EP4716752A2 - Systèmes d'endonucléases - Google Patents

Systèmes d'endonucléases

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Publication number
EP4716752A2
EP4716752A2 EP24811938.0A EP24811938A EP4716752A2 EP 4716752 A2 EP4716752 A2 EP 4716752A2 EP 24811938 A EP24811938 A EP 24811938A EP 4716752 A2 EP4716752 A2 EP 4716752A2
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EP
European Patent Office
Prior art keywords
sequence
seq
nos
endonuclease
identity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP24811938.0A
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German (de)
English (en)
Inventor
Brian C. Thomas
Lisa ALEXANDER
Ian Barr
Christopher Brown
Daniela S.A. Goltsman
Samatar JIRDE
Isabel NOCEDAL
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Metagenomi Therapeutics Inc
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Metagenomi Inc
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Publication of EP4716752A2 publication Critical patent/EP4716752A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • SMART SMall ARchaeal-associaTed nuclease systems.
  • endonuclease effectors are defined by their small size (about 400 aa to about 1050 aa) and the presence of RuvC and HNH catalytic domains.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 90% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease compnsing a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 95% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 99% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 99% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having 100% sequence identity’ to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1323, 1324 and 1329-1346.
  • 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 binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376- 1391, 1392-1414, and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1571, 1591, 1592, 1615, 1625, 1651, 1663, 1672, 1709, 1712, 1713. 1728, 1738, 1764, 1809. 1812, 1884, 1821, 1853, 1893. 1846, 1854, 1878, 1886, 1902, 1890, 1847, 1903, 1890, 1957, 1959, 1960, 1961, 1975, 1988, and 2002.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 1410 or 1960.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1410, 1412. 1953. 1956, 1960, 1961, 1966, 1970, and 1478.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2157, 2159, and 2160.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2017, 2022, 2029, 2031, 2032, 2035, 2044, 2045, 2047, 2048, 2073, 2075, 2090, 2195, 2197, 2198, 2199, 2200, and 2202.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2017, 2022, 2026. 2028, 2029, 2031, 2032, 2035, 2044, 2047. 2054. 2073, 2075, 2090. 2195. 2197, 2198, 2199, 2200. 2202. 2206, 2208, 2211, 2212, and 2216.
  • the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391. 1392-1414, and 1470-2242.
  • modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.
  • the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the modification is in vitro.
  • the modification is in vivo. In some embodiments, the modification is ex vivo.
  • 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 comprise selecting cells comprising the modification.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is an immortalized cell.
  • the cell is an insect cell.
  • the cell is a yeast cell.
  • the cell is a plant cell.
  • the cell is a fungal cell.
  • the cell is a prokaryotic cell.
  • the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, S®, 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.
  • the cell is an engineered cell.
  • the cell is a stable cell.
  • FIGs. 1A - FIG. ID depict phylogenetic trees of SMART I nucleases for ancestral reconstruction of the MG34 (FIG. 1A-1C) and MG102 (FIG. ID) clades.
  • Phylogenetic trees w ere inferred with FastTree or RAxML from MAFFT global (G-INS-i) or local (L-INS-i) multiple sequence alignments.
  • Individual ancestors indicated for each tree by a circle represent different ancestral ages and are based on different phylogenetic trees.
  • FIG. ID shows previously characterized effectors MG102-2, MG102-39. and MG102-42 in the tree.
  • FIGs. 2A - 2B depict that novel SMART I effectors are active nucleases with non-native guides. SMART I effectors were assayed for cleavage activity via a PAM enrichment protocol.
  • FIG. 2A depicts PCR results. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of the single guide RNA from other active MG34 nucleases and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA guided cleavage by the nuclease produced bands at the expected 180 bp size.
  • FIG. 2B depicts NGS sequencing results. NGS sequencing of the bands identified in FIG. 2A were used to generate the PAM sequence and cleavage position relative to the PAM sequence.
  • FIGs. 3A - FIG. 3B depict that that novel SMART I effectors are active nucleases.
  • SMART I ancestral effectors were assayed for cleavage activity via a PAM enrichment protocol.
  • FIG. 3A depicts PCR results. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of the single guide RNA from other active MG34 nucleases and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA guided cleavage by the nuclease produced bands at the expected 180 bp size.
  • Fig. 3B depicts NGS sequencing results. NGS sequencing of the bands identified in FIG. 3A were used to generate the PAM sequence and cleavage position relative to the PAM sequence.
  • FIG. 4A - FIG. 4B illustrates that the novel chimeric SMART I ancestral effectors are active nucleases. Chimeric SMART I ancestral effectors were assayed for cleavage activity via a PAM enrichment protocol.
  • FIG. 4A depicts PCR results. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of the single guide RNA from other active MG34 nucleases and added to a PAM library' (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA guided cleavage by the nuclease produced bands at the expected 180 bp size.
  • FIG. 4B shows NGS sequencing results. NGS sequencing of the bands identified in FIG. 4A for MG34-47 were used to generate the PAM sequence and cleavage position relative to the PAM sequence.
  • FIG. 5 depicts bar plots showing SMART I ancestral effectors recognize diverse PAM sequences.
  • MG34 nucleases were quantitatively assayed for cleavage activity via an in-vitro cleavage assay.
  • the effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the absence (apo) or presence of the single guide RNA from other active MG34 nucleases, and dsDNA target with nRR PAMs (nAA, nAG, nGA, or nGG PAMs) were incubated with ribonucleoprotein complexes to initiate cleavage.
  • IVTT in-vitro transcription/translation
  • Cleavage products were analyzed by nucleic acid electrophoresis and peak area for uncleaved (-3500 bp supercoiled) and cleavage products (-2200 bp linearized) were plotted as a percent of RNA-guided cleavage (y axis).
  • FIGs. 6A - FIG. 6B depict SDS page gel results showing that SMART I ancestral effectors are soluble.
  • FIG. 6A shows purification of MG34-27 and FIG. 6B shows purification of MG34-29.
  • Samples with 2x Laemmli buffer were separated on a Stain-Free 4-20% gradient SDS PAGE gel and visualized by fluorescence imaging.
  • the “Sonication” lanes show the whole cell sample after lysis
  • “Load” shows the contents of the soluble portion of the cell lysate.
  • the lanes Al 1-B10 show the eluate from a gradient elution from 20-300 mM Imidazole.
  • FIG. 1A shows the lanes Al 1-B10 show the eluate from a gradient elution from 20-300 mM Imidazole.
  • FIGs. 7A - FIG. 7B depict activity assay results showing RNA-directed cleavage of plasmids by MG34-27 (FIG. 7A) and MG34-29 (FIG. 7B) with sgRNAs from extant nucleases.
  • MG34-29 50 nM was complexed with excess MG34-1 sgRNA (sgl) and used to cleave a supercoiled plasmid containing an nGG PAM.
  • MG34-27 was complexed with sgRNAs from MG34-1 and MG34-25 (sgl and sg25) and used to cleave four separate PAM containing plasmids.
  • the expected size of the cleaved plasmids is 2200 bp, and the products are indicated by the red oval.
  • FIG. 8 depicts the results of a nuclease activity assay. Nuclease activity was tested by nucleofecting MG34-27 or MG34-29 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the AAVS 1 locus into K562 cells. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of tw o replicates.
  • FIG. 9 depicts the results of a nuclease activity assay. Nuclease activity' was tested by nucleofecting MG34-29 mRNA (500 ng) and increasing amounts of sgRNA (100, 200, 300, or 400 pmol) targeting individual sites in the AAVS1 locus into K.562 cells. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (C7, E7, F7 or G7) and is the average of two replicates.
  • sgRNA 100, 200, 300, or 400 pmol
  • FIGs. 10A-10C depict the identification of ancestral sequences.
