Enzymes with ruvc domains

Abstract

The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.

Description

Attorney Docket No.: MTG-018WO ENZYMES WITH RUVC DOMAINS CROSS-REFERENCE

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

[0002] Described herein, in certain embodiments, are engineered nuclease systems comprising: a) an endonuclease comprising 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 to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease comprises 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 comprises 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 engineered guide polynucleotide comprises a crRNA and a tracrRNA. In some embodiments, the tracrRNA comprises 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 comprises 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 engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 80% identity to a Cas9 endonuclease. In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.

[0003] Described herein, in certain embodiments, are engineered nuclease systems 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 - 1 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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: 215-222.

[0004] Described herein, in certain embodiments, are engineered nuclease systems comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7-11; and b) 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 SEQ ID NO: 52.

[0005] Described herein, in certain embodiments, are engineered nuclease systems comprising: a) an endonuclease comprising 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 147-151.

[0006] Described herein, in certain embodiments, are engineered nuclease systems 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 157-163.

[0007] Described herein, in certain embodiments, are engineered nuclease systems 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 171-192.

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

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

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

[0011] In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a - 2 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease.

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

[0013] In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide.

[0014] In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.

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

[0016] Described herein, in certain embodiments, are methods for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system described herein. In some embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence comprises 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 comprises a sequence of any one of SEQ ID NOs: 251-260, 271-274, and 279-290. In some embodiments, the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.

[0017] Described herein, in certain embodiments, are methods of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system described herein. In some embodiments, the methods further comprises selecting cells comprising the modification.

[0018] Described herein, in certain embodiments, are methods of modifying HAO1 comprising contacting HAO1 using 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, the engineered guide polynucleotide comprising 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 a sequence of any one of SEQ ID NOs: 251-260. In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 261-270. - 3 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0019] Described herein, in certain embodiments, are methods of modifying HAO1 comprising contacting HAO1 using an engineered nuclease system comprising: a) an endonuclease comprising a sequence 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 a sequence of any one of SEQ ID NOs: 271-274. In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 275-278.

[0020] Described herein, in certain embodiments, are methods of modifying TTR comprising contacting TTR using 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, the engineered guide polynucleotide comprising 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 a sequence of any one of SEQ ID NOs: 279-290. In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 291-302.

[0021] Described herein, in certain embodiments, are cells comprising the engineered nuclease system described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell.

[0022] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. - 4 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the 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 by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0024] FIG.1A depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG171-1. FIG.1B depicts the cleavage position 5 nt from the PAM obtained from the in vitro cleavage assay and NGS sequencing.

[0025] FIG.2 depicts analysis of the gene-editing outcomes at the DNA level for editing of human HAO1 by MG21-1 in Hep3B cells.

[0026] FIG.3 depicts analysis of the gene-editing outcomes at the DNA level for editing of human TTR by MG21-1 in Hep3B cells.

[0027] FIG.4 depicts analysis of the gene-editing outcomes at the DNA level for editing of human HAO1 by MG23-1 in Hep3B cells. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0028] The Sequence Listing filed herewith provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions and systems according to the disclosure. Below are exemplary descriptions of sequences therein.

[0029] MG2

[0030] SEQ ID NOs: 15-16 show the peptide sequences of PAM-interacting domains of MG2 nucleases.

[0031] SEQ ID NOs: 53-66 show the nucleotide sequences of target sites of MG2 nucleases.

[0032] MG4

[0033] SEQ ID NOs: 17-22 show the peptide sequences of PAM-interacting domains of MG4 nucleases.

[0034] SEQ ID NOs: 67-76 show the nucleotide sequences of target sites of MG4 nucleases.

[0035] MG5

[0036] SEQ ID NOs: 1-5 show the full-length peptide sequences of MG5 nucleases. - 5 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0037] SEQ ID NOs: 23-27 show the peptide sequences of PAM-interacting domains of MG5 nucleases.

[0038] MG6

[0039] SEQ ID NOs: 28-36 show the peptide sequences of PAM-interacting domains of MG6 nucleases.

[0040] SEQ ID NOs: 77-86 show the nucleotide sequences of target sites of MG6 nucleases.

[0041] MG7

[0042] SEQ ID NO: 51 shows the nucleotide sequence of an MG7 tracrRNA derived from the same locus as an MG7 nuclease.

[0043] SEQ ID NO: 37 shows the peptide sequence of a PAM-interacting domain of an MG7 nuclease.

[0044] SEQ ID NOs: 87-95 show the nucleotide sequences of target sites of MG7 nucleases.

[0045] MG16

[0046] SEQ ID NO: 38 shows the peptide sequence of a PAM-interacting domain of an MG16 nuclease.

[0047] SEQ ID NOs: 96-104 show the nucleotide sequences of target sites of MG16 nucleases.

[0048] MG21

[0049] SEQ ID NOs: 139-146 and 303 show the full-length peptide sequences of MG21 nucleases.

[0050] SEQ ID NOs: 215-222 show the nucleotide sequences of MG21 tracrRNAs derived from the same loci as MG21 nucleases.

[0051] SEQ ID NOs: 223-230 show the nucleotide sequences of MG21 CRISPR repeats.

