Enzymes containing the RUVC domain

Abstract

This disclosure provides an endonuclease enzyme having distinctive domain characteristics, and a method for using such enzyme or a variant thereof.

Description

Background Art

[0001] Cross-reference This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 487,847, filed on March 1, 2023, which is hereby incorporated by reference in its entirety.

Summary of the Invention

[0002] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease containing a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138. In some embodiments, the endonuclease contains a sequence having 100% sequence identity to any one of SEQ ID NOs: 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138. In some embodiments, the manipulated guide polynucleotide contains crRNA and tracrRNA. In some embodiments, the tracrRNA contains a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 215-222, 52, 147-151, 157-163, and 171-192. In some embodiments, the tracrRNA contains a sequence having 100% sequence identity to any one of SEQ ID NOs: 215-222, 52, 147-151, 157-163, and 171-192. In some embodiments, the manipulated guide polynucleotide is a single guide nucleic acid. In some embodiments, the manipulated guide polynucleotide is a dual guide nucleic acid. In some embodiments, the manipulated guide polynucleotide is RNA.In some embodiments, the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease. In some embodiments, the endonuclease has less than 80% identity with respect to Cas9 endonuclease. In some embodiments, the endonuclease is non-covalently bound to the manipulated guide polynucleotide. In some embodiments, the endonuclease is covalently bound to the manipulated guide polynucleotide.

[0003] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 139-146 and 303; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222.

[0004] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7 to 11; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: 52.

[0005] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease containing a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 105-109; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide contains a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 147-151.

[0006] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 110-116; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 157-163.

[0007] In certain embodiments, an engineered nuclease system is described herein, comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 117-138; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 171-192.

[0008] In some embodiments, the manipulated guide polynucleotide is a single guide nucleic acid.

[0009] In some embodiments, the manipulated guide polynucleotide is a dual guide nucleic acid.

[0010] In some embodiments, the manipulated guide polynucleotide is RNA.

[0011] In some embodiments, the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease.

[0012] In some embodiments, the endonuclease has less than 80% identity with the Cas9 endonuclease.

[0013] In some embodiments, the endonuclease is non-covalently bound to the manipulated guide polynucleotide.

[0014] In some embodiments, the endonuclease is covalently bound to the manipulated guide polynucleotide.

[0015] In some embodiments, the endonuclease is fused to the manipulated guide polynucleotide.

[0016] In certain embodiments, methods for modifying a target nucleic acid sequence are described herein, comprising contacting the target nucleic acid sequence using an engineered nuclease system described herein. In some embodiments, modifying a target nucleic acid sequence includes binding, nicking, or cleaving the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence includes genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the modification is in vitro. In some embodiments, the modification is in vivo. In some embodiments, the modification is ex vivo. In some embodiments, the gRNA includes one of the sequences of sequence numbers 251-260, 271-274, and 279-290. In some embodiments, the target nucleic acid sequence includes a sequence having one of the sequences of sequence numbers 261-270, 275-278, and 291-302.

[0017] In certain embodiments, a method for modifying a target nucleic acid sequence in mammalian cells is described herein, comprising contacting the mammalian cells with an engineered nuclease system described herein. In some embodiments, the method further includes selecting cells containing the modification.

[0018] In certain embodiments, a method for modifying HAO1 is described herein, comprising contacting HAO1 with an engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 139-146 and 303; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered guide polynucleotide comprises any one of SEQ ID NOs: 251-260. In some embodiments, the target nucleic acid sequence comprises any one of SEQ ID NOs: 261-270.

[0019] In certain embodiments, a method for modifying HAO1 is described herein, comprising contacting HAO1 with an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to SEQ ID NO: 304; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence. In some embodiments, the engineered guide polynucleotide comprises one of the sequences from SEQ ID NOs: 271 to 274. In some embodiments, the target nucleic acid sequence comprises one of the sequences from SEQ ID NOs: 275 to 278.

[0020] In certain embodiments, a method for modifying a TTR is described herein, comprising contacting the TTR with an engineered nuclease system comprising: a) an endonuclease containing a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 139-146 and 303; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide contains a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered guide polynucleotide contains any one of SEQ ID NOs: 279-290. In some embodiments, the target nucleic acid sequence contains any one of SEQ ID NOs: 291-302.

[0021] In certain embodiments, cells comprising the engineered nuclease system described herein are described herein. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC1, BSC40, BMT10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cells are engineered cells. In some embodiments, the cells are stable cells.

[0022] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be recognized, the present disclosure is capable of other different embodiments, and some details thereof are capable of variation in various obvious respects without departing from the present disclosure. Accordingly, the drawings and the description are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained from the following detailed description that illustrates exemplary embodiments in which the principles of the present disclosure are utilized, and from the appended drawings (also referred to herein as "Figure" and "FIG.").

[0024] [Figure 1A] FIG. 1A shows the sequence logo of the protospacer adjacent motif (PAM) recognized by MG171-1. [Figure 1B] FIG. 1B shows the cleavage positions 5 nt from the PAM obtained from in vitro cleavage assays and NGS sequencing. [Figure 2] FIG. 2 shows the analysis of gene editing results at the DNA level regarding the editing of human HAO1 by MG21-1 in Hep3B cells. [Figure 3] FIG. 3 shows the analysis of gene editing results at the DNA level regarding the editing of human TTR by MG21-1 in Hep3B cells. [Figure 4] FIG. 4 shows the analysis of gene editing results at the DNA level regarding the editing of human HAO1 by MG23-1 in Hep3B cells.

[0025] BRIEF DESCRIPTION OF THE SEQUENCE LISTING The Sequence Listing submitted together with this specification provides exemplary polynucleotide sequences and polypeptide sequences for use in the methods, compositions, and systems according to the present disclosure. The following is an exemplary description of the sequences therein.

[0026] MG2

[0027] SEQ ID NOs: 15 to 16 show the peptide sequences of the PAM interaction domains of the MG2 nuclease.

[0028] SEQ ID NOs: 53 to 66 show the nucleotide sequences of the target sites of the MG2 nuclease.

[0029] MG4

[0030] SEQ ID NOs: 17 to 22 show the peptide sequences of the PAM interaction domains of the MG4 nuclease.

[0031] SEQ ID NOs: 67 to 76 show the nucleotide sequences of the target sites of the MG4 nuclease.

[0032] MG5

[0033] SEQ ID NOs: 1 to 5 show the full-length peptide sequences of the MG5 nuclease.

[0034] SEQ ID NOs: 23 to 27 show the peptide sequences of the PAM interaction domains of the MG5 nuclease.

[0035] MG6

[0036] SEQ ID NOs: 28 to 36 show the peptide sequences of the PAM interaction domains of the MG6 nuclease.

[0037] SEQ ID NOs: 77 to 86 show the nucleotide sequences of the target sites of the MG6 nuclease.

[0038] MG7

[0039] Sequence ID 51 shows the nucleotide sequence of MG7 tracrRNA, which originates from the same gene locus as MG7 nuclease.

[0040] Sequence ID 37 shows the peptide sequence of the PAM interaction domain of MG7 nuclease.

[0041] Sequence IDs 87-95 show the nucleotide sequences of the target site of MG7 nuclease.

[0042] MG16

[0043] Sequence ID 38 shows the peptide sequence of the PAM interaction domain of the MG16 nuclease.

[0044] Sequence IDs 96-104 show the nucleotide sequences of the target site of MG16 nuclease.

[0045] MG21

[0046] Sequence IDs 139-146 and 303 show the full-length peptide sequences of the MG21 nuclease.

[0047] Sequence IDs 215-222 show the nucleotide sequences of MG21 tracrRNA, which originate from the same gene locus as the MG21 nuclease.

[0048] Sequence IDs 223-230 show the nucleotide sequences of the MG21 CRISPR repeat.

[0049] MG23

[0050] Sequence ID 304 shows the full-length peptide sequence of MG23 nuclease.

[0051] MG49

[0052] Sequence ID 39 shows the peptide sequence of the PAM interaction domain of MG49 nuclease.

[0053] MG86

[0054] Sequence ID 6 shows the full-length peptide sequence of the MG86 nuclease.

[0055] Sequence ID 40 shows the peptide sequence of the PAM interaction domain of the MG86 nuclease.

[0056] MG112

[0057] Sequence IDs 7-11 show the full-length peptide sequences of the MG112 nuclease.

[0058] Sequence IDs 41-45 show the peptide sequences of the PAM interaction domain of the MG112 nuclease.

[0059] nnRTCY is the nucleotide sequence of the PAM of the MG112 nuclease.