  • Ancestral intermediate sequences were generated based on a phylogenetic tree which included 441 sequences (FIG. 10A) or 190 sequences (FIG. 10B).
  • Ancestral intermediate sequences were generated by randomly introducing amino acids from one ancestor (First) into a second ancestor using the weights shown in FIG. 10C. as well as the PAML probabilities derived from the original ancestral sequence reconstruction.
  • Up to four different groups of ancestors were generated for the two trees based on ancestral nodes 1 through 4 and the relative weights for each ancestor in the random sampling as given by the “Sampling weight” columns in FIG. 10C.
  • FIG. 11A depicts a cleavage activity assay.
  • MG34 SMART I effectors were assayed for cleavage activity via a PAM enrichment protocol. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of the single guide RNA from the active nuclease MG34-1 and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease produced bands at the expected 180 bp size (arrows). The positive control represents the MG34-1 nuclease with its native guide RNA, while lane numbers represent the MG34 candidate tested (e.g. lane 55 is MG34-55).
  • FIG. 11B depicts a cleavage activity assay.
  • SMART I effectors were assayed for cleavage activity via a PAM enrichment protocol. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of the single guide RNA from the active nuclease MG34-25 and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease produced bands at the expected 180 bp size. Lane numbers represent the MG34 candidate tested (e.g. lane 55 is MG34-55).
  • FIG. 12 depicts NGS sequencing of the bands identified in FIGs. 11A-11B, which were used to generate the preferred PAM sequence and cleavage position relative to the PAM sequence for the active nucleases MG34-71. MG34-72. MG34-73, and MG34-74.
  • FIG. 13 depicts NGS sequencing of the bands identified in FIG. 11 were used to generate the preferred PAM sequence and cleavage position relative to the PAM sequence for the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79.
  • FIG. 14 depicts a cleavage activity’ assay.
  • MG34-35 effector was assayed for cleavage activity 7 via a PAM enrichment protocol. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of a single guide RNA (SEQ ID NOs: 1392-1399) and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease produced bands at the expected 180 bp size.
  • IVTT in-vitro transcription/translation
  • FIG. 15 depicts a cleavage activity 7 assay.
  • MG102 SMART I effectors were assayed for cleavage activity’ via a PAM enrichment protocol. Effectors were expressed in in-vitro transcription/translation (IVTT) reactions in the presence of a single guide RNA and added to a PAM library (dsDNA target). For native effectors (51, 53, 55, and 63) four single guide designs were tested, and for ancestral effectors (64-81), the guides from active candidates MG102-2, MG102-39, and MG102-42 were used. Cleavage products were amplified via ligation to the cut site and subsequent PCR amplification.
  • IVTT in-vitro transcription/translation
  • FIGs. 16A-16E depict NGS sequencing of the bands identified in FIG. 15. which were used to generate the preferred PAM sequence (FIG. 16A) and cleavage position relative to the PAM sequence for the active nucleases MG102-51, MG102-53, MG102-55, MG102-63, MG102-65, MG102-66, MG102-67, MG102-68, MG102-71, MG102-73, MG102-74, MG102- 77, MG102-78, MG102-79, MG102-80 (FIGs. 16B-16E).
  • FIG. 17 depicts MG34-29 mammalian activity at Beta-2 microglobulin (B2M) locus with MG34-1 nGG PAM guides. Nuclease activity was tested by nucleofecting MG34-29 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the B2M locus into K562 cells using an MG34-1 guide scaffold. 192 B2M guides w ere tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of three replicates.
  • B2M beta-2 microglobulin
  • FIG. 18 depicts MG34-29 mammalian activity' at hRosa26 locus with MG34-1 nGG PAM guides. Nuclease activity’ was tested by nucleofecting MG34-29 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the hRosa26 locus into K562 cells using an MG34-1 guide scaffold. 72 hRosa26 guides w ere tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of three replicates.
  • FIG. 19 depicts ASR MG34-29 mammalian activity at AAVS 1 locus with MG34-1 nGG PAM guides. Nuclease activity' was tested by nucleofecting MG34-29 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the AAVS1 locus into K562 cells using an MG34-1 guide scaffold. 192 B2M guides were tested in this study. 96 AAVS1 guides were tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of three replicates.
  • FIG. 20 depicts ASR MG34-29 mammalian activity' with a MG34-1 ABE active guide targeting EMX1.
  • Nuclease activity was tested by nucleofecting MG34-29 mRNA (500 ng) and sgRNA (100. 200, 300, and 400 pmol) targeting individual sites in the AAVS1 (pPE guide) into K562 cells using an MG34-1 guide scaffold. 4 varying locus guides were tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis).
  • FIG. 21 depicts ASR MG34-29 mammalian activity at AAVS1 with MG34-25 scaffold nGG PAM guides.
  • Nuclease activity 7 was tested by nucleofecting MG34-29 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the AAVS1 locus into K562 cells using an MG34-25 guide scaffold.
  • 96 AAVS1 guides were tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of two replicates.
  • FIG. 22 depicts ASR MG34s mammalian activity with 4 AAVS1 locus MG34-1 and 4 MG34-25 scaffold active spacers. Nuclease activity was tested by nucleofecting various MG34 nuclease mRNAs (500 ng) and sgRNA (200 pmol) targeting 4 individual sites in the AAVS1 locus into K562 cells, using MG34-l(left) or MG34-25 (right) guide scaffolds. 4 AAVS1 guides were tested. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (C7, E7, F7 or G7) and guide scaffold combination.
  • C7, E7, F7 or G7 specific spacer
  • FIG. 23 depicts ASR MG34-72 mammalian activity at AAVS1 with MG34-25 scaffold nGG PAM guides. Nuclease activity was tested by nucleofecting MG34-72 mRNA (500 ng) and sgRNA (200 pmol) targeting individual sites in the AAVS1 locus into K562 cells using an MG34-25 guide scaffold. 96 AAVS1 guides were tested. After 72 hours, cells were harvested and prepped for NGS to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (X axis) and is the average of two replicates.
  • FIG. 24 depicts MG102 mammalian activity with MG102-53 nAR AAVS1 guides. Nuclease activity was tested by nucleofecting MG102-53 (500 ng) with its own nAR PAM sgRNAs (450 pmol), targeting individual sites in the AAVS1 locus into K562 cells. 96 AAVS1 guides were tested. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (x axis).
  • FIG. 25 depicts ASR MG102-68 mammalian activity with MG102-39 nRC PAM guides. Nuclease activity' was tested by nucleofecting MG102-68 mRNA (500 ng) with sgRNA (450 pmol) targeting individual sites in the AAVS1 locus into K562 cells, using the MG102-39 guide scaffold at nRC PAM sites. 96 AAVS1 guides were tested. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (x axis).
  • FIG. 26 depicts ASR MG102 mammalian activity' with MG102-2 nRC PAM active spacers and varying MG102 guide scaffolds. Nuclease activity was tested by nucleofecting MG102 mRNAs (500 ng) with sgRNA (450 pmol) targeting individual sites in the AAV SI locus into K562 cells. Guides scaffolds were from either MG102-2, MG102-39, or MG102-53 guides and spacers had been previously validated with MG102-2 nuclease. 24 AAVS1 guides were tested. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (x axis).
  • FIG. 27 depicts ASR MG102-71 mammalian activity with MG102-39 nRC PAM guides. Nuclease activity was tested by nucleofecting MG102-71 mRNA (500 ng) with sgRNA (450 pmol) targeting individual sites in the AAVS1 locus into K562 cells, using the MG102-39 guide scaffold at nRC PAM sites. 96 AAVS1 guides were tested. After 72 hours, cells were harvested and NGS libraries were prepared to assess editing efficiency. Each bar represents editing efficiency at the site targeted by the specific spacer (x axis).
  • SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312 show the full-length peptide sequences of MG33 nucleases.
  • SEQ ID NOs: 199 and 669-670 show the nucleotide sequence of a tracrRNA predicted to function with an MG33 nuclease.
  • SEQ ID NOs: 201 and 1003-1005 show the nucleotide sequences of predicted singleguide RNA (sgRNA) sequences predicted to function with an MG33 nuclease. “N”’s denote variable residues and non-N-residues represent the scaffold sequence.
  • SEQ ID NOs: 1023-1028 show PAM sequences compatible with MG33 nucleases.
  • SEQ ID NOs: 1045-1054 show CRISPR repeats of MG33 nucleases described herein.
  • SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 show the full-length peptide sequences of MG34 nucleases.
  • SEQ ID NOs: 200 and 1348 show the nucleotide sequence of a tracrRNA predicted to function with an MG34 nuclease.
  • SEQ ID NOs: 202, 203, 613-616, and 1369 show the nucleotide sequences of predicted single-guide RNA (sgRNA) sequences predicted to function with an MG34 nuclease. “N”s denote variable residues and non-N-residues represent the scaffold sequence. [0059] SEQ ID NOs: 1023-1028 and 1441-1450 show PAM sequences compatible with MG34 nucleases.
  • sgRNA single-guide RNA
  • SEQ ID NOs: 1055-1057, and 1349 show CRISPR repeats of MG34 nucleases described herein.
  • SEQ ID NOs: 1392-1414 show the nucleotide sequences of MG34 single guide RNAs.
  • SEQ ID NOs: 1470- 2242 show the nucleotide sequences of MG34 chemically synthesized/modified sgRNAs.
  • SEQ ID NOs: 1470-1485 show the nucleotide sequences of MG34-35 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 1486-1489 show the nucleotide sequences of MG34-25 sgRNAs targeting AAVS1.
  • SEQ ID NO: 1490-1493 shows show the nucleotide sequences of MG34-1 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 1494-1685 show the nucleotide sequences of MG34-l sgRNAs targeting B2M.
  • SEQ ID NOs: 1686-1757 show the nucleotide sequences of MG34-l sgRNAs targeting hRosa26.
  • SEQ ID NOs: 1758-1806 show the nucleotide sequences of MG34-1 sgRNAs targeting TRAC.
  • SEQ ID NO: 1807 shows the nucleotide sequence of MG34-1 sgRNA targeting VISTA enhancer hs267 regulatory pPE633.
  • SEQ ID NO: 1808 shows the nucleotide sequence of MG34-1 sgRNA targeting Sharpr-MPRA regulatory region 15312 pPE634.
  • SEQ ID NO: 1809 shows the nucleotide sequence of MG34-1 sgRNA targeting EMX1 Intron pPE641.
  • SEQ ID NO: 1810 shows the nucleotide sequence of MG34-1 sgRNA targeting HSPA12A gene (Hsp70 member 12A) pPE635.
  • SEQ ID NOs: 1811- 1906 shows the nucleotide sequences of MG34-1 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 1907-2002 shows the nucleotide sequence of MG34-25 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2003-2098 show the nucleotide sequences of MG102-39 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2099-2194 show the nucleotide sequences of MG102-53 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2195-2202 show the nucleotide sequences of MG102-2 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2203-2210 show the nucleotide sequences of MG102-39 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2211-2218 show the nucleotide sequences of MG102-53 sgRNAs targeting AAVS1.
  • SEQ ID NOs: 2219-2226 show the nucleotide sequences of MG102- 2 sgRNAs targeting TRAC.
  • SEQ ID NOs: 2227-2234 show the nucleotide sequences of MG102-39 sgRNAs targeting TRAC.
  • SEQ ID NOs: 2235-2242 show the nucleotide sequences of MG102-53 sgRNAs targeting TRAC.
  • SEQ ID Nos: 25-198, 221-459, 489-580, 617-668, and 674-675 show the full-length peptide sequences of MG35 nucleases.
  • SEQ ID Nos: 460-461 show the nucleotide sequences of MG35 tracrRNAs derived from the same loci as MG35 nucleases.
  • SEQ ID NOs: 462, 676, and 1229-1230 show CRISPR repeats of MG35 nucleases described herein.
  • SEQ ID NOs: 677-686, 1006-1012, and 1231-1259 show the nucleotide sequences of MG35 single guide RNAs.
  • SEQ ID NOs: 687-974 show the nucleotide sequences of MG35 single guide RNA encoding sequences.
  • SEQ ID NOs: 1029-1034 show PAM sequences compatible with MG35 nucleases.
  • SEQ ID NOs: 1172-1228 show the nucleotide sequences of loci encoding MG35 nucleases described herein.
  • SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346 show the full- length peptide sequences of MG102 nucleases.
  • SEQ ID NOs: 672-673, 1327-1328, and 1370-1372 show the nucleotide sequences of MG102 tracrRNAs derived from the same loci as MG102 nucleases.
  • SEQ ID NOs: 205-220 show the sequences of example nuclear localization sequences (NLSs) that, in some embodiments, are appended to nucleases according to the disclosure.
  • NLSs nuclear localization sequences
  • SEQ ID NOs: 1013-1022 and 1376-1391 show the nucleotide sequences of MG102 single guide RNAs.
  • SEQ ID NOs: 1035-1044 and 1451-1469 show PAM sequences compatible with MG102 nucleases.
  • SEQ ID NOs: 1058-1072,1325-1326, and 1373-1375 show CRISPR repeats of MG102 nucleases described herein.
  • SEQ ID NO: 1171 shows the nucleotide sequence of a locus encoding an MG102 nuclease described herein.
  • SEQ ID NO: 975 shows the full-length peptide sequence of an MG143 nuclease.
  • SEQ ID NOs: 1073 shows a CRISPR repeat of an MG143 nuclease described herein.
  • SEQ ID NOs: 976-979 and 1274-1288 show the full-length peptide sequences of MG144 nucleases.
  • SEQ ID NOs: 1074-1077 show CRISPR repeats of MG144 nucleases described herein. MG145 nucleases
  • SEQ ID NO: 980 shows the full-length peptide sequence of an MG145 nuclease.
  • SEQ ID NOs: 1078 shows a CRISPR repeat of an MG145 nuclease described herein.
  • SEQ ID NOs: 1079-1082 and 1145-1166 show the DNA sequences of TRAC target sites.
  • SEQ ID Nos: 1083-1086 and 1123-1144 show the nucleotide sequences of sgRNAs engineered to function with an MG102 nuclease in order to target TRAC.
  • SEQ ID NOs: 1167-1168 show- the nucleotide sequences of sgRNAs engineered to function with an MG33 nuclease in order to target TRAC.
  • SEQ ID Nos: 1169-1170 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 1087-1104 show' the nucleotide sequences of sgRNAs engineered to function with an MG102 nuclease in order to target AAVS1.
  • SEQ ID NOs: 1105-1122 show the DNA sequences of AAVS1 target sites.
  • SEQ ID NOs: 2270-2330 show' the nucleotide sequences of target sites in the genome.
  • 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.
  • nucleotide refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurnng nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • 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.
  • derivatives include, for example,
  • nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • 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 -carboxy fluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA).
  • ROX 6-carboxy-X-rhodamine
  • DABYL 4-(4'dimethylaminophenylazo) benzoic acid
  • EDANS 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid
  • 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, [dRl 10]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, 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.
  • nucleotide encompasses chemically modified nucleotides.
  • An exemplary chemically-modified nucleotide is biotin-dNTP.
  • 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).
  • polynucleotide oligonucleotide
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded 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), ribozy mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA).
  • 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),
  • nucleic acid probes and primers.