[0052] MG23

[0053] SEQ ID NO: 304 shows the full-length peptide sequence of an MG23 nuclease.

[0054] MG49

[0055] SEQ ID NO: 39 shows the peptide sequence of a PAM-interacting domain of an MG49 nuclease.

[0056] MG86

[0057] SEQ ID NO: 6 shows the full-length peptide sequence of an MG86 nuclease.

[0058] SEQ ID NO: 40 shows the peptide sequence of a PAM-interacting domain of an MG86 nuclease.

[0059] MG112

[0060] SEQ ID NOs: 7-11 show the full-length peptide sequences of MG112 nucleases. - 6 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0061] SEQ ID NOs: 41-45 show the peptide sequences of PAM-interacting domains of MG112 nucleases.

[0062] nnRTCY is the nucleotide sequence of a PAM of an MG112 nuclease.

[0063] SEQ ID NO: 50 shows the nucleotide sequence of an sgRNA engineered to function with an MG112 nuclease.

[0064] SEQ ID NO: 52 show the nucleotide sequence of an MG7 tracrRNA derived from the same loci as MG112 nucleases above.

[0065] MG116

[0066] SEQ ID NOs: 12-14 show the full-length peptide sequences of MG116 nucleases.

[0067] SEQ ID NOs: 46-48 show the peptide sequences of PAM-interacting domains of MG116 nucleases.

[0068] MG171

[0069] SEQ ID NOs: 105-109 show the full-length peptide sequences of MG171 nucleases.

[0070] SEQ ID NOs: 147-151 show the nucleotide sequences of MG171 tracrRNAs derived from the same loci as MG171 nucleases.

[0071] SEQ ID NOs: 152-156 show the nucleotide sequences of MG171 CRISPR repeats.

[0072] SEQ ID NOs: 231-240 show the nucleotide sequences of sgRNAs engineered to function with an MG171 nuclease.

[0073] MG177

[0074] SEQ ID NOs: 110-116 show the full-length peptide sequences of MG177 nucleases.

[0075] SEQ ID NOs: 157-163 show the nucleotide sequences of MG177 tracrRNAs derived from the same loci as MG177 nucleases.

[0076] SEQ ID NOs: 164-170 show the nucleotide sequences of MG177 CRISPR repeats.

[0077] SEQ ID NOs: 241-250 show the nucleotide sequences of sgRNAs engineered to function with an MG171 nuclease.

[0078] MG183

[0079] SEQ ID NOs: 117-138 show the full-length peptide sequences of MG183 nucleases.

[0080] SEQ ID NOs: 171-192 show the nucleotide sequences of MG183 tracrRNAs derived from the same loci as MG183 nucleases.

[0081] SEQ ID NOs: 193-214 show the nucleotide sequences of MG183 CRISPR repeats.

[0082] HAO1 Targeting with MG21-1

[0083] SEQ ID NOs: 251-260 show the nucleotide sequences of sgRNAs engineered to function with an MG21-1 nuclease in order to target human HAO1. - 7 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0084] SEQ ID NOs: 261-270 show the DNA sequences of human HAO1 target sites.

[0085] HAO1 Targeting with MG23-1

[0086] SEQ ID NOs: 271-274 show the nucleotide sequences of sgRNAs engineered to function with an MG31-1 nuclease in order to target human HAO1.

[0087] SEQ ID NOs: 275-278 show the DNA sequences of human HAO1 target sites.

[0088] TTR Targeting with MG21-1

[0089] SEQ ID NOs: 279-290 show the nucleotide sequences of sgRNAs engineered to function with an MG21-1 nuclease in order to target human TTR.

[0090] SEQ ID NOs: 291-302 show the DNA sequences of human TTR target sites. DETAILED DESCRIPTION

[0091] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0092] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0093] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0094] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA.

[0095] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the - 8 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0096] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0097] The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels of nucleotides include but are not limited 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). Specific examples of fluorescently labeled nucleotides include [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 available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, - 9 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Arlington Heights, IL; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6- dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′- dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, 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 Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically-modified nucleotide is biotin-dNTP. Non- limiting 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).

[0098] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi- stranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-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 a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and / or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, - 10 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0099] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. 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 with or without secondary or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, refer 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 a chemical moiety not naturally present on the amino acid. The term “amino acid” includes both D-amino acids and L-amino acids.

[0100] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element. The effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, a regulatory element, which may comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0101] A “functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence. A biological activity of a DNA sequence includes its ability to influence expression in a manner attributed to the full-length sequence. - 11 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0102] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring. An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring a new function or property. An “engineered” system comprises at least one engineered component.

[0103] The term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA. tracrRNA interacts with the CRISPR (cr) RNA to form a guide nucleic acid (e.g., guide RNA or gRNA) that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid.

[0104] As used herein, a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated 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 can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to 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. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment referred to as a “nucleic acid- - 12 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO targeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.” A nucleic acid-targeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”

[0105] As used herein, the term “complex” refers to a joining of at least two components. The two components may each retain the properties / activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0106] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov) ; CLUSTALW with parameters of the Smith- Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters.