[0060] Sequence ID 50 shows the nucleotide sequence of an sgRNA engineered to function with the MG112 nuclease.

[0061] Sequence ID 52 shows the nucleotide sequence of MG7 tracrRNA, which originates from the same gene locus as the MG112 nuclease mentioned above.

[0062] MG116

[0063] Sequence IDs 12-14 show the full-length peptide sequences of the MG116 nuclease.

[0064] Sequence IDs 46-48 show the peptide sequences of the PAM interaction domain of the MG116 nuclease.

[0065] MG171

[0066] Sequence IDs 105-109 show the full-length peptide sequences of the MG171 nuclease.

[0067] Sequence IDs 147-151 show the nucleotide sequences of MG171 tracrRNA, which originate from the same gene locus as the MG171 nuclease.

[0068] Sequence IDs 152-156 show the nucleotide sequences of the MG171 CRISPR repeat.

[0069] Sequence IDs 231-240 show the nucleotide sequences of sgRNAs engineered to function with the MG171 nuclease.

[0070] MG177

[0071] Sequence IDs 110-116 show the full-length peptide sequences of the MG177 nuclease.

[0072] Sequence IDs 157-163 show the nucleotide sequences of MG177 tracrRNA, which originates from the same gene locus as the MG177 nuclease.

[0073] Sequence IDs 164-170 show the nucleotide sequences of the MG177 CRISPR repeat.

[0074] Sequence IDs 241-250 show the nucleotide sequences of sgRNAs engineered to function with the MG171 nuclease.

[0075] MG183

[0076] Sequence IDs 117-138 show the complete long peptide sequences of the MG183 nuclease.

[0077] Sequence IDs 171-192 show the nucleotide sequences of MG183 tracrRNA, which originate from the same gene locus as the MG183 nuclease.

[0078] Sequence IDs 193-214 show the nucleotide sequences of the MG183 CRISPR repeat.

[0079] HAO1 targeting using MG21-1

[0080] Sequence IDs 251–260 show the nucleotide sequences of sgRNAs engineered to function with the MG21-1 nuclease to target human HAO1.

[0081] Sequence IDs 261-270 show the DNA sequences of the human HAO1 target site.

[0082] HAO1 targeting using MG23-1

[0083] Sequence IDs 271-274 show the nucleotide sequences of sgRNAs engineered to function with the MG31-1 nuclease to target human HAO1.

[0084] Sequence IDs 275-278 show the DNA sequences of the human HAO1 target site.

[0085] TTR targeting using MG21-1

[0086] Sequence IDs 279–290 show the nucleotide sequences of sgRNAs engineered to function with the MG21-1 nuclease to target human TTR.

[0087] Sequence IDs 291-302 show the DNA sequences of human TTR target sites. [Modes for carrying out the invention]

[0088] While various embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Numerous variations, alterations, and substitutions can be conceived by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used.

[0089] Whenever the terms “at least,” “greater than,” or “greater than or equal to” precede the first number in a set of two or more numbers, the terms “at least,” “greater than,” or “greater than or equal to” apply to each of the numbers in that set. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0090] Whenever the terms “less than,” “less than,” or “less than or equal to” precede the first number in a set of two or more numbers, the terms “less than,” “less than,” or “less than or equal to” apply to each of the numbers in that set. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0091] The implementation of some of the methods disclosed herein utilizes techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise indicated.

[0092] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context otherwise explicitly indicates. Furthermore, to the extent that the terms "including," "includes," "having," "has," and "with," or their variations thereof, are used in either the detailed description and / or the claims, such terms are intended to be as inclusive as the term "comprising."

[0093] The terms “about” or “approximately” mean a range of acceptable error for a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., on the limits of the measuring system. For example, “about” may mean a range of one or more standard deviations according to practices in the art. Alternatively, “about” may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0094] As used in this disclosure, the term “nucleotide” refers to a base-sugar-phosphate combination. Nucleotides intended to be nucleotides include naturally occurring and synthetic nucleotides. A nucleotide is the monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates, such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, e.g., dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used in this disclosure, the term nucleotide also includes dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or may be labeled in a detectable manner, such as by using optically detectable moieties (e.g., fluorophores) or moieties containing quantum dots. Examples of detectable labels include radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Examples of fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). A specific example of fluorescently labeled nucleotides is Perkin in Foster City, California.[R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP and [dROX]ddTTP; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP (FluoroLink Cy3-dUTP) and FluoroLink Cy5-dUTP (FluoroLink Cy5-dUTP); fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Indiana; and chromosome-labeled nucleotides available from Molecular Probes, Eugene, Oregon: BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and TexasRed-12-dUTP is an example. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically modified nucleotide is biotin-dNTP. Non-exclusive examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0095] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, which are either deoxyribonucleotides or ribonucleotides or analogs thereof, in single-stranded, double-stranded, or multi-stranded forms. Polynucleotides as intended include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene fragments, multiple gene loci (single loci) determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In the context of polynucleotides, where T is mentioned, T refers to U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to a given cell and / or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., modified backbone, sugar, or nucleic acid base). Where present, modifications to the nucleotide structure are conferred before or after the assembly of the polymer. Non-limiting examples of modifications include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein bound to sugar), thiol-containing nucleotides, biotin-bound nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, quosin, and wyosine. The sequence of a nucleotide may be interrupted by non-nucleotide components.

[0096] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of at least two amino acid residues joined by peptide bonds. These terms do not imply a specific length of the polymer and are not intended to imply or distinguish whether a peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or whether it is naturally occurring. The terms apply to amino acid polymers containing at least one modified amino acid, as well as naturally occurring amino acid polymers. In some embodiments, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins and proteins (e.g., domains) with or without secondary or tertiary structures. The terms also encompass amino acid polymers modified by any other operations, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with labeling components. As used in this disclosure, the term “amino acid” refers to natural and non-natural amino acids, including but not limited to modified amino acids. Modified amino acids include amino acids that have been chemically modified to include a group or chemical moiety that is not naturally present on the amino acid. The term "amino acid" includes both D-amino acids and L-amino acids.

[0097] As used herein, “operably linked,” “operable linkage,” “operatively linked,” or their grammatical equivalents refer to the arrangement of genetic elements, such as promoters, enhancers, and polyadenylation sequences, where the action (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element may, but does not have to be, the same type as the action of the first genetic element. For example, if the movement of the first element causes the activation of the second element, the two genetic elements are operably linked. For example, if a regulatory element, which may include a promoter sequence and / or an enhancer sequence, helps initiate the transcription of a coding sequence, the regulatory element is operatively linked to the coding region. Intervening residues may exist between the regulatory element and the coding region, as long as this functional relationship is maintained.

[0098] A “functional fragment” of a DNA or protein sequence refers to a fragment that possesses biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to influence expression in a manner attributable to the full-length sequence.

[0099] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to objects modified by human intervention. For example, these terms refer to polynucleotides or polypeptides that do not exist in nature. Engineered peptides have, but do not require, low sequence identity with respect to naturally occurring human proteins (e.g., less than 50%, less than 25%, less than 10%, less than 5%, less than 1%). For example, the VPR domain and VP64 domain are synthetic transactivation domains. Non-limiting examples include: nucleic acids modified by altering their sequence to a sequence that does not occur in nature; nucleic acids modified by ligating them to nucleic acids that are not related in nature so that the ligated product possesses a function not present in the original nucleic acid; engineered nucleic acids synthesized in vitro using sequences that do not exist in nature; proteins modified by altering their amino acid sequence to a sequence that does not exist in nature; engineered proteins that acquire new functions or properties. An “engineered” system includes at least one engineered component.

[0100] The terms "tracrRNA" or "tracr sequence" refer to trans-activated CRISPR RNA. TracrRNA can interact with CRISPR(cr)RNA to hybridize to a target nucleic acid, thereby forming a guide nucleic acid (e.g., guide RNA or gRNA) that directs the relevant nuclease to the target nucleic acid.

[0101] As used herein, “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that can hybridize to a target nucleic acid, thereby directing the relevant nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid may include crRNA or tracrRNA, or a combination of both. The term guide nucleic acid encompasses engineered guide nucleic acids and programmable guide nucleic acids that specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. A double-stranded target polynucleotide chain that is complementary to the guide nucleic acid and hybridizes with it is called the complementary chain. A double-stranded target polynucleotide chain that is complementary to the complementary chain and therefore not complementary to the guide nucleic acid is called the non-complementary chain. A guide nucleic acid having a polynucleotide chain is called a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is called a “double guide nucleic acid.” Unless otherwise specified, the term “guide nucleic acid” is inclusive and refers to both single guide nucleic acids and dual guide nucleic acids. A guide nucleic acid may include a segment referred to as a “nucleic acid targeting segment,” “nucleic acid targeting sequence,” or “spacer.” A nucleic acid targeting segment may include a subsegment referred to as a “protein-binding segment,” “protein-binding sequence,” or “Cas protein-binding segment.”