  • 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, di deoxynucleotides, 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, inosine, thiouridine, pseudouridine, dihydro uridine, queuosine, and wyosine.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • peptide polypeptide
  • protein protein
  • peptide bonds 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 cases, 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 tertian- structure (e.g.. domains).
  • 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.
  • amino acid and amino acids 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.
  • amino acid includes both D-amino acids and L-amino acids.
  • non-native refers to a nucleic acid or polypeptide sequence that is non-naturally occurring.
  • Non-native refers to a non-naturally occurring nucleic acid or polypeptide sequence that comprises modifications such as mutations, insertions, or deletions.
  • the term non-native encompasses fusion nucleic acids or polypeptides that encodes or exhibits an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) of the nucleic acid or polypeptide sequence to which the non-native sequence is fused.
  • a non-native nucleic acid or polypeptide sequence includes those linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.
  • operably linked refers 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.
  • two genetic elements are operably linked if movement of the first element causes an activation of the second element.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • optically aligned refers to an alignment of two amino acid sequences that give the highest percent identity score or maximizes the number of matched residues.
  • tracrRNA or “tracr sequence” means trans-activating CRISPR RNA.
  • tracrRNA interacts with the CRISPR (cr) RNA to form guide (g) RNA in type II and subtype V- B CRISPR-Cas systems. If the tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%. 60%. 70%. 80%. 90%. 95%. or 100% sequence identity and/or sequence similarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes, S. aureus').
  • a wild type exemplary tracrRNA sequence e.g., a tracrRNA from S. pyogenes, S. aureus'.
  • tracrRNA may refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera.
  • the term tracrRNA encompasses a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g. a tracrRNA from ⁇ S’. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • a tracrRNA sequence has at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides.
  • Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.
  • 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.
  • 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 acidtargeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.”
  • a nucleic acidtargeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”
  • 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.
  • RuvC III domain refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC I, RuvC II, and RuvC III).
  • a RuvC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g.. Pfam HMM PF18541 for RuvC III).
  • HMMs Hidden Markov Models
  • HNH domain refers to an endonuclease domain having characteristic histidine and asparagine residues.
  • An HNH domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).
  • HMMs Hidden Markov Models
  • bridge helix domain or “BH domain” refers to an arginine- rich helix domain present in Cas enzymes that plays an important role in initiating cleavage activity upon binding of target DNA.
  • recognition domain or “REC domain” refers to a domain thought to interact with the repeat: anti-repeat duplex of the gRNA and to mediate the formation of a Cas endonuclease/gRNA complex.
  • WED domain refers to a domain (e.g., present in a Cas protein) interacting primarily with repeat: anti-repeat duplex of the sgRNA and PAM duplex.
  • a WED domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Elidden Markov Models (HMMs) built based on documented domain sequences.
  • HMMs Elidden Markov Models
  • PAM interacting domain or “PI domain” refers to a domain found in Cas enzymes positioned in the endonuclease-DNA-complex to recognize the PAM sequence on the non-complementary DNA strand of the guide RNA.
  • 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.
  • a complex comprises an endonuclease and a guide polynucleotide.
  • V A, C, or G.
  • Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species offers the potential to drastically increase the number of new CRISPR/Cas systems documented and speed the discovery 7 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.
  • CRISPR/Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes.
  • 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 (30-40bp) 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-adj acent motif (PAM) sequence within a defined vicinity 7 of the target seed (the PAM usually being a sequence not commonly represented within the host genome).
  • PAM protospacer-adj acent motif
  • CRISPR-Cas sy stems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.
  • Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes, and comprise Types I, III, and IV.
  • Type I CRISPR-Cas systems are considered of moderate complexity in terms of components.
  • 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).
  • PAM protospacer-adjacent motif
  • This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) protein component of the crRNA- directed nuclease complex.
  • Cas I nucleases function primarily as DNA nucleases.
  • Type III CRISPR systems may be characterized by the presence of a central nuclease, known as CaslO, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits.
  • CaslO central nuclease
  • RAMP repeat-associated mysterious protein
  • the mature crRNA is processed from a pre- crRNA using a Cas6-like enzyme.
  • type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).
  • Type IV CRISPR-Cas systems possess an effector complex that comprises a highly reduced large subunit nuclease (csfl), 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.
  • csfl highly reduced large subunit nuclease
  • csf3 two genes for RAMP proteins of the Cas5
  • csf2 Cas7
  • Class 2 CRISPR-Cas systems have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.
  • Type II CRISPR-Cas systems are considered the simplest in terms of components.
  • 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 (e.g, Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA.
  • Cas II nucleases are DNA nucleases.
  • Type II effectors generally exhibit a structure comprising 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.
  • Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Casl2) structure similar to that of Type II effectors, 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 to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are DNA nucleases.
  • Casl2 nuclease effector
  • Type V enzymes e.g., Cas 12a
  • Cas 12a some Type V enzymes 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.
  • Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC- like domains, the single polypeptide effector of Type VI systems (e.g., Casl3) 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.
  • Type VI systems e.g., C2C2C2
  • class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease/genome editing applications.
  • 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.
  • 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 (compare top and bottom panel of FIG. 2).
  • a linker e.g., GAAA
  • sgRNA single fused synthetic guide RNA
  • 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 e.g., MG Enzymes
  • engineered nuclease systems comprising a small endonuclease.
  • the endonuclease comprises a RuvC-1 domain or a RuvC domain. In some embodiments, the endonuclease comprises an HNH domain. In some embodiments, the endonuclease comprises a RuvC domain and an HNH domain. In some embodiments, the endonuclease comprises an arginine rich region comprising an RRxRR motif or a domain with PF 14239 homology. In some embodiments, the endonuclease comprises a REC domain. In some embodiments, the endonuclease comprises a BH (Bridge Helix) domain. In some embodiments, the endonuclease comprises a WED (wedge) domain.
  • the endonuclease comprises a PI (PAM interacting) domain.
  • the endonuclease is configured to be selective for a target adjacent motif (TAM) sequence comprising any one of ANGG (SEQ ID NO: 1029).
  • TAM target adjacent motif
  • ANGG SEQ ID NO: 1029
  • NARAA SEQ ID NO: 1030
  • ATGAAA SEQ ID NO: 1031
  • ATGA SEQ ID NO: 1032
  • WTGG SEQ ID NO: 1033
  • the endonuclease is from an uncultivated microorganism. In some embodiments, the endonuclease is a Cas endonuclease. In some embodiments, the endonuclease is a class 2 endonuclease. In some embodiments, the endonuclease is a class 2, type II Cas endonuclease.
  • the endonuclease has a molecular weight of about 120 kDa or less, about 110 kDa or less, about 100 kDa or less, about 90 kDa or less, about 80 kDa or less, about 70 kDa or less, about 60 kDa or less, about 50 kDa or less, about 40 kDa or less, about 30 kDa or less, about 20 kDa or less, or about 10 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 120 kDa or less.
  • the endonuclease has a molecular weight of about 110 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 100 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 90 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 80 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 70 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 60 kDa or less.
  • the endonuclease has a molecular weight of about 50 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 40 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 30 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 20 kDa or less. In some embodiments, the endonuclease has a molecular weight of about 10 kDa or less.
  • the endonuclease is not a Cas9 endonuclease, a Casl4 endonuclease, a Casl2a endonuclease, a Casl2b endonuclease, a Cas 12c endonuclease, a Casl2d endonuclease, a Casl2e endonuclease, a Casl3a endonuclease, a Casl3b endonuclease, a Casl3c endonuclease, or a Cas 13d endonuclease.
  • the endonuclease has less than less than 80% identity, less than 75% identity, less than 70% identity, less than 65% identity, less than 60% identity, less than 55% identity, or less than 50% identity to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 80% identity to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 75% identity to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 70% identity to a Cas9 endonuclease.