[0107] Included in the current disclosure are variants of any of the enzymes described herein with one or more conservative amino acid substitutions. Such conservative 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 accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins - 13 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g. non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with 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%, at least about 99% identity to any one of the endonuclease protein sequences described herein (e.g. MG2, MG4, MG5, MG6, MG7, MG16, MG21, MG23, MG49, MG86, MG112, MG116, MG171, MG177, or MG183 family endonucleases described herein). In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease are not disrupted. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of at least one of the residues predicted as essential. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of all of the residues predicted as essential.

[0108] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another: 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) Overview

[0109] The discovery of new Cas enzymes with unique functionality and structure may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving - 14 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR / Cas systems known and speed the discovery of new oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX / CasY CRISPR systems from metagenomic analysis of natural microbial communities.

[0110] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40 bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins / enzymes. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.

[0111] Class 1 CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.

[0112] Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex - 15 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO called Cascade, which also comprises a nuclease (Cas3) that protein component of the crRNA- directed nuclease complex. Cas 1 nucleases function primarily as DNA nucleases.

[0113] Type III CRISPR systems may be characterized by the presence of a central nuclease, known as Cas10, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre- crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0114] Type IV CRISPR-Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.

[0115] Class II CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.

[0116] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature Cas9 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 adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC- like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0117] Type V CRISPR-Cas systems are characterized by a nuclease effector (Cas12) structure similar to that of Type II / Cas9, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself (Cas12) to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again known as DNA nucleases. Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., - 16 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cas12a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.

[0118] Type VI CRISPR-Cas systems are unique in that they appear to be the only class so far known as RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (Cas13) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to not need a tracrRNA for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.

[0119] Because of their simpler architecture, Class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease / genome editing applications.

[0120] One of the early adaptations of such a system for in vitro use involved (i) recombinantly-expressed, purified full-length Cas9 (e.g., a Class 2, Type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature ~42 nt crRNA bearing a ~20 nt 5’ sequence complementary to the target DNA sequence desired to be cleaved followed by a 3’ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg2+. A later improved, engineered system involved the crRNA of (ii) joined to the 5’ end of (iii) by a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.

[0121] Such engineered 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 with 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 an sgRNA (having a 5’ sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence joined to a 3’ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter). MG Enzymes

[0122] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease and an engineered guide polynucleotide. 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 - 17 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO domain. In some embodiments, the endonuclease is a double strand nuclease. In some embodiments, the endonuclease is catalytically dead. In some embodiments, the endonuclease is a double strand nuclease. In some embodiments, the endonuclease is modified. In some embodiments, the endonuclease is modified resulting in an endonuclease with nickase activity.. In some embodiments, the modified endonuclease is a site-directed nickase

[0123] In some embodiments, the endonuclease is a MG2 endonuclease. In some embodiments, the endonuclease is a MG4 endonuclease. In some embodiments, the endonuclease is a MG5 endonuclease. In some embodiments, the endonuclease is a MG6 endonuclease. In some embodiments, the endonuclease is a MG7 endonuclease. In some embodiments, the endonuclease is a MG16 endonuclease. In some embodiments, the endonuclease is a MG21 endonuclease. In some embodiments, the endonuclease is a MG23 endonuclease. In some embodiments, the endonuclease is a MG49 endonuclease. In some embodiments, the endonuclease is a MG86 endonuclease. In some embodiments, the endonuclease is a MG112 endonuclease. In some embodiments, the endonuclease is a MG116 endonuclease. In some embodiments, the endonuclease is a MG171 endonuclease. In some embodiments, the endonuclease is a MG177 endonuclease. In some embodiments, the endonuclease is a MG183 endonuclease.

[0124] In some embodiments, the engineered nuclease system is discovered through metagenomic sequencing. In some embodiments, the metagenomic sequencing is conducted on samples collected from various environments. In some embodiments, the environment is a human microbiome, an animal microbiome, an environment with high temperatures, an environment with low temperatures, or sediment.

[0125] In some embodiments, the endonuclease is a MG5 endonuclease (i.e., SEQ ID NOs: 1- 5). In some embodiments, the endonuclease comprises 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: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about - 18 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 85% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1-5.

[0126] In some embodiments, the endonuclease is a MG21 endonuclease (i.e., SEQ ID NOs: 139-146 and 303). In some embodiments, the endonuclease comprises 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: 139- 146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID - 19 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO NOs: 139-146 and 303. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 139-146 and 303.

[0127] In some embodiments, the endonuclease is a MG23 endonuclease (i.e., SEQ ID NO: 304). In some embodiments, the endonuclease comprises 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 comprises a sequence having at least about 70% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 304. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 304.

[0128] In some embodiments, the endonuclease is a MG86 endonuclease (i.e., SEQ ID NO: 6). In some embodiments, the endonuclease comprises 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 comprises a sequence having at least about 70% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 6. In some embodiments, the endonuclease - 20 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO comprises a sequence having at least about 90% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 6. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 6.

[0129] In some embodiments, the endonuclease is a MG112 endonuclease (i.e., SEQ ID NOs: 7-11). In some embodiments, the endonuclease comprises 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: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 7-11. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 7-11.