[0102] As used in this disclosure, the term “complex” refers to the conjugation of at least two components. Each of the two components may retain properties / activities it had before forming the complex, or may acquire properties as a result of forming the complex. Conjugation includes, but is not limited to, covalent bonds, non-covalent bonds (i.e., hydrogen bonds, ionic interactions, van der Waals interactions, and hydrophobic bonds), the use of linkers, fusion, or any other preferred method. The components intended for the complex include polynucleotides, polypeptides, or combinations thereof. For example, the complex includes an endonuclease and a guide polynucleotide.

[0103] In the context of two or more nucleic acid or polypeptide sequences, the terms “sequence identity” or “percent identity” refer to two (e.g., in pairwise alignment) or more (e.g., in multiple sequence alignment) sequences that are identical, or have a specific percentage of identical amino acid residues or nucleotides, when compared and aligned to the maximum extent possible across a local or global comparison window, as measured using a sequence comparison algorithm. A suitable sequence comparison algorithm for polypeptide sequences is, for example, using a BLOSUM62 score matrix with parameters of word length (W) = 3, expectation (E) = 10, and gap costs set with existence = 11 and extension = 1, and applying a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues. Examples include BLASTP using adjustment; BLASTP using a PAM30 score matrix with a word length (W) of 2, an expected value (E) of 1,000,000, and a gap cost of 9 for open gaps and 1 for extended gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in a set of BLAST available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using parameters of the Smith-Waterman homology search algorithm with a match of 2, a mismatch of -1, and a gap of -1; MUSCLE using default parameters; MAFFT using parameters of a retree of 2 and a maximum iteration of 1000; and Novafold using default parameters.

[0104] A variant of any of the enzymes described herein having one or more conserved amino acid substitutions is included in this disclosure. Such conserved substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R-chain length to each other. In addition, or alternatively, conserved substitutions can be identified by locating interspecies mutated amino acid residues (e.g., non-conserved residues) without altering the fundamental function of the encoded protein, by comparing aligned sequences of homologous proteins from different species. Such conserved-substituted variants are at least about 20%, at least about 25%, at least about 30%, at least about 35%, and at least one of the endonuclease protein sequences described herein (e.g., endonucleases of the MG2, MG4, MG5, MG6, MG7, MG16, MG21, MG23, MG49, MG86, MG112, MG116, MG171, MG177, or MG183 families described herein). The variants may include variants having approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, and at least approximately 99% identity. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants may include sequences with substitutions such that the activity of key active site residues of the endonuclease is not disrupted. In some embodiments, any functional variant of any of the proteins described herein lacks at least one substitution of essentially predicted residues. In some embodiments, any functional variant of any of the proteins described herein lacks all of the essentially predicted residue substitutions.

[0105] Lists of conserved substitutions that result in functionally similar amino acids are available from various references (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993))). The following eight groups each contain amino acids that are conserved substitutions with each other. 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), leucine (L), methionine (M), valine (V), 6) Phenylalanine (F), tyrosine (Y), tryptophan (W), 7) Serine (S), threonine (T), and 8) Cysteine ​​(C), Methionine (M)

[0106] overview The discovery of novel Cas enzymes with unique functionalities and structures could further transform deoxyribonucleic acid (DNA) editing technology, potentially improving speed, specificity, functionality, and ease of use. Compared to the predicted proliferation of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems in microorganisms and the complete diversity of microbial species, the literature contains relatively few functionally characterized CRISPR / Cas enzymes. This is partly because, under laboratory conditions, a vast number of microbial species cannot be easily cultured. Metagenomic sequencing from ecological niches in natural environments representing a large number of microbial species could dramatically increase the number of known novel CRISPR / Cas systems and potentially accelerate the discovery of new oligonucleotide editing functions. A fruitful recent example of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0107] The CRISPR / Cas system is an RNA-directed nuclease complex described as functioning as an adaptive immune system in microorganisms. In their natural context, the CRISPR / Cas system arises at the operon or locus of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which generally comprises two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences, encoding an RNA-based targeting element; and (ii) an ORF encoding Cas, which encodes a nuclease polypeptide directed by the RNA-based targeting element together with an accessory protein / enzyme. Efficient nuclease targeting of specific target nucleic acid sequences generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer adjacency motif (PAM) sequence within a defined vicinity of the target seed (the PAM is typically a sequence not normally expressed in the host genome). Depending on the precise function and configuration of the system, CRISPR-Cas systems are typically organized into two classes, five types, and 16 subtypes based on shared functional characteristics and evolutionary similarities.

[0108] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes and include types I, III, and IV.

[0109] The type I CRISPR-Cas system is considered to have moderate complexity in terms of its components. In the type I CRISPR-Cas system, an array of RNA targeting elements is transcribed as long precursor crRNAs (pre-crRNAs) processed at repeat elements, releasing short mature crRNAs that direct the nuclease complex to a nucleic acid target if followed by a suitable short consensus sequence called a protospacer adjacent motif (PAM). This processing occurs via the endoribonuclease subunit (Cas6) of a large endonuclease complex called a cascade, which also contains the nuclease (Cas3), a protein component of the crRNA-directing nuclease complex. The Cas1 nuclease primarily functions as a DNA nuclease.

[0110] Type III CRISPR systems may be characterized by the presence of a central nuclease known as Cas10, along with RAMP (repeat-associated mysterious protein) containing a Csm or Cmr protein subunit. Similar to type I systems, mature crRNA is processed from pre-crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA double strands (such as the DNA strand used as a template for RNA polymerase).

[0111] The type IV CRISPR-Cas system has an effector complex consisting of a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins from the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted smaller subunit. Such systems are typically found in endogenous plasmids.

[0112] Class II CRISPR-Cas systems generally have a single polypeptide multi-domain nuclease effector and include types II, V, and VI.

[0113] The Type II CRISPR-Cas system is considered the simplest in terms of its components. In the Type II CRISPR-Cas system, processing the CRISPR array into mature crRNA does not require the presence of a special endonuclease subunit, but rather a small transcoded crRNA (tracrRNA) having a region complementary to the repeat sequence of the array. This tracrRNA interacts with both its corresponding effector nuclease (Cas9) and the repeat sequence to form a precursor dsRNA structure, which is then cleaved by endogenous ribonuclease III to produce a mature Cas9 effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are known as DNA nucleases. The Cas9 effector has a characteristic structure consisting of a RuvC-like endonuclease domain that employs a ribonuclease H fold having an unrelated HNH nuclease domain inserted into the fold of the RuvC-like nuclease domain. The RuvC-like domain is involved in cleaving the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is involved in cleaving the substituted DNA strand.

[0114] The V-type CRISPR-Cas system is characterized by a nuclease effector (Cas12) structure similar to that of the type II / Cas9 system, containing a RuvC-like domain. Like type II, most (but not all) V-type CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA; however, unlike type II systems which require ribonuclease III to cleave pre-crRNA into multiple crRNAs, V-type systems can cleave pre-crRNA using the effector nuclease itself (Cas12). Similar to type II CRISPR-Cas systems, V-type CRISPR-Cas systems are again known as DNA nucleases. Unlike type II CRISPR-Cas systems, some V-type enzymes (e.g., Cas12a) appear to possess robust single-strand nonspecific deoxyribonuclease activity, activated by primary crRNA-oriented cleavage of a double-stranded target sequence.

[0115] The type VI CRISPR-Cas system is unique in that it appears to be the only class of RNA-guided RNA endonucleases known to date. Instead of a RuvC-like domain, the single polypeptide effector (Cas13) of the type VI system contains two HEPN ribonuclease domains. Unlike both the type II and type V systems, the type VI system also appears not to require tracrRNA to process pre-crRNA into crRNA. However, similar to the type V system, some type VI systems (e.g., C2C2) appear to possess robust single-strand nonspecific nuclease (ribonuclease) activity, activated by the first crRNA-directed cleavage of the target RNA.

[0116] Due to their simpler structure, Class 2 CRISPR-Cas have been the most widely adopted for manipulation and development as designer nucleases / genome editing applications.