  • the endonuclease has less than less than 65% identity to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 60% identity to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 55% identity 7 to a Cas9 endonuclease. In some embodiments, the endonuclease has less than less than 50% identity to a Cas9 endonuclease.
  • the endonuclease comprises a sequence with at least 70%. at least 75%. at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322- 1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 1-198, 221-459. 463-612. 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity’ to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002. 1322-1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity- to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975- 1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity- to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 81% sequence identity- to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 83% sequence identity- to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 86% sequence identity’ to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 87% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 89% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 93% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 95% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 96% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity’ to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 98% sequence identity' to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440.
  • the endonuclease is a MG33 nuclease.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289- 1312. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1. 463-486. 981-988. and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity’ to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity' to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity' to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 86% sequence identity' to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988. and 1289-1312.
  • the endonuclease comprises a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1. 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 92% sequence identity’ to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1. 463-486. 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 95% sequence identity to anyone of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity- to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the endonuclease comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity to anyone of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312.
  • the nuclease is a MG34 nuclease.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 70% sequence identity to any- one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity- to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity- to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347. 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440.
  • the endonuclease is a MG35 nuclease.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 83% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 86% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity’ to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675.
  • the endonuclease comprises a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity’ to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675.
  • the endonuclease comprises a sequence with at least 95% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675.
  • the endonuclease comprises a sequence with at least 98% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity’ to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675.
  • the endonuclease is a MG102 nuclease.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 70% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 80% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324. and 1329-1346.
  • the endonuclease comprises a sequence with at least 83% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity' to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324. and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 86% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324. and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 89% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 92% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324. and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 95% sequence identity' to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324. and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity' to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324. and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity’ to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.
  • the endonuclease is a MG144 nuclease.
  • the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 80% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 976-979 and 1274- 1288. In some embodiments, the endonuclease comprises a sequence with at least 83% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity' to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 976-979 and 1274- 1288. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • the endonuclease comprises a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 976-979 and 1274- 1288. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288. In some embodiments, the endonuclease comprises a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 80% identity 7 to any one of SEQ ID NOs: 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1324 and 1329-1346. In some embodiments, the endonuclease comprises a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity 7 to any one of SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440. In some embodiments, the endonuclease comprises a sequence having at least 90% identity to 1350-1368 and 1415-1440. In some embodiments, the endonuclease comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 95% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 99% identity 7 to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 99% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having 100% sequence identity 7 to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1323, 1324 and 1329-1346.
  • the present disclosure provides an endonuclease described herein configured to induce a double stranded break proximal to said target locus 5' to a protospacer adjacent motif (PAM).
  • the endonuclease induces a double-stranded break 6-8 nucleotides from the PAM or 7 nucleotides from the PAM.
  • the present disclosure provides an endonuclease described herein configured to induce a singlestranded break proximal to said target locus 5' to a protospacer adjacent motif (PAM).
  • the endonuclease induces a single-stranded break 6-8 nucleotides from the PAM or 7 nucleotides from the PAM.
  • an endonuclease configured to induce a single-stranded break comprises an inactivating mutation in one or more catalytic residues of an endonuclease described herein.
  • the endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease.
  • NLS comprises a sequence selected from SEQ ID NOs: 205-220 and 2243-2268.
  • the NLS comprises a sequence of any one of SEQ ID NOs: 205-220 and 2243- 2268, 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%.
  • the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 85% identity 7 to SEQ ID NOs: 205-220 and 2243-2268.
  • the NLS comprises a sequence having at least about 90% identity' to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 93% identity' to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 205-220 and 2243-2268.
  • the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 205-220 and 2243-2268. In some embodiments, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 205-220 and 2243-2268.
  • Table 1 Examples NLS Sequences that are used with Cas effectors according to the present disclosure.
  • the endonucleases described herein are variants thereof with sequence identity to particular domains.
  • the domain is an arginine rich domain (e.g., a domain with PF14239 homology), a REC (recognition) domain, a BH (bridge helix) domain, a WED (wedge) domain, a PI (PAM-interacting) domain, a PF 14239 homology' domain, or any other domain described herein.
  • residues encompassing one or more of these domains is identified in a protein by alignment to one of the proteins below (e.g, when one of the proteins below and the protein of interest are optimally aligned), wherein the residue boundaries for example domains are described.
  • endonuclease systems comprising (a) an endonuclease disclosed herein, and (b) an engineered guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • a guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • gRNA guide ribonucleic acid
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • the engineered guide polynucleotide is configured to form a complex with the endonuclease.
  • the engineered guide polynucleotide comprises a spacer sequence.
  • the spacer sequence is configured to hybridize to a target nucleic acid sequence.
  • the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • the guide polynucleotide targets a gene or locus in a cell.
  • the guide polynucleotide targets a gene or locus in a mammalian cell.
  • 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.
  • the target gene or target locus is TRAC.
  • the target gene or target locus is AAVS1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259. 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242 or a sequence having at least 90%.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%. at least about 30%. at least about 35%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677- 974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470- 2242.
  • the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672- 673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672- 673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672- 673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 199-203. 460-461. 613-616, 669-670, 672-673, 677- 974, 1003-1022. 1231-1259. 1327-1328, 1348, 1369-1372, 1376-1391. 1392-1414, and 1470- 2242.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259. 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414. and 1470-2242 or a sequence having at least 90%.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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%.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 80% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392- 1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identity to any one of SEQ ID NOs: 199- 203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669- 670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328. 1348, 1369-1372, 1376-1391, 1392- 1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 96% identity' to any one of SEQ ID NOs: 199- 203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identity’ to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669- 670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392- 1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identity to any one of SEQ ID NOs: 199- 203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328. 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231- 1259, 1327-1328, 1348, 1369-1372, 1376-1391. 1392-1414, and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376- 1391, 1392-1414, and 1470-2242.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470- 2242 or a sequence having at least 90%. 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID Nos: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1571, 1591, 1592, 1615, 1625, 1651, 1663, 1672, 1709, 1712, 1713, 1728, 1738, 1764, 1809, 1812, 1884, 1821, 1853, 1893, 1846, 1854, 1878, 1886, 1902, 1890, 1847, 1903, 1890, 1957, 1959, 1960, 1961, 1975, 1988, and 2002.
  • the engineered guide polynucleotide comprises a sequence having at least 90%. 95%. 97%. 98%. 99%.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to SEQ ID NOs: 1410 or 1960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1410 or 1960.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1410, 1412. 1953. 1956, 1960, 1961. 1966, 1970, and 1478. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1410, 1412, 1953. 1956, 1960, 1961, 1966, 1970, and 1478.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2157, 2159, and 2160. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2157, 2159, and 2160.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2017, 2022, 2029, 2031, 2032, 2035, 2044, 2045, 2047, 2048, 2073, 2075, 2090, 2195, 2197, 2198, 2199, 2200, and 2202.
  • the engineered guide polynucleotide comprises a sequence having at least 90%, 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID NOs: 2017. 2022. 2029,
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2017, 2022, 2026. 2028, 2029, 2031,
  • the engineered guide polynucleotide comprises a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2017, 2022, 2026, 2028, 2029, 2031, 2032, 2035, 2044, 2047, 2054, 2073, 2075, 2090, 2195, 2197, 2198, 2199, 2200, 2202, 2206, 2208, 2211, 2212, and 2216.
  • the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372. 1376-1391.
  • the engineered guide polynucleotide comprises a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391. 1392-1414, and 1470-2242.
  • the guide polynucleotide e.g., SEQ ID NOs: 199. 201, 669-670, and 1003-1005
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 199, 201. 669-670, and 1003-1005.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.