[0130] In some embodiments, the endonuclease is a MG116 endonuclease (i.e., SEQ ID NOs: 12-14). In some embodiments, the endonuclease comprises a sequence having at least about 30%, - 21 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 12-14. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 12-14.

[0131] In some embodiments, the endonuclease is a MG171 endonuclease (i.e., SEQ ID NOs: 105-109). In some embodiments, the endonuclease comprises 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. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID - 22 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 105-109. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 105-109.

[0132] In some embodiments, the endonuclease is a MG177 endonuclease (i.e., SEQ ID NOs: 110-116). In some embodiments, the endonuclease comprises 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. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 110-116. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 110-116.

[0133] In some embodiments, the endonuclease is a MG183 endonuclease (i.e., SEQ ID NOs: 117-138). In some embodiments, the endonuclease comprises a sequence having at least about - 23 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 117-138. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 117-138.

[0134] In some embodiments, the endonuclease comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is at an N-terminus of the endonuclease. In some embodiments, the NLS is at a C-terminus of the endonuclease. In some embodiments, the NLS is at an N-terminus and a C-terminus of the endonuclease.

[0135] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 305- 350, or 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 SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: - 24 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 305-350. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 305-350. In some embodiments, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 305-350. Table 1: Example NLS Sequences that can be used with Cas Effectors According to the Disclosure Source NLS amino acid sequence SEQ ID NO:- 25 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Source NLS amino acid sequence SEQ ID NO:Guide Polynucleotides - 26 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0136] In some embodiments, the engineered nuclease system disclosed herein comprises an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.

[0137] In some embodiments, the guide RNAs comprise various structural elements including but not limited to: a spacer sequence which binds to the protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the guide RNA comprises a crRNA comprising a spacer sequence. In some embodiments, the guide RNA comprises a tracrRNA or a modified tracrRNA.

[0138] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to SEQ ID NO: 51. In - 27 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to SEQ ID NO: 51.

[0139] In some embodiments, the tracrRNA comprises 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 SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a - 28 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO sequence having at least about 99% identity to SEQ ID NO: 51. In some embodiments, the tracrRNA comprises a sequence having 100% identity to SEQ ID NO: 51.

[0140] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 215-222. In some embodiments, - 29 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 215-222.

[0141] In some embodiments, the tracrRNA comprises 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 SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 215-222. - 30 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0142] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence - 31 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO comprising at least about 60-100 consecutive nucleotides having 100% identity to SEQ ID NO: 52.

[0143] In some embodiments, the tracrRNA comprises 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 SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having at least about 99% identity to SEQ ID NO: 52. In some embodiments, the tracrRNA comprises a sequence having 100% identity to SEQ ID NO: 52.

[0144] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to any one of SEQ ID NOs: 147-151. In some embodiments, - 32 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 147-151.

[0145] In some embodiments, the tracrRNA comprises 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%, - 33 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 147-151.

[0146] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 157-163. In some embodiments, - 34 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 157-163.

[0147] In some embodiments, the tracrRNA comprises 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%, - 35 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 157-163.

[0148] In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 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 to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 171-192. In some embodiments, - 36 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 171-192.

[0149] In some embodiments, the tracrRNA comprises 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 SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 70% - 37 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 171-192.

[0150] In some embodiments, the engineered nuclease system disclosed herein comprises an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.

[0151] In some embodiments, the engineered guide polynucleotide (i.e., SEQ ID NO: 50) functions with a MG112 nuclease. In some embodiments, the engineered guide polynucleotide comprises 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 SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least - 38 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO about 75% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to SEQ ID NO: 50. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to SEQ ID NO: 50.

[0152] In some embodiments, the engineered guide polynucleotide (i.e., SEQ ID NOs: 231- 240) functions with a MG171 nuclease. In some embodiments, the engineered guide polynucleotide comprises 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 SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered - 39 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 231-240. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 231-240.

[0153] In some embodiments, the engineered guide polynucleotide (i.e., SEQ ID NOs: 241- 250) functions with a MG177 nuclease. In some embodiments, the engineered guide polynucleotide comprises 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 SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ - 40 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 241-250. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 241-250.

[0154] In some embodiments, the engineered guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the engineered guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter-nucleoside linkers of the engineered guide polynucleotide, or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0155] In some embodiments, the engineered guide polynucleotide comprises modifications to a ribose sugar or nucleobase. In some embodiments, the engineered guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring - 41 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids.

[0156] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than -H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’-4’ biradical bridged) nucleosides. Examples of 2’-substituted modified nucleosides comprise, 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 comprises 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).

[0157] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, the engineered guide polynucleotide comprises only modified sugars. In certain embodiments, the engineered guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2’-O- methoxyethyl group. In some embodiments, the engineered guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications.

[0158] In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises - 42 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0159] In some embodiments, the engineered guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the engineered guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the engineered guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the engineered guide polynucleotide is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the engineered guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length. MG Systems

[0160] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease comprising and an engineered guide polynucleotide. In some embodiments, the engineered guide polynucleotide comprises a tracrRNA. In some embodiments, the engineered guide polynucleotide comprises a guide nucleic acid (e.g., gRNA). In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0161] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 215-222. In some - 43 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 215-222. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 139-146 and 303 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 215-222.