[0117] One of the early adaptations of such a system for in vivo use is (i) recombinantly expressed purified full-length Cas9 (e.g., class 2 type II Cas enzyme) isolated from Streptococcus pyogenes SF370, (ii) purified mature crRNA of about 42 nt carrying a 5' sequence of about 20 nt complementary to the target DNA sequence to be cleaved, followed by a 3' tracr binding sequence (total crRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence), (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg 2+ The system was subsequently improved and manipulated, and involved a crRNA of (ii) linked to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) capable of directing Cas9 to a target on its own.

[0118] Such manipulated systems can be adapted for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g., a class 2 type II Cas enzyme) under a suitable mammalian promoter having a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal), and (ii) an ORF encoding sgRNA (having a 5' sequence beginning with G, followed by a 3' tracr binding sequence, a 20nt complementary targeting nucleic acid sequence, a linker, and a tracrRNA sequence) under a suitable polymerase III promoter (e.g., U6 promoter).

[0119] MG Enzyme In certain embodiments, an engineered nuclease system comprising an endonuclease and an engineered guide polynucleotide is described herein. In some embodiments, the endonuclease is a class 2, type II endonuclease. In some embodiments, the endonuclease comprises a RuvC_III domain. In some embodiments, the endonuclease comprises an HNH domain. In some embodiments, the endonuclease is a double-stranded nuclease. In some embodiments, the endonuclease has lost its activity. In some embodiments, the endonuclease is a double-stranded nuclease. In some embodiments, the endonuclease is modified. In some embodiments, the endonuclease is modified to result in an endonuclease having nickas activity. In some embodiments, the modified endonuclease is a site-specific nickas.

[0120] In some embodiments, the endonuclease is MG2 endonuclease. In some embodiments, the endonuclease is MG4 endonuclease. In some embodiments, the endonuclease is MG5 endonuclease. In some embodiments, the endonuclease is MG6 endonuclease. In some embodiments, the endonuclease is MG7 endonuclease. In some embodiments, the endonuclease is MG16 endonuclease. In some embodiments, the endonuclease is MG21 endonuclease. In some embodiments, the endonuclease is MG23 endonuclease. In some embodiments, the endonuclease is MG49 endonuclease. In some embodiments, the endonuclease is MG86 endonuclease. In some embodiments, the endonuclease is MG112 endonuclease. In some embodiments, the endonuclease is MG116 endonuclease. In some embodiments, the endonuclease is MG171 endonuclease. In some embodiments, the endonuclease is MG177 endonuclease. In some embodiments, the endonuclease is MG183 endonuclease.

[0121] In some embodiments, the manipulated nuclease system is discovered via metagenomic sequencing. In some embodiments, metagenomic sequencing is performed on samples collected from various environments. In some embodiments, these environments include the human microbiome, animal microbiome, high-temperature environments, low-temperature environments, or sediment.

[0122] In some embodiments, the endonuclease is MG5 endonuclease (i.e., SEQ ID NOs. 1-5). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 70% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 75% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with any one of sequence numbers 1 to 5. In some embodiments, the endonuclease contains a sequence having at least about 99% identity with any one of sequence numbers 1 to 5.In some embodiments, the endonuclease contains a sequence that has 100% identity with any one of sequence numbers 1 to 5.

[0123] In some embodiments, the endonuclease is MG21 endonuclease (i.e., SEQ ID NOs. 139-146 and 303). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 75% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 97% identity to one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having at least about 98% identity to one of sequence numbers 139-146 and 303.In some embodiments, the endonuclease contains a sequence having at least about 99% identity to any one of sequence numbers 139-146 and 303. In some embodiments, the endonuclease contains a sequence having 100% identity to any one of sequence numbers 139-146 and 303.

[0124] In some embodiments, the endonuclease is MG23 endonuclease (i.e., SEQ ID NO: 304). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, 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: 304. In some embodiments, the endonuclease contains a sequence having at least about 70% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 75% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 80% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 85% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 90% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 95% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 96% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 97% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 98% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having at least about 99% identity to SEQ ID NO: 304. In some embodiments, the endonuclease contains a sequence having 100% identity to SEQ ID NO: 304.

[0125] In some embodiments, the endonuclease is MG86 endonuclease (i.e., SEQ ID NO: 6). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, 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: 6. In some embodiments, the endonuclease contains a sequence having at least about 70% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 75% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 80% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 85% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 90% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 95% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 96% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 97% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 98% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having at least about 99% identity to SEQ ID NO: 6. In some embodiments, the endonuclease contains a sequence having 100% identity to SEQ ID NO: 6.

[0126] In some embodiments, the endonuclease is the MG112 endonuclease (i.e., SEQ ID NOs. 7-11). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 70% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 75% identity to any one of SEQ ID NOs. 7-11. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with any one of sequence numbers 7-11. In some embodiments, the endonuclease contains a sequence having at least about 99% identity with any one of sequence numbers 7 to 11.In some embodiments, the endonuclease contains a sequence that has 100% identity with any one of sequence numbers 7 to 11.

[0127] In some embodiments, the endonuclease is the MG116 endonuclease (i.e., SEQ ID NOs. 12-14). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. 12-14 contains a sequence having at least about 70% identity to any one of SEQ ID NOs. 12-14 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. 12-14 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with any one of sequence numbers 12-14. In some embodiments, the endonuclease contains a sequence having at least about 99% identity with one of sequence numbers 12-14.In some embodiments, the endonuclease contains a sequence that has 100% identity with any one of sequence numbers 12-14.

[0128] In some embodiments, the endonuclease is MG171 endonuclease (i.e., SEQ ID NOs. 105-109). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. 105-109 contains a sequence having at least about 70% identity to any one of SEQ ID NOs. 105-109 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. 105-109 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with any one of sequence numbers 105 to 109. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with any one of sequence numbers 105 to 109.In some embodiments, the endonuclease contains a sequence having at least about 99% identity to one of sequence numbers 105-109. In some embodiments, the endonuclease contains a sequence having 100% identity to one of sequence numbers 105-109.

[0129] In some embodiments, the endonuclease is MG177 endonuclease (i.e., SEQ ID NOs. 110-116). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. 110-116 contains a sequence having at least about 70% identity to any one of SEQ ID NOs. 110-116 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. 110-116 contains a sequence having at least about 75% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with any one of sequence numbers 110-116.In some embodiments, the endonuclease contains a sequence having at least about 99% identity to any one of sequence numbers 110-116. In some embodiments, the endonuclease contains a sequence having 100% identity to any one of sequence numbers 110-116.

[0130] In some embodiments, the endonuclease is MG183 endonuclease (i.e., SEQ ID NOs. 117-138). In some embodiments, the endonuclease contains a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the endonuclease contains a sequence having at least about 80% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 85% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 90% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 95% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 96% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 97% identity with one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having at least about 98% identity with one of sequence numbers 117-138.In some embodiments, the endonuclease contains a sequence having at least about 99% identity to one of sequence numbers 117-138. In some embodiments, the endonuclease contains a sequence having 100% identity to one of sequence numbers 117-138.

[0131] In some embodiments, the endonuclease includes a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the endonuclease. In some embodiments, the NLS is located at the C-terminus of the endonuclease. In some embodiments, the NLS is located at both the N-terminus and the C-terminus of the endonuclease.

[0132] In some embodiments, the NLS includes a sequence that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of the sequences from sequence numbers 305 to 350. In some embodiments, the NLS includes a sequence that has at least about 80% identity with sequence numbers 305 to 350. In some embodiments, the NLS includes a sequence that has at least about 85% identity with sequence numbers 305 to 350. In some embodiments, the NLS includes sequences that have at least about 90% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 91% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 92% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 93% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 94% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 95% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 96% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 97% identity to sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 98% identity with sequence numbers 305-350. In some embodiments, the NLS includes sequences that have at least about 99% identity with sequence numbers 305-350.In some embodiments, the NLS includes sequences that have 100% identity with sequence numbers 305-350.

[0133] [Table 1-1]

[0134] [Table 1-2]

[0135] Guide polynucleotides In some embodiments, the engineered nuclease systems disclosed herein include engineered guide polynucleotides, such as guide ribonucleic acid (gRNA), single gRNA, or double guide RNA.

[0136] In some embodiments, the guide RNA includes, but is not limited to, a spacer sequence that binds to a protospacer sequence (target sequence), crRNA, and optional tracrRNA, comprising a variety of structural elements. In some embodiments, the guide RNA includes crRNA containing a spacer sequence. In some embodiments, the guide RNA includes tracrRNA or modified tracrRNA.

[0137] In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with respect to SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 70% identity with respect to SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 90% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 91% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 92% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence comprising at least about 60 to 100 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 51.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 96% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 97% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 98% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 99% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA includes a sequence comprising at least about 60 to 100 consecutive nucleotides having 100% identity with SEQ ID NO: 51.

