one of claims 196-203, wherein the repRNA further comprises a ribozyme site.

205. The composition or system of claim 204, wherein the ribozyme site is a hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), or glmS ribozyme site, or a variant thereof.

206. The composition or system of any one of claims 199-205, wherein the ribozyme site is an HDV ribozyme site.

207. The composition or system of any one of claims 204-206, wherein the ribozyme site is upstream of one or more exons and / or introns of the repRNA.

208. A system for targeting a nucleic acid for trans-splicing, the system comprising: (a) the endonuclease of any one of claims 1 to 108 and an RNA molecule comprising a sequence complementary to one strand of a target nucleic acid molecule; and (b) a repair RNA (repRNA) sequence comprising: (i) one or more exons and / or introns; (ii) splice donor and / or splice acceptor.

209. The system of claim 208, wherein the RNA molecule is a gRNA.

210. The system of claim 208 or 209, wherein the endonuclease is not linked to, associated with, and / or fused to an RNA binding protein.

211. The composition or system of any one of claims 208-210, wherein the repRNA is operably linked to one or more gRNAs.

212. A composition comprising an endonuclease and having an amino acid sequence that is at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO:

3.

213. The composition of claim 212, having one or more substitutions at positions corresponding to D38X, A59X, G172X, T236X, T319X, H375X, H419X, T424X, E529X, T541X, G562X, K564X, D569X, A586X, N641X, D642X, S647X, D721X, R779X, K13X, K566X, G554X, A35X, E110X, G314X, K114X, D498X, I86X, V57X, H249X, R704X of SEQ ID NO: 3, wherein the substitution is defined by X and wherein X is any amino acid.

214. The composition of claim 213, wherein X is an essential or non-essential amino acid.

215. The composition of claim 213 or claim 214, wherein X is a hydrophilic or hydrophobic amino acid.

216. The composition of claim 215, wherein X is a hydrophilic amino acid.

217. The composition of claim 216, wherein X is a polar and positively charged hydrophilic amino acid.

218. The composition of claim 217, wherein X is selected from arginine (R) or lysine (K).

219. The composition of claim 216, wherein X is a polar and neutrally charged hydrophilic amino acid.

220. The composition of claim 219, wherein X is selected from the group consisting of asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

221. The composition of claim 216, wherein X is a polar and negatively charged hydrophilic amino acid.

222. The composition of claim 221, wherein X is selected from aspartic acid (D) or glutamic acid (E).

223. The composition of claim 216, wherein X is an aromatic, polar, positively charged, hydrophilic amino acid.

224. The composition of claim 223, wherein X is histidine (H).

225. The composition of claim 215, wherein X is a hydrophobic amino acid.

226. The composition of claim 225, wherein X is a hydrophobic, aliphatic amino acid.

227. The composition of claim 226, wherein X is selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), or valine (V).

228. The composition of claim 225, wherein X is a hydrophobic, aromatic amino acid.

229. The composition of claim 228, wherein X is selected from phenylalanine (F), tryptophan (W), or tyrosine (Y).

230. The composition of any one of claims 212-229, comprising one or more of the following substitutions: a hydrophilic residue other than aspartic acid (D) at position corresponding to 38; a hydrophobic residue other than alanine (A) at position corresponding to 59; a hydrophobic residue other than glycine (G) at the position corresponding to 172; a hydrophilic residue other than threonine (T) at position corresponding to 236; a hydrophilic residue other than threonine (T) at the position corresponding to 319; a hydrophilic residue other than histidine (H) at the position corresponding to 375; a hydrophilic residue other than histidine (H) at the position corresponding to 419; a hydrophilic residue other than threonine (T) at the position corresponding to 424; a hydrophilic residue other than glutamic acid (E) at the position corresponding to 529; a hydrophilic residue other than threonine (T) at the position corresponding to 541; a hydrophobic residue other than glycine (G) at the position corresponding to 562; a hydrophilic residue other than lysine (K) at the position corresponding to 564; a hydrophilic residue other than aspartic acid (D) at the position corresponding to 569; a hydrophobic residue other than alanine (A) at the position corresponding to 586; a hydrophilic residue other than asparagine (N) at position corresponding to 641; a hydrophilic residue other than aspartic acid (D) at the position corresponding to 642; a hydrophilic residue other than serine (S) at position corresponding to 647; a hydrophilic residue other than aspartic acid (D) at the position corresponding to 721; a hydrophilic residue other than arginine (R) at the position corresponding to 779; a hydrophilic residue other than lysine (K) at position corresponding to 13; a hydrophilic residue other than lysine (K) at the position corresponding to 566; a hydrophobic residue other than glycine (G) at the position corresponding to 554; a hydrophobic residue other than alanine (A) at position corresponding to 35; a hydrophilic residue other than glutamic acid (E) at the position corresponding to 110; a hydrophobic residue other than glycine (G) at the position corresponding to 314; a hydrophilic residue other than lysine (K) at the position corresponding to 114; a hydrophilic residue other than aspartic acid (D) at the position corresponding to 498; a hydrophobic residue other than isoleucine (I) at the position corresponding to 86; a hydrophobic residue other than valine (V) at position corresponding to 57; a hydrophilic residue other than histidine (H) at the position corresponding to 249; and A hydrophilic residue other than arginine (R) at the position corresponding to 704.

231. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises D38F.

232. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises A59V.

233. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises G172L.

234. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises T236L.

235. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises T319I.

236. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises H375L.

237. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises H419Y.

238. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises T424F.

239. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises E529L.

240. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises T541L.

241. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises G562Y.

242. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises K564M.

243. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises D569L.

244. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises A586I.

245. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises N641F.

246. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises D642L.

247. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises S647L.

248. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises D721L.

249. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises R779I.

250. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises K13R.

251. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises K566R.

252. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises G554H.

253. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises A35N.

254. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises E110T.

255. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises G314Q.

256. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises K114P.

257. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises D498P.

258. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises I86P.

259. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises V57E.

260. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises H249W.

261. The composition of any one of claims 212-230, wherein the endonuclease of SEQ ID NO: 3 comprises R704F.

262. The composition of any one of claims 212-261, wherein the endonuclease of SEQ ID NO: 3 comprises D38F and A59V.

263. The composition of any one of claims 212-262, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, and G172L.

264. The composition of any one of claims 212-263, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, and T236L.

265. The composition of any one of claims 212-264, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, and T319I.

266. The composition of any one of claims 212-265, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, and H375L.

267. The composition of any one of claims 212-266, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, and H419Y.

268. The composition of any one of claims 212-267, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, and T424F.

269. The composition of any one of claims 212-268, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, and E529L.

270. The composition of any one of claims 212-269, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, and T541L.

271. The composition of any one of claims 212-270, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, and G562X.

272. The composition of any one of claims 212-271, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, and K564M.

273. The composition of any one of claims 212-272, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, and D569L.

274. The composition of any one of claims 212-273, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, and A586I.

275. The composition of any one of claims 212-274, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, and N641F.

276. The composition of any one of claims 212-275, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, and D642L.

277. The composition of any one of claims 212-276, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, and S647L.

278. The composition of any one of claims 212-277, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, and D721L.

279. The composition of any one of claims 212-278, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, and R779I.

280. The composition of any one of claims 212-279, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, and K13R.

281. The composition of any one of claims 212-280, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, aK564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, and K566R.

282. The composition of any one of claims 212-281, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, and G554H.

283. The composition of any one of claims 212-282, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N.

284. The composition of any one of claims 212-283, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, and A35N.

285. The composition of any one of claims 212-284, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, and E110T.

286. The composition of any one of claims 212-285, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, and G314Q.

287. The composition of any one of claims 212-286, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, and K114P.

288. The composition of any one of claims 212-287, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, and D498P.

289. The composition of any one of claims 212-288, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, and I86P.

290. The composition of any one of claims 212-289, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, and V57E.

291. The composition of any one of claims 212-290, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, and H249W.

292. The composition of any one of claims 212-291, wherein the endonuclease of SEQ ID NO: 3 comprises D38F, A59V, G172L, T236L, T319I, H375L, H419Y, T424F, E529L, T541L, G562X, K564M, D569L, A586I, N641F, D642L, S647L, D721L, R779I, K13R, K566R, G554H, A35N, E110T, G314Q, K114P, D498P, I86P, V57E, H249W, and R704F.

293. A composition as described in any of claims 196-292, wherein the composition comprises a gRNA, a repRNA and a Cas nuclease operably linked to a single promoter or a bidirectional promoter.

294. A composition as described in claim 293, wherein the gRNA and repRNA are located on a first side of the bidirectional promoter, and the Cas nuclease is located on a second side of the bidirectional promoter.

295. A method for detecting and / or quantifying nucleic acids in a sample, the method comprising contacting the sample with a composition as described in any one of claims 212-294.

296. The method of claim 295, wherein the nucleic acid is a target and / or reporter nucleic acid.

297. The method of claim 295 or 296, wherein the method comprises detecting a reporter signal generated upon endonuclease cleavage.

298. The method of claim 297, wherein the reporter signal is a fluorescent signal.

299. The method of claim 297, wherein the endonuclease has incidental cleavage activity.

300. A method for targeted trans-splicing of pre-mRNA in a cell, the method comprising contacting the cell with a composition or system as described in any one of claims 199-294.

301. A method for targeted trans-splicing of pre-mRNA in a cell, the method comprising contacting the cell with a composition or system as described in any one of claims 199-294.

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