  • the guide polynucleotide is encoded by a sequence having at least about 96% identity 7 to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 199. 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identify to any one of SEQ ID NOs: 199, 201. 669-670, and 1003-1005.
  • the guide polynucleotide is encoded by a sequence having at least about 99% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identify’ to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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% identify to any one of SEQ ID NOs: 199, 201. 669-670.
  • the guide polynucleotide hybridizes or targets a sequence complementary' having at least about 80% identify’ to any one of SEQ ID NOs: 199, 201, 669- 670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ having at least about 90% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary 7 having at least about 96% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identify to any one of SEQ ID NOs: 199. 201, 669-670, and 1003-1005.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identify to any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identify to any one of SEQ ID NOs: 199. 201, 669-670, and 1003- 1005. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the guide polynucleotide (e.g., SEQ ID NOs: 200, 202, 203, 613- 616, 1348, 1369, and 1392-1414) is configured to form a complex with a MG34 nuclease.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369. and 1392-1414 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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: 200, 202, 203.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity’ to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 200, 202, 203. 613-616. 1348, 1369, and 1392-1414.
  • the guide polynucleotide is encoded by a sequence having at least about 90% identity’ to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 200, 202, 203. 613-616. 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity’ to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 200, 202, 203. 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity’ to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 200, 202, 203. 613- 616, 1348, 1369, and 1392-1414. [0164] In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 1348 and 1392-1414 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1348 and 1392-1414.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity' to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity' to any one of SEQ ID NOs: 1348 and 1392- 1414.
  • the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1348 and 1392-1414.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 80% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616. 1348. 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary- having at least about 85% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 200, 202.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity to any one of SEQ ID NOs: 200. 202, 203, 613-616. 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 96% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identity to any one of SEQ ID NOs: 200.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identity’ to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identity to any one of SEQ ID NOs: 200. 202, 203, 613-616. 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 200, 202, 203, 613- 616, 1348, 1369, and 1392-1414.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 80% identity to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identity’ to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 1348 and 1392- 1414.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity’ to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ having at least about 96% identity to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' having at least about 97% identity’ to any one of SEQ ID NOs: 1348 and 1392- 1414.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identity to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' having at least about 99% identity’ to any one of SEQ ID NOs: 1348 and 1392- 1414. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' having 100% identity to any one of SEQ ID NOs: 1348 and 1392-1414.
  • the guide polynucleotide (e.g., SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259) is configured to form a complex with a MG35 nuclease.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 460-461, 677- 974, 1006-1012, and 1231-1259 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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: 460-461. 677-974.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity 7 to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 460-461. 677-974. 1006-1012. and 1231-1259.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 80% identity to any one of SEQ ID NOs: 460-461, 677- 974, 1006-1012, 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012. and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231- 1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 96% identity 7 to any one of SEQ ID NOs: 460-461, 677- 974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identity to any one of SEQ ID NOs: 460-461. 677-974, 1006-1012. and 1231-1259.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identity' to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231- 1259. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006- 1012, and 1231-1259.
  • the guide polynucleotide targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 2270-2330.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 2270-2330.
  • the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 2270-2330.
  • the guide polynucleotide e.g., SEQ ID NOs: 672-673, 1013- 1022, 1327-1328, 1370-1372, and 1376-1391
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 672-673, 1013- 1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 672-673, 1013-1022. 1327-1328.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 672-673, 1013-1022. 1327-1328, 1370-1372, and 1376- 1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 672-673, 1013-1022. 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 1376-1391 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1376-1391.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1376-1391.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1376- 1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1376-1391.
  • the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1376- 1391. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary' having at least about 80% identity to any one of SEQ ID NOs: 672-673, 1013- 1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370- 1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity to any one of SEQ ID NOs: 672- 673, 1013-1022, 1327-1328. 1370-1372, and 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 96% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376- 1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identity to any one of SEQ ID NOs: 672-673, 1013- 1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 98% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328. 1370- 1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 672-673, 1013- 1022, 1327-1328, 1370-1372, and 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 80% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 85% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 90% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 95% identity' to any one of SEQ ID NOs: 1376-1391.
  • the guide polynucleotide hybridizes or targets a sequence complementary having at least about 96% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 97% identity to any one of SEQ ID NOs: 1376- 1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' having at least about 98% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having at least about 99% identity to any one of SEQ ID NOs: 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary having 100% identity to any one of SEQ ID NOs: 1376-1391.
  • the target gene is TRAC.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 1083-1086, 1123-1144. and 1167-1168 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 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%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1083-1086. 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity' to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1083-1086. 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide is encoded by a sequence having at least about 97% identity’ to any one of SEQ ID NOs: 1083-1086, 1 123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1083-1086, 1 123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRAC gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1083-1086, 1123-1144. and 1167-1168.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1083-1086, 1123- 1144, and 1167-1168.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having 100% identity to any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168.
  • the guide polynucleotide hybridizes or targets a sequence within the TRAC gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1079- 1082, 1145-1166, and 1169-1170 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1079-1082, 1 145-1 166, and 1169-1170.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166. and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166. and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170.
  • the target gene is AAVS1.
  • the guide polynucleotide is encoded by any one of SEQ ID NOs: 1087-1104 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%. at least about 30%, at least about 35%.
  • the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1087- 1104.
  • the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity’ to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the AAVS1 gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1087-1104 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1087-1 104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 1087-1104.
  • the guide polynucleotide hybridizes or targets a sequence within the AAV S 1 gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1105- 1122 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1105-1122.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1105-1122.
  • the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1105-1122.
  • the sy stems provided herein comprise one or more guide polynucleotides.
  • the guide polynucleotide comprises a sense sequence.
  • the guide polynucleotide comprises an anti-sense sequence.
  • the guide polynucleotide comprises nucleotide sequences other than the region complementary' to or substantially complementary' to a region of a target sequence.
  • a crRNA is part or considered part of a guide polynucleotide, or is comprised in a guide polynucleotide, e.g., a crRNA:tracrRNA chimera.
  • the guide polynucleotide comprises synthetic nucleotides or modified nucleotides.
  • the guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodiester.
  • all of the inter-nucleoside linkers of the guide polynucleotide, or contiguous nucleotide sequence thereof, are modified.
  • the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter-nucleoside linkers. In some embodiments, the guide polynucleotide comprises I, 2. 3, 4, 5. 6, 7, 8. 9. 10. or more than 10 modified inter-nucleoside linkers (e.g.. phosphorothioate inter-nucleoside linkage).
  • the guide polynucleotide comprises modifications to a ribose sugar or nucleobase.
  • the 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.
  • 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 which ty pically lacks a bond between the C2 and C3 carbons (e.g., UNA).
  • the sugar- modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids.
  • the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety 7 , for example peptide nucleic acids (PNA) or morpholino nucleic acids.
  • the guide polynucleotide comprises one or more modified sugars.
  • 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.
  • substituents are introduced at the 2’, 3', 4’. 5’ positions, or combinations thereof.
  • nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., substituted nucleosides.
  • a 2’ sugar modified nucleoside in some embodiments, is a nucleoside that has a substituent other than H or -OH at the T position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2'-4’ biradical bridged) nucleosides.
  • 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 nucleoside.
  • the modification in the ribose group comprises a modification at the 2’ position of the ribose group.
  • 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).
  • the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In some embodiments, the 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-methyl. In some embodiments, the modified sugar comprises a 2’-fluoro. In some embodiments, the modified sugar comprises a 2’-O- methoxyethyl group. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5.
  • the guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter-nucleoside linkers and greater than about 10%, 25%, 50%, 75%, or 90% modified sugars.