[0138] In some embodiments, the tracrRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 70% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 75% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 80% identity with SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 85% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 90% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 91% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 92% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 93% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 94% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 95% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 96% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence having at least about 97% identity to SEQ ID NO: 51.In some embodiments, the tracrRNA contains a sequence that is at least about 98% identical to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence that is at least about 99% identical to SEQ ID NO: 51. In some embodiments, the tracrRNA contains a sequence that is 100% identical to SEQ ID NO: 51.

[0139] In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of the SEQ ID NOs. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 90% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 91% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 92% identity with any one of sequence numbers 215 to 222.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 93% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 94% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 95% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 96% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 97% identity with any one of SEQ ID NOs. 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 98% identity with any one of sequence numbers 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 99% identity with any one of sequence numbers 215 to 222. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having 100% identity with any one of sequence numbers 215 to 222.

[0140] In some embodiments, the tracrRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 70% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 75% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence that has at least about 80% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 85% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 90% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 91% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 92% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 93% identity with one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 94% identity with one of sequence numbers 215-222.In some embodiments, the tracrRNA contains a sequence that has at least about 95% identity to one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 96% identity to one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 97% identity to one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 98% identity to one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has at least about 99% identity to one of sequence numbers 215-222. In some embodiments, the tracrRNA contains a sequence that has 100% identity to one of sequence numbers 215-222.

[0141] In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with respect to SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 70% identity with respect to SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 90% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 91% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 92% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence comprising at least about 60 to 100 consecutive nucleotides having at least about 93% identity with SEQ ID NO: 52.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 94% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 95% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 96% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 97% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 98% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 99% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA includes a sequence comprising at least about 60 to 100 consecutive nucleotides having 100% identity with SEQ ID NO: 52.

[0142] In some embodiments, the tracrRNA contains a sequence that has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that has at least about 70% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that has at least about 75% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that has at least about 80% identity with SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 85% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 90% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 91% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 92% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 93% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 94% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 95% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 96% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 97% identical to SEQ ID NO: 52.In some embodiments, the tracrRNA contains a sequence that is at least about 98% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is at least about 99% identical to SEQ ID NO: 52. In some embodiments, the tracrRNA contains a sequence that is 100% identical to SEQ ID NO: 52.

[0143] In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of the SEQ ID NOs. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 90% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 91% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 92% identity with any one of sequence numbers 147 to 151.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 93% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 94% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 95% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 96% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 97% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides having at least about 98% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides having at least about 99% identity with any one of sequence numbers 147 to 151. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides having 100% identity with any one of sequence numbers 147 to 151.

[0144] In some embodiments, the tracrRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 70% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 75% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence that has at least about 80% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 85% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 90% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 91% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 92% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 93% identity with one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 94% identity with one of sequence numbers 147-151.In some embodiments, the tracrRNA contains a sequence that has at least about 95% identity to one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 96% identity to one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 97% identity to one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 98% identity to one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has at least about 99% identity to one of sequence numbers 147-151. In some embodiments, the tracrRNA contains a sequence that has 100% identity to one of sequence numbers 147-151.

[0145] In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of the SEQ ID NOs. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 90% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 91% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 92% identity with any one of sequence numbers 157 to 163.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 93% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 94% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 95% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 96% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 97% identity with any one of SEQ ID NOs. 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides that have at least about 98% identity with any one of sequence numbers 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides that have at least about 99% identity with any one of sequence numbers 157 to 163. In some embodiments, the tracrRNA includes a sequence containing at least about 60 to 100 consecutive nucleotides that have 100% identity with any one of sequence numbers 157 to 163.

[0146] In some embodiments, the tracrRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 70% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 75% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence that has at least about 80% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 85% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 90% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 91% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 92% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 93% identity with one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 94% identity with one of sequence numbers 157-163.In some embodiments, the tracrRNA contains a sequence that has at least about 95% identity to one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 96% identity to one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 97% identity to one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 98% identity to one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has at least about 99% identity to one of sequence numbers 157-163. In some embodiments, the tracrRNA contains a sequence that has 100% identity to one of sequence numbers 157-163.

[0147] In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of the SEQ ID NOs. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 75% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 80% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 85% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 90% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 91% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence comprising at least 60 to 100 consecutive nucleotides having at least about 92% identity with any one of sequence numbers 171 to 192.In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 93% identity with any one of SEQ ID NOs. 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 94% identity with any one of SEQ ID NOs. 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 95% identity with any one of SEQ ID NOs. 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 96% identity with any one of SEQ ID NOs. 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least 97% identity with any one of SEQ ID NOs. 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 98% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having at least about 99% identity with any one of sequence numbers 171 to 192. In some embodiments, the tracrRNA includes a sequence containing at least 60 to 100 consecutive nucleotides having 100% identity with any one of sequence numbers 171 to 192.

[0148] In some embodiments, the tracrRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 70% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence having at least about 75% identity with any one of SEQ ID NOs. In some embodiments, the tracrRNA contains a sequence that has at least about 80% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 85% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 90% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 91% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 92% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 93% identity with one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 94% identity with one of sequence numbers 171-192.In some embodiments, the tracrRNA contains a sequence that has at least about 95% identity to one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 96% identity to one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 97% identity to one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 98% identity to one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has at least about 99% identity to one of sequence numbers 171-192. In some embodiments, the tracrRNA contains a sequence that has 100% identity to one of sequence numbers 171-192.

[0149] In some embodiments, the engineered nuclease systems disclosed herein include engineered guide polynucleotides, such as guide ribonucleic acid (gRNA), single gRNA, or double guide RNA.

[0150] In some embodiments, the manipulated guide polynucleotide (i.e., SEQ ID NO: 50) functions with the MG112 nuclease. In some embodiments, the manipulated guide polynucleotide contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide contains a sequence having at least about 70% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide contains a sequence having at least about 75% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 80% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 85% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 90% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 91% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 92% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 93% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 94% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 95% identity with SEQ ID NO: 50.In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 96% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 97% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 98% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 99% identity with SEQ ID NO: 50. In some embodiments, the manipulated guide polynucleotide includes a sequence having 100% identity with SEQ ID NO: 50.

[0151] In some embodiments, the manipulated guide polynucleotides (i.e., SEQ ID NOs. 231-240) function with the MG171 nuclease. In some embodiments, the manipulated guide polynucleotides contain sequences having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the manipulated guide polynucleotides contain sequences having at least about 70% identity to any one of SEQ ID NOs. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 75% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 80% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 85% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 90% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 91% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 92% identity to one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 93% identity to one of SEQ ID NOs. 231-240.In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 94% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 95% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 96% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 97% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 98% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 99% identity to any one of SEQ ID NOs. 231-240. In some embodiments, the manipulated guide polynucleotide includes a sequence having 100% identity to any one of SEQ ID NOs. 231-240.

[0152] In some embodiments, the manipulated guide polynucleotides (i.e., SEQ ID NOs. 241-250) function with the MG177 nuclease. In some embodiments, the manipulated guide polynucleotides contain sequences having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity to any one of SEQ ID NOs. In some embodiments, the manipulated guide polynucleotides contain sequences having at least about 70% identity to any one of SEQ ID NOs. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 75% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 80% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 85% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 90% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 91% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 92% identity to one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 93% identity to one of SEQ ID NOs. 241-250.In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 94% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 95% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 96% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 97% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 98% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having at least about 99% identity to any one of SEQ ID NOs. 241-250. In some embodiments, the manipulated guide polynucleotide includes a sequence having 100% identity to any one of SEQ ID NOs. 241-250.

[0153] In some embodiments, the manipulated guide polynucleotide comprises a synthetic or modified nucleotide. In some embodiments, the manipulated guide polynucleotide comprises one or more internucleoside linkers modified from natural phosphodiesters. In some embodiments, all or the entire sequence of internucleoside linkers of the manipulated guide polynucleotide is modified. For example, in some embodiments, the internucleoside linkages include sulfur (S), such as phosphorothioate internucleoside linkages.