[0162] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 7-11 and an - 44 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO engineered polynucleotide comprising a sequence having at least about 70% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 75% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 80% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 85% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 90% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 95% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 96% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 97% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 98% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising a sequence having at least about 99% identity to SEQ ID NO: 52. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 7-11 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 52. - 45 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0163] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide - 46 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 147-151. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 105-109 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 147-151.

[0164] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of - 47 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 157-163. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 110-116 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 157-163.

[0165] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least - 48 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO about at least about 97% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 171-192. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 117-138 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 171-192. Cells

[0166] Described herein, in certain embodiments, is a cell comprising the systems described herein.

[0167] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NS0), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or a Heliothis virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., a E. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell. - 49 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0168] In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or derivative thereof. Delivery and Vectors

[0169] Disclosed herein, in some embodiments, are nucleic acid sequences encoding an engineered nuclease system disclosed herein.

[0170] In some embodiments, the nucleic acid encoding the engineered nuclease system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.

[0171] In some embodiments, the nucleic acid encoding the engineered nuclease system is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), P1-derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: 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, pRI 101-AN DNA, pCambia2301,pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0172] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, 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 a U6 promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is encoded by a sequence of any one of SEQ ID NOs: 190-191, or 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%, - 50 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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 of any one of SEQ ID NOs: 190-191.

[0173] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a 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 an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.

[0174] 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-rh10, 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, 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.

[0175] 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 - 51 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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.

[0176] 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 - 52 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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.

[0177] In some embodiments, the nucleic acid encoding the engineered nuclease system is delivered by a non-nucleic acid-based 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 associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0178] In some embodiments, the fusion protein or genome editing system is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein or genome editing system is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes a fusion protein or genome editing system. For example, a cell is transduced (e.g., with a virus encoding a fusion protein or genome editing system), or transfected (e.g., with a plasmid encoding a fusion protein or genome editing system) with a nucleic acid that encodes a fusion protein or genome editing system, or the translated fusion protein or genome editing system. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing a fusion protein or genome editing system or containing a fusion protein or genome editing system are transduced or transfected with one or more gRNA molecules, for example, when the fusion protein or genome editing system comprises a CRISPR nuclease. In some embodiments, a plasmid expressing a fusion protein or genome editing system is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing. - 53 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0179] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91 / 17424 and WO 91 / 16024. In some embodiments, the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0180] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817. Methods of Use

[0181] Described herein, in certain embodiments, are methods for modifying a target nucleic acid comprising 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.

[0182] In some embodiments, the methods are used to introduce a modification in the genome of a cell. In some embodiments, the modification is an insertion, deletion, or mutation. In some embodiments, the methods are used to introduce site-directed insertions, deletions, and / or mutations in the genome of a cell (for example an insertion and a mutation). In some embodiments, the methods are used in combination with a nucleic acid template to facilitate site- directed insertions into the genome of a cell.

[0183] In some embodiments, the cell is a human cell. In some embodiments, the cell genome or a vector comprised 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.

[0184] In some embodiments, the engineered guide polynucleotide targets a gene in a cell. In some embodiments, the engineered guide polynucleotide targets a gene in a mammalian cell. In - 54 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO some embodiments, the mammalian cell is a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, or a human cell.

[0185] In some embodiments, the target gene is HAO1. In some embodiments, the gRNA comprises 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 SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 251-260 and 271-274. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 251-260 and 271-274. - 55 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0186] In some embodiments, the gRNA hybridizes or targets a sequence complementary to a 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 to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 70% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 75% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 80% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 85% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 90% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 91% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 92% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 93% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 94% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 95% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 96% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 97% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 98% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the - 56 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having at least about 99% identity to any one of SEQ ID NOs: 261-270 and 275-278. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a HAO1 sequence having 100% identity to any one of SEQ ID NOs: 261-270 and 275-278.

[0187] In some embodiments, the target gene is TTR. In some embodiments, the gRNA comprises 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 SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 279-290. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 279-290.

[0188] 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 - 57 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 70% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 75% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 80% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 85% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 90% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 91% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 92% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 93% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 94% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 95% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 96% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 97% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 98% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having at least about 99% identity to any one of SEQ ID NOs: 291-302. In some embodiments, the gRNA hybridizes or targets a sequence complementary to a TTR sequence having 100% identity to any one of SEQ ID NOs: 291-302. - 58 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO EXAMPLES Example 1 – In silico identification of Type II CRISPR effectors

[0189] Putative Type II CRISPR effectors were identified. The resulting homologs were filtered to include those with an e-value ≤ 1 x 10-5, a length ≥ 500 aa, and an associated CRISPR loci predicted using minCED. The effectors were dereplicated at 99% amino acid identity (AAI), globally aligned, and a phylogenetic tree was constructed.