  • the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified inter- nucleoside linkers (e.g., phosphorothioate inter-nucleoside linkage) and 1, 2, 3. 4, 5, 6. 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).
  • modified inter- nucleoside linkers e.g., phosphorothioate inter-nucleoside linkage
  • modified sugars e.g., comprising a 2’-O-methyl or 2’-fluoro
  • the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary’ to a eukaryotic genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.
  • the guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the 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 guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70.
  • the guide RNA structure comprises an RNA sequence predicted to comprise a hairpin.
  • the hair pin comprises a stem and a loop.
  • the stem comprises at least 12 pairs, at least 14 pairs, at least 16 pairs or at least 18 pairs of ribonucleotides.
  • the stem comprises at least 12 pairs of ribonucleotides.
  • the stem comprises at least 14 pairs of ribonucleotides.
  • the stem comprises at least 16 pairs of ribonucleotides.
  • the stem comprises at least 18 pairs of ribonucleotides.
  • the guide RNA structure further comprises a second stem and a second loop.
  • the second stem comprises at least 5 pairs, at least 6 pairs, at least 7 pairs, at least 8 pairs, at least 9 pairs or at least 10 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 5 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 6 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 7 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 8 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 9 pairs of ribonucleotides. In some embodiments, the second stem comprises at least 10 pairs of ribonucleotides.
  • the guide RNA structure further comprises an RNA structure and this RNA structure comprises at least two hairpins.
  • the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a prokary otic genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary' to a bacterial genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to an archaeal genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary' to a eukaryotic genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a fungal genomic sequence.
  • the guide ribonucleic acid sequence is complementary' to a plant genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a mammalian genomic sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a human genomic sequence.
  • MG Endonuclease Systems comprising an endonuclease and an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1-198, 221-459. 463-612. 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002. 1322-1324,1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity' to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002. 1322-1324, 1329-1347, 1350- 1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 90% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 95% identity to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease comprising a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 99% identity 7 to 1350-1368 and 1415-1440.
  • the endonuclease comprises a sequence having at least 99% identity to any one of SEQ ID NOs: 1324 and 1329-1346.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440.
  • the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1323. 1324 and 1329-1346.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347. 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to anyone of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity- to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 1322-1324. 1329- 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346. 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity' to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity 7 to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 1324. 1329-1346, 1347, 1350-1368. and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368. and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 21324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 1324. 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 21324, 1329-1346. 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity’ to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368. and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 1324. 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity’ to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 1324, 1329-1346, 1347. 1350-1368. and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668. and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity to anyone of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 25-198. 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668. and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity- to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity' to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence ith at least 90% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668. and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity’ to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 581-612.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324. and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 581-612.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 581-612.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324. and 1329- 1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity to anyone of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity- to any one of SEQ ID NOs: 581-612. 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity 7 to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any 7 one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 581-612.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity- to anyone of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity- to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity' to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 70% sequence identity to any 7 one of SEQ ID NOs: 976-979 and 1274- 1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 75% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 80% sequence identity' to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 81% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 82% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 83% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 84% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 86% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 87% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 88% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 89% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 90% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 91% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 92% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 93% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 94% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 95% sequence identity’ to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 96% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 97% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 98% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 99% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • the engineered nuclease system comprises an endonuclease comprising a sequence with at least 100% sequence identity to any one of SEQ ID NOs: 976-979 and 1274-1288 and an engineered guide polynucleotide configured to form a complex with the endonuclease.
  • 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 binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677- 974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-198, 221-459, 463- 612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674- 675, 975-1002.
  • an engineered guide polynucleotide configured to form a complex with the endonuclease
  • the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327- 1328, 1348, 1369-1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity’ to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369- 1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324.1329-1347.
  • an engineered guide polynucleotide configured to form a complex with the endonuclease
  • the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669- 670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328. 1348, 1369-1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415- 1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677- 974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-198, 221-459. 463- 612, 617-668, 674-675, 975-1002. 1322-1324,1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 199-203, 460-461. 613-616, 669-670, 672-673, 677-974, 1003-1022.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-198. 221-459.
  • an engineered guide polynucleotide configured to form a complex with the endonuclease
  • the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327- 1328, 1348, 1369-1372, 1392-1414. and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity’ to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324,1329-1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369- 1372, 1392-1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002. 1322-1324.1329-1347. 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 199-203, 460-461, 613-616, 669- 670, 672-673, 677-974, 1003-1022, 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID Nos: 1-198, 221-459, 463-612, 617-668, 674- 675, 975-1002.
  • an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 199-203, 460-461, 613- 616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1392- 1414, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1. 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1.
  • an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 199. 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 199. 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 199, 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1, 463- 486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 199. 201, 669-670, and 1003- 1005.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 199. 201, 669-670, and 1003-1005.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 2-24, 487- 488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 75% identity' to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313- 1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 2-24. 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321. 1347. 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313- 1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 200. 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313- 1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368. and 1415-1440 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 460-461, 677-974. 1006-1012. and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 25-198, 221-459, 489- 580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity 7 to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668. and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 25-198. 221-459. 489-580. 617-668. and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 460-461. 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 460- 461, 677-974, 1006-1012, and 1231-1259.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity 7 to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 85% identity' to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273. 1322- 1324, and 1329-1347 and an engineered guide polynucleotide configured to form a complex with the endonuclease, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.
  • the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348. 1369-1372. 1376- 1391, 1392-1414, and 1470-2242. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 92% sequence identity 7 to any one of SEQ ID NOs: 1327- 1328, 1348, 1369-1372, 1376-1391, 1392-1414. and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 93% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 94% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1327- 1328, 1348, 1369-1372, 1376-1391, 1392-1414. and 1470-2242. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.
  • the engineered guide polynucleotide comprises a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 98% sequence identity’ to any one of SEQ ID NOs: 1327- 1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 99% sequence identity' to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391.
  • the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392- 1414, and 1470-2242.
  • the engineered nuclease system further comprises a singlestranded DNA repair template. In some embodiments, the engineered nuclease system further comprises a double-stranded DNA repair template. In some embodiments, the single- or doublestranded DNA repair template comprises from 5' to 3' a first homology arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid sequence. In some embodiments, the single- or double-stranded DNA repair template comprises from 5' to 3' a synthetic DNA sequence of at least 10 nucleotides.
  • the single- or doublestranded DNA repair template comprises from 5' to 3' a second homology arm comprising a sequence of at least 20 nucleotides 3' to the target sequence.
  • the single- or double-stranded DNA repair template comprises from 5' to 3': a first homology arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, or a second homology arm comprising a sequence of at least 20 nucleotides 3' to the target sequence.
  • the first homology arm comprises a sequence of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60. at least 70. at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides.
  • the first homology arm comprises a sequence of at least 10 nucleotides.
  • the first homology arm comprises a sequence of at least 20 nucleotides.
  • the first homology 7 arm comprises a sequence of at least 30 nucleotides.
  • the first homology arm comprises a sequence of at least 40 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 50 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 60 nucleotides. In some embodiments, the first homology 7 arm comprises a sequence of at least 70 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 80 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 90 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 100 nucleotides.
  • the first homology arm comprises a sequence of at least 110 nucleotides. In some embodiments, the first homology 7 arm comprises a sequence of at least 120 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 130 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 140 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 150 nucleotides. In some embodiments, the first homology 7 arm comprises a sequence of at least 175 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 200 nucleotides.
  • the first homology arm comprises a sequence of at least 250 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 300 nucleotides. In some embodiments, the first homology 7 arm comprises a sequence of at least 400 nucleotides. In some embodiments, the first homology 7 arm comprises a sequence of at least 500 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 750 nucleotides. In some embodiments, the first homology arm comprises a sequence of at least 1000 nucleotides. [0215] In some embodiments, the engineered nuclease system further comprises a source of Mg 2+ .