[0154] In some embodiments, the manipulated guide polynucleotide comprises modifications to the ribose sugar or nucleic acid base. In some embodiments, the manipulated guide polynucleotide comprises one or more nucleosides containing a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., Loc nucleic acid (LNA)), or an unbound ribose ring typically lacking a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is substituted with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0155] In some embodiments, the manipulated guide polynucleotide contains one or more modified sugars. In some embodiments, the sugar modification includes modifications by altering substituents on the ribose ring to non-hydrogen groups or to naturally occurring 2'-OH groups in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2', 3', 4', or 5' positions, or in combination thereof. In some embodiments, the nucleoside having a modified sugar moiety includes 2'-modified nucleosides, e.g., 2'-substituted nucleosides. In some embodiments, 2'-sugar-modified nucleosides are nucleosides having substituents other than -H or -OH at the 2' position (2'-substituted nucleosides), or include 2'-linked biradicals, as well as 2'-substituted nucleosides and LNA (2'-4' biradical-bridged) nucleosides. Examples of 2'-substituted nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification in the ribose group involves a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0156] In some embodiments, the manipulated guide polynucleotide contains one or more modified sugars. In some embodiments, the manipulated guide polynucleotide contains only modified sugars. In some embodiments, the manipulated guide polynucleotide contains about 10%, 25%, 50%, 75%, or more than 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar contains a 2'-O-methoxyethyl group. In some embodiments, the manipulated guide polynucleotide contains both internucleoside linker modification and nucleoside modification.

[0157] In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the manipulated guide polynucleotide includes a sequence complementary to a human genomic polynucleotide sequence.

[0158] In some embodiments, the manipulated guide polynucleotide has a length of 30 to 250 nucleotides. In some embodiments, the manipulated guide polynucleotide has a length of more than 90 nucleotides. In some embodiments, the manipulated guide polynucleotide has a length of less than 245 nucleotides. In some embodiments, the manipulated guide polynucleotide has a length of 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240 nucleotides, or more than 240 nucleotides. In some embodiments, the manipulated guide polynucleotides are approximately 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-120, 30-140, 30-160, 30-180, 30-200, 30-220, 30-240, 50-60, 50-70, 50-80, 50-90, and 50- The number of nucleotides is approximately 100, 50-120, 50-140, 50-160, 50-180, 50-200, 50-220, 50-240, 100-120, 100-140, 100-160, 100-180, 100-200, 100-220, 100-240, 160-180, 160-200, 160-220, or 160-240.

[0159] MG System In certain embodiments, an engineered nuclease system comprising an endonuclease and an engineered guide polynucleotide is described herein. In some embodiments, the engineered guide polynucleotide comprises tracrRNA. In some embodiments, the engineered guide polynucleotide is a guide nucleic acid (e.g., gRNA). When referring to polynucleotides, T means U (uracil) in RNA and T (thymine) in DNA.

[0160] In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 70% identity with one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least about 70% identity with one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 75% identity with one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 75% identity with one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 80% identity with one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 80% identity with one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 85% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least about 85% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 90% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 90% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 95% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 95% identity to any one of SEQ ID NOs: 215-222.In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 97% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 139-146 and 303, and a manipulated polynucleotide containing a sequence having at least 98% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease having at least about 99% identity to one of sequence numbers 139-146 and 303, and a manipulated polynucleotide having at least about 99% identity to one of sequence numbers 215-222. In some embodiments, the manipulated nuclease system comprises an endonuclease having 100% identity to one of sequence numbers 139-146 and 303, and a manipulated polynucleotide having 100% identity to one of sequence numbers 215-222.

[0161] In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 70% identity with SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 75% identity with SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 80% identity with SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 85% identity with any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 85% identity with SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 90% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 90% identity to SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 95% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least 95% identity to SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least 96% identity to SEQ ID NOs: 52.In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 97% identity to SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 98% identity to SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 99% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing a sequence having at least about 99% identity to SEQ ID NOs: 52. In some embodiments, the manipulated nuclease system includes an endonuclease containing 100% identity to any one of SEQ ID NOs: 7-11, and a manipulated polynucleotide containing 100% identity to SEQ ID NOs: 52.

[0162] In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 85% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 85% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 90% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least 90% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 95% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least 95% identity to any one of SEQ ID NOs: 147-151.In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 105-109, and a manipulated polynucleotide containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the manipulated nuclease system comprises an endonuclease having at least about 99% identity to one of sequence numbers 105-109, and a manipulated polynucleotide having at least about 99% identity to one of sequence numbers 147-151. In some embodiments, the manipulated nuclease system comprises an endonuclease having 100% identity to one of sequence numbers 105-109, and a manipulated polynucleotide having 100% identity to one of sequence numbers 147-151.

[0163] In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 85% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 85% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 90% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least 90% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 95% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least 95% identity with any one of SEQ ID NOs: 157-163.In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 96% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 96% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 97% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least about 97% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 98% identity with any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide containing a sequence having at least 98% identity with any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system comprises an endonuclease having at least about 99% identity to any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide having at least about 99% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the manipulated nuclease system comprises an endonuclease having 100% identity to any one of SEQ ID NOs: 110-116, and a manipulated polynucleotide having 100% identity to any one of SEQ ID NOs: 157-163.

[0164] In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least about 70% identity with any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 75% identity with any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least 75% identity with any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 80% identity with any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least 80% identity with any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 85% identity to one of sequence numbers 117-138, and a manipulated polynucleotide containing a sequence having at least about 85% identity to one of sequence numbers 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 90% identity to one of sequence numbers 117-138, and a manipulated polynucleotide containing a sequence having at least 90% identity to one of sequence numbers 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 95% identity to one of sequence numbers 117-138, and a manipulated polynucleotide containing a sequence having at least 95% identity to one of sequence numbers 171-192.In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least about 96% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least about 97% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system includes an endonuclease containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide containing a sequence having at least about 98% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system comprises an endonuclease having at least about 99% identity to any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide having at least about 99% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the manipulated nuclease system comprises an endonuclease having 100% identity to any one of SEQ ID NOs: 117-138, and a manipulated polynucleotide having 100% identity to any one of SEQ ID NOs: 171-192.

[0165] cell In certain embodiments, cells comprising the system described herein are described herein.

[0166] In some embodiments, the cells include eukaryotic cells (e.g., plant cells, animal cells, protist cells, or fungal cells), mammalian cells (Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK), human fetal kidney (HEK), mouse myeloma (NS0), or human retinal cells), immortalized cells (e.g., HeLa cells, COS cells, HEK-293T cells, MDCK cells, 3T3 cells, PC12 cells, Huh7 cells, HepG2 cells, K562 cells, N2a cells, or SY5Y cells), insect cells (e.g., armyworm (Spodoptera frugiperda) cells, nettle moth (Trichoplusia ni) cells, fruit fly (Drosophila melanogaster) cells, S2 cells, or tobacco budworm (Heliothis virescens) cells), and yeast cells (e.g., budding yeast (Saccharomyces)). These include cells of *Cerevisiae*, *Cryptococcus*, or *Candida*, plant cells (e.g., parenchyma cells, plaque cells, or sclerophyll cells), fungal cells (e.g., *Saccharomyces cerevisiae* cells, *Cryptococcus*, or *Candida* cells), or prokaryotic cells (e.g., *E. coli* cells, *Streptococcus* bacterial cells, *Streptomyces* soil bacterial cells, or archaeal cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0167] In some embodiments, the cells are A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC1, BSC40, BMT10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0168] Delivery and vector In some embodiments, nucleic acid sequences encoding the manipulated nuclease systems disclosed herein are described herein.

[0169] In some embodiments, the nucleic acid encoding the manipulated nuclease system is DNA, such as linear DNA, plasmid DNA, or minicircle DNA. In some embodiments, the nucleic acid encoding the manipulated nuclease system is RNA, such as mRNA.

[0170] In some embodiments, the nucleic acid encoding the manipulated nuclease system is delivered by a nucleic acid vector. In some embodiments, the nucleic acid vector is a plasmid (e.g., a circular DNA molecule that can autonomously replicate inside a cell), a cosmid (e.g., a pWE or sCos vector), an artificial chromosome, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a phagemid, a phage derivative, a bacmid, or a virus. In some embodiments, the nucleic acid vectors include pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, and pRI 101-AN. The selection is made from a list consisting of DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0171] In some embodiments, the nucleic acid vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of mini-promoters, inducible promoters, constitutive promoters, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synapsin, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is the U6 promoter. In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter is coded by any one sequence from sequence numbers 190 to 191, or by a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity to any one of sequence numbers 190 to 191.

[0172] In some embodiments, the nucleic acid vector is a virus. In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vacciniavirus, or retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vacciniavirus. In some embodiments, the virus is a retrovirus.

[0173] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh 10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, The herpesvirus is AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0174] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof.In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0175] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0176] In some embodiments, the nucleic acid encoding the manipulated nuclease system is delivered by a non-nucleic acid delivery system (e.g., a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is bound to a lipid. In some embodiments, the lipid-bound nucleic acid is encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via binding molecules that bind to both the liposome and the nucleic acid, confined within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, containing micelles or complexed with micelles, or otherwise bound to a lipid. In some embodiments, the nucleic acid is contained in lipid nanoparticles (LNPs).