[0190] Type II effectors were identified from MG171, MG177, MG183, and MG21 families (SEQ ID NOs: 105-146). All the Type II effectors have the catalytic residues required for activity and range in length between 1,064 and 1,154 aa. Example 2 – sgRNA design and in vitro activity of MG171 and MG177 nucleases

[0191] sgRNA Design

[0192] TracrRNAs were determined. TracrRNAs (SEQ ID NOs: 147-151, 157-163, 171-192, and 215-222) and repeats (SEQ ID NOs: 152-156,164-170,193-214, and 223-230) were folded, trimmed, and connected with a tetraloop sequence GAAA, or TTCG if GAAA altered the fold to form sgRNAs. The effectors were screened with eight sgRNA designs (SEQ ID NOs: 231-250) consisting of a short and long scaffold, 40% and 67% GC content spacers, and 20 nt and 24 nt spacers.

[0193] In vitro activity assay

[0194] CRISPR effector nucleases were expressed in an in vitro expression system using 5 nM of a PCR-generated DNA template. After expression, the nuclease reaction mixtures were diluted 10-fold and subjected to a nuclease reactions 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 digested PAM plasmids in the nuclease reactions were cleaned using size selection purification beads and eluted in TE buffer. The digested PAM plasmids (15 nM) were blunt-end ligated to double-stranded adapter oligos (150 nM) with T4 ligase in 1X T4 ligase buffer. The ligated product was sequenced using 150 bp single read amplicon sequencing. The resulting reads were filtered by a quality score >20. PAMs were identified by mapping the reads to the PAM plasmid backbones requiring a perfect match. SeqLogos of the PAMs were generated and PAM sequences were determined by the height of each nucleotide. The cut site was identified by calculating the distance between the PAM and the ligated adapter.

[0195] The in vitro cleavage assay 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 - 59 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO CRISPR effector nucleases. An exemplary PAM and the cleavage position relative to the PAM for the MG171-1 effector nuclease are shown in FIG.1. Example 3 – In cell editing activity of MG nucleases

[0196] mRNA synthesis

[0197] The nucleic acids coding for MG21-1 or MG23-1 were cloned into pUC19 plasmids including an RNA-pol T7 promoter, 5’ and 3’ UTRs, and a 100 nt polyA tail. To linearize the plasmid, 100 µg of plasmid was digested with SapI. 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 reactions, 1 µg of linearized plasmid DNA was added to a 20 µL reaction 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 was incubated at 50 °C for 1 hr and the resulting transcribed mRNA was purified.

[0198] Mammalian cell activity assay

[0199] Hep3B cells were cultured in EMEM + 10% FBS at 37 °C and 5% CO2. Cells were nucleofected with either MG21-1 or MG23-1 mRNA along with the indicated chemically- synthesized guide RNAs targeting either HAO1 or TTR (500 ng mRNA / 150 pmol guide) in Hep3B cells (100,000) using an electroporator.72 hr post-nucleofection, cells were harvested and genomic DNA extracted. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA using Q5 High-Fidelity DNA polymerase. Amplicons were sequenced and analyzed for gene editing.