[0177] In some embodiments, the fusion protein or genome editing system is introduced into cells in any preferred manner, either stably or transiently. In some embodiments, the fusion protein or genome editing system is transfected into cells. In some embodiments, cells are transfected or transfected with a nucleic acid construct encoding the fusion protein or genome editing system. For example, cells are transfected (e.g., with a virus encoding the fusion protein or genome editing system) or transfected with the fusion protein or genome editing system, or the nucleic acid encoding the translated fusion protein or genome editing system (e.g., with a plasmid encoding the fusion protein or genome editing system). In some embodiments, the transfection is stably or transiently transfected. In some embodiments, cells expressing or containing the fusion protein or genome editing system are transfected or transfected with one or more gRNA molecules, for example, when the fusion protein or genome editing system contains a CRISPR nuclease. In some embodiments, plasmids expressing fusion proteins or genome editing systems are introduced into cells by electroporation, transient (e.g., lipofection), stable genome integration (e.g., piggybac), and viral transduction (e.g., lentivirus or AAV), or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into cells as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. For example, methods for delivering polypeptides and / or RNPs into cells by electroporation or cell compression are known in the art.

[0178] Exemplary methods for nucleic acid delivery include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced incorporation of DNA. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and SF Cell Line 4D-Nucleofector X Kit® (Lonza)). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those described in International Publications 91 / 17424 and 91 / 16024. In some embodiments, delivery is to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration). In some embodiments, the nucleic acid is contained within liposomes or nanoparticles that specifically target host cells.

[0179] Additional methods for delivering nucleic acids to cells are known to those skilled in the art. See, for example, U.S. Patent Application Publication No. 2003 / 0087817.

[0180] How to use In certain embodiments, methods for modifying a target nucleic acid are described herein, including providing an engineered nuclease system disclosed herein. In some embodiments, the engineered nuclease system comprises an endonuclease and an engineered guide polynucleotide. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid is double-stranded DNA. In some embodiments, the target nucleic acid is single-stranded.

[0181] In some embodiments, the method is used to introduce modifications into the genome of a cell. In some embodiments, the modifications are insertions, deletions, or mutations. In some embodiments, the method is used to introduce site-specific insertions, deletions, and / or mutations (e.g., insertions and mutations) into the genome of a cell. In some embodiments, the method is used in combination with a nucleic acid template to facilitate site-specific insertions into the genome of a cell.

[0182] In some embodiments, the cells are human cells. In some embodiments, the cell genome or the vector contained in the cell is modified. In some embodiments, the cell genome is modified ex vivo. In some embodiments, the cell genome is modified in vivo.

[0183] In some embodiments, the manipulated guide polynucleotide targets genes in cells. In some embodiments, the manipulated guide polynucleotide targets genes in mammalian cells. In some embodiments, the mammalian cells are those of pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, or humans.

[0184] In some embodiments, the target gene is HAO1. In some embodiments, the gRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 70% identity with any one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 75% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 80% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 85% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 90% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 91% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 92% identity with one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence that has at least about 93% identity with one of sequence numbers 251-260 and 271-274.In some embodiments, the gRNA contains a sequence having at least about 94% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 95% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 96% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 97% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 98% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence having at least about 99% identity to one of sequence numbers 251-260 and 271-274. In some embodiments, the gRNA contains a sequence that has 100% identity with any one of sequence numbers 251-260 and 271-274.

[0185] In some embodiments, the gRNA hybridizes or targets a sequence complementary to an HAO1 sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 70% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 75% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 80% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 85% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 90% identity with one of the sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 91% identity with one of the sequence numbers 261-270 and 275-278.In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 92% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 93% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 94% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 95% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 96% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 97% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 98% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence having at least about 99% identity with one of sequence numbers 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to the HAO1 sequence that has 100% identity with any one of sequence numbers 261-270 and 275-278.

[0186] In some embodiments, the target gene is a TTR. In some embodiments, the gRNA contains a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity to any one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence having at least about 70% identity to any one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence having at least about 75% identity to any one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 80% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 85% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 90% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 91% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 92% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 93% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 94% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 95% identity with one of sequence numbers 279-290.In some embodiments, the gRNA contains a sequence that has at least about 96% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 97% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 98% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has at least about 99% identity with one of sequence numbers 279-290. In some embodiments, the gRNA contains a sequence that has 100% identity with one of sequence numbers 279-290.

[0187] In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, 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%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 70% identity with any one of SEQ ID NOs. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 75% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 80% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 85% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 90% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 91% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence that has at least approximately 92% identity with one of sequence numbers 291-302.In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 93% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 94% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 95% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 96% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 97% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 98% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 99% identity with one of sequence numbers 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having 100% identity with one of sequence numbers 291-302. [Examples]

[0188] Example 1-II Identification of a CRISPR Effector in Silico The presumed type II CRISPR effect pedal was identified. e value ≤ 1 × 10⁻⁶ 5 The obtained homologs were filtered to include homologs with a length ≥ 500 aa and the relevant CRISPR locus predicted using minCED. These effectors were dereplicated with 99% amino acid identity (AAI) and comprehensively aligned to construct a phylogenetic tree.

[0189] Type II effectors were identified from the MG171, MG177, MG183, and MG21 families (SEQ ID NOs: 105-146). All type II effectors possess the catalytic residue necessary for activity and have a length range of 1,064-1,154 aa.

[0190] Example 2 - sgRNA design and in vitro activity of MG171 and MG177 nucleases sgRNA design

[0191] TracrRNAs were determined. TracrRNAs (sequence numbers 147-151, 157-163, 171-192, and 215-222) and repeats (sequence numbers 152-156, 164-170, 193-214, and 223-230) were folded, trimmed, and ligated to tetraloop sequences GAAA, or TTCG if GAAA altered the fold, to form sgRNAs. Effectors were screened with eight sgRNA designs (sequence numbers 231-250) consisting of short and long scaffolds, 40% and 67% GC content spacers, and 20nt and 24nt spacers.

[0192] In vitro activity assay

[0193] CRISPR effector nucleases were expressed in an in vitro expression system using a 5 nM PCR-generated DNA template. After expression, the nuclease reaction mixture was diluted 10-fold and subjected to a nuclease reaction for 1 hour in a mixture containing 5 nM protospacer-adjacent motif (PAM) library DNA plasmid and 50 nM sgRNA in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCl2. The PAM plasmid digested by the nuclease reaction was washed with size-selective purification beads and eluted in TE buffer. The digested PAM plasmid (15 nM) was blunt-end ligated to a double-stranded adapter oligo (150 nM) containing T4 ligase in 1 × T4 ligase buffer. The ligated product was sequenced using 150 bp single-read amplicon sequencing. The obtained reads were filtered by a quality score of >20. PAM was identified by mapping readouts to a PAM plasmid backbone requiring a perfect match. A PAM sequence logo (SeqLogo) was generated, and the PAM sequence was determined by the height of each nucleotide. The cleavage site was identified by calculating the distance between the PAM and the ligated adapter.

[0194] In vitro cleavage assays demonstrated that MG171-1, MG171-2, MG171-3, MG171-4, MG171-5, MG177-2, MG177-3, MG177-4, MG177-5, and MG177-6 are active type II CRISPR effector nucleases. An exemplary PAM and cleavage site for the MG171-1 effector nuclease are shown in Figure 1.

[0195] Example 3 - Intracellular editing activity of MG nuclease mRNA synthesis

[0196] Nucleic acids encoding MG21-1 or MG23-1 were cloned into a pUC19 plasmid containing an RNA-pol T7 promoter, 5'UTR and 3'UTR, and a 100nt poly-A tail. 100 μg of plasmid was digested with SapI to linearize it. The plasmid was purified with phenol / chloroform and precipitated with 70% ethanol. The DNA pellet was resuspended in 20 μL of nuclease-free water. For in vitro transcription, 1 μg of linearized plasmid DNA was added to 20 μL of reaction mixture containing 1X reaction buffer (40 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 50 mM NaCl, 2.5 mM spermidine, 1 mM DTT) and 750 units of Hi-T7 RNA polymerase. The reaction mixture was incubated at 50°C for 1 hour, and the resulting transcription mRNA was purified.

[0197] Mammalian cell activity assay

[0198] Hep3B cells were cultured in EMEM + 10% FBS at 37°C and 5% CO2. Cells were nucleofected using either MG21-1 or MG23-1 mRNA with either HAO1 or TTR (500 ng mRNA / 150 pmol guide) in Hep3B cells (100,000 cells) using an electroporator. Cells were harvested 72 hours after nucleofection and genomic DNA was extracted. Individual target sequences for each guide RNA were amplified using Q5 High-Fidelity DNA Polymerase, using PCR primers suitable for use in NGS-based DNA sequencing. The amplicons were sequenced and analyzed for gene editing.

[0199] For both HAO1 and TTR, MG21-1 resulted in up to 97% indels (Figures 2 and 3), while MG23-1 resulted in up to 63% indels (Figure 4). These results demonstrate that MG21-1 and MG23-1 have the ability to reliably edit genes at therapeutically relevant loci.

[0200] [Table 2-1]

[0201] [Table 2-2]

[0202] [Table 2-3]

[0203] [Table 2-4]

[0204] [Table 2-5]

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[0206] [Table 2-7]

[0207] [Table 2-8]

[0208] [Table 2-9]

[0209] Table 2-10

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[0278] Table 2-79

[0279] Table 2-80

[0280] While preferred embodiments of the Disclosure are illustrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. The Disclosure is not intended to be limited by the specific examples provided herein. Although the Disclosure is described with reference to the preceding specification, the descriptions and illustrations of embodiments of the Disclosure are not intended to be constrained. Numerous variations, alterations, and substitutions will be recalled herein by those skilled in the art without departing from the Disclosure. Furthermore, it should be understood that all aspects of the Disclosure are not limited to the specific descriptions, configurations, or relative proportions described herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in practicing the Disclosure. Therefore, the Disclosure is intended to encompass any such alternatives, modifications, alterations, or equivalents. The subsequent claims are intended to define the scope of the Disclosure, and that the methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. A manipulated nuclease system, a) Endonucleases containing a sequence having at least 80% sequence identity with any one of sequence numbers 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.

2. The manipulated nuclease system according to claim 1, wherein the endonuclease comprises a sequence having at least 90% sequence identity with any one of sequence numbers 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138.

3. The manipulated nuclease system according to claim 1, wherein the endonuclease contains a sequence having 100% sequence identity with any one of sequence numbers 1-5, 139-146, 303, 304, 6, 7-11, 12-14, 105-109, 110-116, and 117-138.

4. The manipulated nuclease system according to any one of claims 1 to 3, wherein the manipulated guide polynucleotide comprises crRNA and tracrRNA.

5. The manipulated nuclease system according to claim 4, wherein the tracrRNA includes a sequence having at least 90% sequence identity with any one of sequence numbers 215-222, 52, 147-151, 157-163, and 171-192.

6. The manipulated nuclease system according to claim 4, wherein the tracrRNA contains a sequence having 100% sequence identity with any one of sequence numbers 215-222, 52, 147-151, 157-163, and 171-192.

7. The manipulated nuclease system according to claims 1 to 6, wherein the manipulated guide polynucleotide is a single guide nucleic acid.

8. The manipulated nuclease system according to claims 1 to 6, wherein the manipulated guide polynucleotide is a double guide nucleic acid.

9. The manipulated nuclease system according to any one of claims 1 to 8, wherein the manipulated guide polynucleotide is RNA.

10. The operated nuclease system according to any one of claims 1 to 9, wherein the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease.

11. The manipulated nuclease system according to any one of claims 1 to 10, wherein the endonuclease has less than 80% identity with Cas9 endonuclease.

12. The manipulated nuclease system according to any one of claims 1 to 11, wherein the endonuclease is non-covalently bound to the manipulated guide polynucleotide.

13. The manipulated nuclease system according to any one of claims 1 to 11, wherein the endonuclease is covalently bound to the manipulated guide polynucleotide.

14. A manipulated nuclease system, a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 139-146 and 303, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs. 215 to 222.

15. A manipulated nuclease system, a) An endonuclease containing a sequence having at least 80% sequence identity with any one of sequence numbers 7 to 11, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with SEQ ID NO:

52.

16. A manipulated nuclease system, a) An endonuclease containing a sequence having at least 80% sequence identity with any one of sequence numbers 105 to 109, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 147 to 151.

17. A manipulated nuclease system, a) an endonuclease containing a sequence having at least 80% sequence identity with any one of sequence numbers 110 to 116, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of sequence numbers 157 to 163.

18. A manipulated nuclease system, a) An endonuclease containing a sequence having at least 80% sequence identity with any one of sequence numbers 117 to 138, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 171 to 192.

19. The manipulated nuclease system according to claims 14 to 18, wherein the manipulated guide polynucleotide is a single guide nucleic acid.

20. The manipulated nuclease system according to claims 14 to 18, wherein the manipulated guide polynucleotide is a double guide nucleic acid.

21. The manipulated nuclease system according to any one of claims 14 to 20, wherein the manipulated guide polynucleotide is RNA.

22. The operated nuclease system according to any one of claims 1 to 20, wherein the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease.

23. The manipulated nuclease system according to any one of claims 14 to 22, wherein the endonuclease has less than 80% identity with Cas9 endonuclease.

24. The manipulated nuclease system according to any one of claims 14 to 23, wherein the endonuclease is non-covalently bound to the manipulated guide polynucleotide.

25. The manipulated nuclease system according to any one of claims 14 to 23, wherein the endonuclease is covalently bound to the manipulated guide polynucleotide.

26. The manipulated nuclease system according to any one of claims 14 to 23, wherein the endonuclease is fused to the manipulated guide polynucleotide.

27. A method for modifying a target nucleic acid sequence, comprising contacting the target nucleic acid sequence with an engineered nuclease system according to any one of claims 1 to 26.

28. The method according to claim 27, wherein modifying the target nucleic acid sequence includes binding, nicking, or cleaving the target nucleic acid sequence.

29. The method according to any one of claims 27 to 28, wherein the target nucleic acid sequence includes genomic DNA, viral DNA, viral RNA, or bacterial DNA.

30. The method according to any one of claims 27 to 29, wherein the modification is performed in vitro.

31. The method according to any one of claims 27 to 29, wherein the modification is in vivo.

32. The method according to any one of claims 27 to 29, wherein the modification is exvivo.

33. The method according to any one of claims 27 to 32, wherein the gRNA is encoded by a sequence having one of sequence numbers 251-260, 271-274, and 279-290.

34. The method according to any one of claims 27 to 32, wherein the target nucleic acid sequence includes a sequence having one of sequence numbers 261 to 270, 275 to 278, and 291 to 302.

35. A method for modifying a target nucleic acid sequence in mammalian cells, comprising contacting the mammalian cells with an engineered nuclease system according to any one of claims 1 to 26.

36. The method according to claim 35, further comprising selecting cells containing the above-mentioned modifications.

37. A method for modifying HAO1, a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 139-146 and 303, b) A method comprising contacting HAO1 using an engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs. 215 to 222.

38. The method according to claim 37, wherein the manipulated guide polynucleotide is encoded by a sequence having any one of sequence numbers 251 to 260.

39. The method according to claim 37, wherein the target nucleic acid sequence includes a sequence having any one of sequence numbers 261 to 270.

40. A method for modifying HAO1, a) An endonuclease containing a sequence having at least 80% sequence identity with sequence number 304, b) A method comprising contacting HAO1 using an engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

41. The method according to claim 40, wherein the manipulated guide polynucleotide is encoded by a sequence having any one of sequence numbers 271 to 274.

42. The method according to claim 40, wherein the target nucleic acid sequence includes a sequence having any one of sequence numbers 275 to 278.

43. A method for modifying TTR, a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 139-146 and 303, b) A method comprising contacting a TTR using an engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215 to 222.

44. The method according to claim 43, wherein the manipulated guide polynucleotide is encoded by a sequence having any one of sequence numbers 279 to 290.

45. The method according to claim 43, wherein the target nucleic acid sequence includes a sequence having any one of sequence numbers 291 to 302.

46. A cell comprising the manipulated nuclease system according to any one of claims 1 to 26.

47. The cell according to claim 46, wherein the cell is a eukaryotic cell.

48. The cell according to claim 46, wherein the cell is a mammalian cell.

49. The cell according to claim 46, wherein the cell is an immortalized cell.

50. The cell according to claim 46, wherein the cell is an insect cell.

51. The cell according to claim 46, wherein the cell is a yeast cell.

52. The cell according to claim 46, wherein the cell is a plant cell.

53. The cell according to claim 46, wherein the cell is a fungal cell.

54. The cell according to claim 46, wherein the cell is a prokaryotic cell.

55. The cell according to claim 46, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC1, BSC40, BMT10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

56. The cell according to claim 46, wherein the cell is an engineered cell.

57. The cell according to claim 46, wherein the cell is a stable cell.

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