[0200] Up to 97% indels were observed with MG21-1 for both HAO1 and TTR (FIGs.2 and 3), while MG23-1 resulted in up to 63% indels (FIG.4). These results demonstrate the ability of MG21-1 and MG23-1 to robustly edit genes at therapeutically relevant loci. - 60 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO SEQUENCE LISTING Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: L T P P E L Q R D T Y K T T V S R A L L W R A E P D L T A T A G K L A K N R F V R D A P P S D I- 61 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Q E P D S G V N R R L S Q A Q F Y EF N E A D V D D D PS E H F V L R P T T E R K C NI K G G K K A V K A T A- 62 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: V Y F K A G F S S R R L Y E G H H S K V RI L E E F A H EL IL A R Q E I Y L K E S D IQ V R A V R V G N F- 63 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A K R D R P P D R GP S L G V L T E G P D L IE Q G N Q F A T P H D R K P L A V L E Y V A G V P R- 64 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: L D M S E I E E S F T D S T N F E L I K K E T K N N P V I N F Y E L G L K F P F S F L Y K N T PE T Y- 65 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: TE V Y N N P IH Q I N K T G V F T S L T Y E Y QI K L T T Y S T Y S H T N E E D Y A L D E C L R R G- 66 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A N N R Y D N T I V EE R R LI E K M I Q K F T S Q P F N K Y N D N PI N RL I A T F L G K H K K K N M K- 67 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: V N Q D E D K N KI N S C M D K D E A D Q I E SI W H K V K K C Y V V P L E IE K Q D F E I A N D D D Y K- 68 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: I T A C P M F L I E L NI K P F V P R P L L L D W Y E A Q V I A I P V R Q N GF V W E Y G G L D L T R Y K- 69 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A G W G P R R G E Y V T V T G E G A A H G K L TL R I A E E N H SS E V G Q LL D R R R V IQ V D A G HP R H- 70 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K D Q L L T I IK R N IS T W A C A K V A N P I S F D E C P S A L R I K L D K I- 71 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: C V D E I A R R F T K L D K P I E H R S Q R P G- 72 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: I I I R F R L L E S Q S S M G P S H D V IF M F G S S G D- 73 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: RS FI P K R F P K S M F G R L H T Q M T T K L S L G LP N T K K- 74 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: N N S PS R N Y L E SI E N F K K V K R D LF NI P K K D K S K- 75 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: G Y D N RI A L I N A P F A A R A G A C G A C U A C- 76 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO:- 77 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO:- 78 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO:- 79 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO:- 80 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: E R G D T R M Q L C Y L- 81 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: E GF A S H E A R K D R D E C T L P KS V F P D T AE R G D R V E E E F H T L Y E G L V S N R A A F R V- 82 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Q N G V A N T T E R A K R V S L V R K P F A M S D K G N N L K C L V Y D K Y M N IT V E R G L A T- 83 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K E V KI T G T D G S H E L E K D R D A R R A T A D D S T E R G L A T K E V K T E TL K HI Y V E E K D- 84 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: FR Y A T E H E R D K FS K D T C A T A K L A L R L L P E D V E R P V S K R L D A E G A A V R F TI G D- 85 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Q T M R S ES Y W R GS K V L V L G G R W Q N A D G A T P T P R RF T R P E R L K L L KS S Y W E P A V T A- 86 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Y G R E E E T R W S R Q T Q Q A A E D V R G A R T N T E Y II LP R P F IE A I Q IG L E T A K K G D E A K- 87 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: R P N K I QI S T E L E F A S T G V S R R R L K A K L V TL F F RT D R L L L K Q L A R G S R P R L G S L- 88 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K R R L L IE L E E P E R K IL R K R L N P P T A R V D Q R N L P D A L Y F I L N E H I L G KI E S A R S IS- 89 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K Y R Y W I E S G V A I D SR E N V S PT R G L Q Y A IF R R A N L N G E S I R S L H D D A R F ID K R M R P L L P- 90 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: S R P K A E G V Q M T S L L S A G Q D T S V W L L IP C M R N E W V A F L K P A E G MI R SP P A V N- 91 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: D L N L D A D S Q D MI R E L A L R A S R G R R S V A S I K L F Y A R N IS M E S P N L T N E E W IP EI L A- 92 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K K G F V P E K R L P R R R A K E V L A Y NP F K E V E C I W E I A F D R H L A L EP Y R L P A L V PE- 93 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: E A K Q A E L G E S A M V L A H L W K H L R E LF A P K L PI E Y H SI R I D V F W F C T A FP R M A R Y N- 94 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: S P F Y R T L V IS L S I E S F M R N R AI D G H A IF R R K N IN P F E I E R Q H N K S V S TS K D K K A Y PI- 95 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: E S P Q R S L V E P QI LL N W K I Q V FR A K K P L Q L D TS Q E G E T GI H G G LI E A S L A G M L P M L EL- 96 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: S L PT P N L N D A D D S N VI R H P K A K K R PT R IE A Q R E H S S A A L K A V VI E I G K Q R VF G S S- 97 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Y K L I R N Q PE S L L E L D K P P D L A S R L A R N S P F Q G F S TT S D Y K D F PI Q S P A L L K L- 98 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: E L A R K L W R F E V I Q Q I G S FS V PP G T M D R R F N LI E P K V R C D A P N FT L V K G K R N R- 99 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Q L R I R D E S T EP H S G L P K P RI D E Y A L Q R L T F V I E L HI D S R R P R H M R A K S H D- 100 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: G SI R E L V F I LF C L D L A K S D V E K D P R K S P HS T L P K S T A K I P A N L E Y N I P T G S RF- 101 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: V SI K I A E Y V F L W R IT N AI F N A A N I A A R G P E L E F R R K D N R GF D Q A E D V A Q V L I R H R C- 102 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: N AI A K H R D T R G P E L V R S G I R II N G P E Y K E G E K W E RI W E V V G V E V T V PT S LL L Q G- 103 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: G N P R AI L K G H V VI RE Q IL D E S V IE L V IS T S M I R L V S L L Q A S F I K T G H F R P R I G R- 104 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Y D K Q Y D S G DI V S F V A T L L P E G S R G R R G G IF G S R S V I Y H R R V KS RI L Q L K A- 105 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: L G R M L K V P S SP T D D F A IL S D P N L V G R D Q A R P Y IR Y A AS G Y T I Y PY A Q L K V S G G Y E S K E- 106 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A K F E G S E R T V G P I N A I Y K T V G R G L A G Q E R E R T E E EE K V T LF V E D K V E D- 107 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: P L N A T D G D V V N T R A RT P N N FE E F H S A V L P N R D R T K F L K I A E D E K Y K F F A R L- 108 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A V A G D E E E Y IL P D L V A S K K D K L R T L P Y L K GI A D D S G C A K R E T S M G T R Y S F C E K S- 109 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: D E SE V R H N V A T G K F EL D H F A D R E Y R R P C A E D I K L V G L R E L N M IT R P E V- 110 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: F K N V L R G R I V A K D D L K E K TE I K IN K L I K Q E L R K P IL E E K SS G Y C L K T A G T A G- 111 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: T A G T A G T A G T T T T T A C C T T A C G C T C C G C C C G T C G- 112 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: G G G T T C A G T A G C C T G G G A C G T C G C T C A A T A T- 113 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: T C G T A T T C G T A G T T G T T T T T T C A A T T G C A G A A T T C T T C T- 114 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: A T T C G C C C C T T C T T T C C T T T- 115 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: C C T T T T A A T C G T A G T G T G T G T T A G T A G T T- 116 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: T T T T T T A C U U A A G G G C U G G G C U G G G C U G G G- 117 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: C U A A C U U U C G G C G U G C U G U C A U C U A U A U G C G U U A U A Cr C r A rG rG- 118 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: U r G r Ur Ar G r G r Ur Ar C rC r A rG rG C r G r Ur Ar G r G r Ur Ar G r G r Ur Ar Ur C r A rG rG G r G r Ur- 119 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Ar Ar Cr U r Gr Gr- 120 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: C rC r U r C U r A r G r A r A r G r G r A r G r- 121 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Ar rC r A rG rG Gr C r A rG rG A r G r Ur Ar rC rC r A rG rG- 122 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Cr Cr U r Gr Gr C rC r A rG rG C rC r A rG rG A r G r Ur Ar Cr C r A rG rG C r G r Ur Ar C rC r A rG rG U r G r Ur- 123 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: Ar- 124 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: S A E L A Y K P A T L L D I G S Y Q K V Q L- 125 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: H R I K L G D Q G S F T R K N D D H D Q C I K L TF L G N G L LT G K R L V E K L DI S E L L Q S A K- 126 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO Cat. SEQ Descriptio Type Organism Other Sequence ID n Information NO: K S D S T F IV T- 127 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO

[0201] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. - 128 - IPTS / 126962209.1

Claims

Attorney Docket No.: MTG-018WO CLAIMS WHAT IS CLAIMED IS:

1. An engineered nuclease system comprising: a) an endonuclease comprising 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 to hybridize to a target nucleic acid sequence.

2. The engineered nuclease system of claim 1, wherein the endonuclease comprises 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.

3. The engineered nuclease system of claim 1, wherein the endonuclease comprises 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.

4. The engineered nuclease system of any one of claims 1-3, wherein the engineered guide polynucleotide comprises a crRNA and a tracrRNA.

5. The engineered nuclease system of claim 4, wherein the tracrRNA comprises 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.

6. The engineered nuclease system of claim 4, wherein the tracrRNA comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 215-222, 52, 147-151, 157-163, and 171-192.

7. The engineered nuclease system of claims 1-6, wherein the engineered guide polynucleotide is a single guide nucleic acid.

8. The engineered nuclease system of claims 1-6, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

9. The engineered nuclease system of any one of claims 1-8, wherein the engineered guide polynucleotide is RNA.

10. The engineered nuclease system of any one of claims 1-9, wherein the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. - 129 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 11. The engineered nuclease system of any one of claims 1-10, wherein the endonuclease has less than 80% identity to a Cas9 endonuclease.

12. The engineered nuclease system of any one of claims 1-11, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.

13. The engineered nuclease system of any one of claims 1-11, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

14. 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222.

15. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7-11; and b) 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 SEQ ID NO:

52.

16. An engineered nuclease system comprising: a) an endonuclease comprising 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 147-151.

17. An engineered nuclease system 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 157-163. - 130 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 18. An engineered nuclease system 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 171-192.

19. The engineered nuclease system of claims 14-18, wherein the engineered guide polynucleotide is a single guide nucleic acid.

20. The engineered nuclease system of claims 14-18, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

21. The engineered nuclease system of any one of claims 14-20, wherein the engineered guide polynucleotide is RNA.

22. The engineered nuclease system of any one of claims 1-20, wherein the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas 12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease.

23. The engineered nuclease system of any one of claims 14-22, wherein the endonuclease has less than 80% identity to a Cas9 endonuclease.

24. The engineered nuclease system of any one of claims 14-23, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.

25. The engineered nuclease system of any one of claims 14-23, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

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

27. A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of any one of claims 1-26.

28. The method of claim 27, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.

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

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

31. The method of any one of claims 27-29, wherein the modification is in vivo. - 131 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 32. The method of any one of claims 27-29, wherein the modification is ex vivo.

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

34. The method of any one of claims 27-32, wherein the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.

35. A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of any one of claims 1-26.

36. The method of claim 35, further comprising selecting cells comprising the modification.

37. A method of modifying HAO1 comprising contacting HAO1 using 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222.

38. The method of claim 37, wherein the engineered guide polynucleotide is encoded by a sequence having any one of SEQ ID NOs: 251-260.

39. The method of claim 37, wherein the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270.

40. A method of modifying HAO1 comprising contacting HAO1 using an engineered nuclease system comprising: a) an endonuclease comprising a sequence 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.

41. The method of claim 40, wherein the engineered guide polynucleotide is encoded by a sequence having any one of SEQ ID NOs: 271-274.

42. The method of claim 40, wherein the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 275-278.

43. A method of modifying TTR comprising contacting TTR using an engineered nuclease system comprising: - 132 - IPTS / 126962209.1Attorney Docket No.: MTG-018WO 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, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 215-222.

44. The method of claim 43, wherein the engineered guide polynucleotide is encoded by a sequence having any one of SEQ ID NOs: 279-290.

45. The method of claim 43, wherein the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 291-302.

46. A cell comprising the engineered nuclease system of any one of claims 1-26.

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

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

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

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

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

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

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

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

55. The cell of claim 46, wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

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

57. The cell of claim 46, wherein the cell is a stable cell. - 133 - IPTS / 126962209.1

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