  • the present disclosure provides an endonuclease system described herein configured to cause a chemical modification of a nucleotide base yvithin or proximal to a target locus targeted by the endonuclease system.
  • chemical modification of a nucleotide base refers to modification of the chemical moiety involved in base-pairing rather than modification of the sugar or phosphate portion of the nucleotide.
  • the chemical modification comprises deamination of an adenosine or a cytosine nucleotide.
  • the endonuclease to which the base editor is fused or coupled comprises a deactivating mutation in at least one catalytic residue of the endonuclease (e.g., in the RuvC domain).
  • the base editor is fused N- or C-terminally to said endonuclease or linked via chemical conjugation.
  • base editors include any adenosine or cytosine deaminases, comprising but not limited to Adenosine Deaminase RNA Specific 1 (AD ARI), Adenosine Deaminase RNA Specific 2 (ADAR2), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1 (APOBEC1), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 2 (APOBEC2), Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3A (APOBEC3A).
  • AD ARI Adenosine Deaminase RNA Specific 1
  • ADAR2 Adenosine Deaminase RNA Specific 2
  • APOBEC1 Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 1
  • APOBEC2 Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 2
  • APOBEC3A Apolipoprotein B MRNA Edit
  • the base editor comprises a yeast, eukaryotic, mammalian, or human base editor.
  • the present disclosure provides an endonuclease system described herein configured to cause a chemical modification of histone within or proximal to a target locus targeted by the endonuclease system.
  • endonuclease systems configured to cause a chemical modification of a histone comprise an endonuclease having a histone editor coupled or fused in frame to said endonuclease.
  • the histone editor is coupled or fused N- or C-terminally to the endonuclease.
  • the chemical modification comprise methylation, acetylation, demethylation, or deacetylation.
  • the endonuclease to which the histone editor is fused or coupled comprises a deactivating mutation in at least one catalytic residue of the endonuclease (e.g., in the RuvC domain).
  • the histone editor comprises a histone methyltransferase (e.g., ASH1L, DOT1L, EHMT1, EHMT2, EZH1. EZH2. MLL, MLL2, MLL3, MLL4, MLL5, NSD1.
  • PRDM2 SET, SETBP1, SETD1A, SETD1B, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETD9, SETDB1, SETDB2, SETMAR, SMYD1, SMYD2, SMYD3, SMYD4, SMYD5, SUV39H1, SUV39H2.
  • SUV420HI, or SUV420H2) a histone demethylase (e.g., the KDM1, KDM2, KDM3, KDM4.
  • KDM5, or KDM6 families KDM5, or KDM6 families
  • a histone acetyltransferase e.g., GNATs or HAT family acetyltransferases
  • a histone deacetylase e.g., HDAC1, HDAC2, HDAC 3, HDAC4. HDAC5.
  • the histone editor comprises a yeast, eukaryotic, mammalian, or human histone editor.
  • nucleic acid sequences encoding an engineered nuclease system comprising an endonuclease and an engineered guide polynucleotide or components of the engineered nuclease system.
  • the nucleic acid encoding the endonuclease system or components thereof is a DNA. for example a linear DNA. a plasmid DNA, or a minicircle DNA.
  • the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.
  • the nucleic acid encoding the endonuclease sy stem or components thereof is delivered by a nucleic acid-based vector.
  • 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).
  • cosmid e.g., pWE or sCos vectors
  • HAC human artificial chromosome
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosome
  • Pl -derived artificial chromosomes (PAC) Pl -derived artificial chromosomes
  • PAC Pl -derived artificial chromosomes
  • phagemid phage derivative
  • bacmid or virus.
  • 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, pEFla-mCherry-Nl vector, pEFla-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
  • the nucleic acid-based vector comprises a promoter.
  • the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof.
  • the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, pl9, p40, Synapsin, CaMKII, GRK.1, and derivatives thereof.
  • the promoter is a U6 promoter.
  • the promoter is a CAG promoter.
  • the nucleic acid-based vector is a virus.
  • 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.
  • the virus is an alphavirus.
  • the virus is a parvovirus.
  • the virus is an adenovirus.
  • the virus is an AAV.
  • the virus is a baculovirus.
  • 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.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV- rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B.
  • AAV-PHP-EB AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03.
  • the herpesvirus is HSV type 1. HSV-2, VZV, EBV, CMV, HHV-6. HHV-7, or HHV-8.
  • the virus is AAV 1 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 AAV 10 or a derivative thereof. In some embodiments, the virus is AAV 11 or a derivative thereof.
  • the virus is AAV 12 or a derivative thereof. In some embodiments, the virus is AAV 13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO 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.
  • the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the vims 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.
  • the vims is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the vims is AAV-HSC1 or a derivative thereof. In some embodiments, the vims is AAV-HSC2 or a derivative thereof. In some embodiments, the vims 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 vims is AAV-HSC6 or a derivative thereof. In some embodiments, the vims is AAV-HSC7 or a derivative thereof.
  • 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 vims is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC 11 or a derivative thereof. In some embodiments, the virus is AAV-HSC 12 or a derivative thereof. In some embodiments, the virus is AAV-HSC 13 or a derivative thereof. In some embodiments, the vims is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC 15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof.
  • the virus is AAV-DJ/8 or a derivative thereof.
  • the vims is AAV-Myo or a derivative thereof.
  • the vims is AAV-NP40 or a derivative thereof.
  • the virus is AAV-NP59 or a derivative thereof.
  • the virus is AAV-NP22 or a derivative thereof.
  • the virus is AAV-NP66 or a derivative thereof.
  • the virus is AAV-HSC16 or a derivative thereof. [0225]
  • the virus is HSV-1 or a derivative thereof.
  • the virus is HSV-2 or a derivative thereof.
  • the vims is VZV or a derivative thereof. In some embodiments, the vims is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.
  • the nucleic acid encoding the engineered nuclease system or components thereof 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.
  • 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.
  • the nucleic acid is comprised in a lipid nanoparticle (LNP).
  • the engineered nuclease system or components thereof is introduced into the cell in any suitable way, either stably or transiently.
  • the engineered nuclease system or components thereof is transfected into the cell.
  • the cell is transduced or transfected with a nucleic acid construct that encodes the engineered nuclease system or components thereof.
  • a cell is transduced (e.g., with a virus encoding the engineered nuclease system or components thereof), or transfected (e.g...
  • the transduction is a stable or transient transduction.
  • cells expressing the engineered nuclease system or components thereof or containing the engineered nuclease system or components thereof are transduced or transfected with one or more gRNA molecules, for example, when the engineered nuclease system or components thereof comprises a CRISPR nuclease.
  • a plasmid expressing the engineered nuclease system or components thereof is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lenti virus or AAV) or other methods known to those of skill in the art.
  • the gene editing system is introduced into the cell as one or more polypeptides.
  • 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.
  • Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes. liposomes, immunoliposomes, poly cation 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.
  • lipofection reagents are sold commercially (e g., TransfectamTM, LipofectinTM and SF Cell Line 4D-Nucleofector X KitTM (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.
  • the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g, in vivo administration).
  • the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.
  • the present disclosure provides a cell comprising a vector or a nucleic acid described herein.
  • the cell expresses a gene editing system or parts thereof.
  • the cell is a human cell.
  • the cell is genome edited ex vivo.
  • the cell is genome edited in vivo.
  • Described herein, in certain embodiments, is a cell comprising the systems or vectors described herein.
  • 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).
  • 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).
  • an immortalized cell e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY 5Y cell
  • an insect cell e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or aHeliothis virescens cell
  • a yeast cell e.g., a Saccharomyces cerevisiae cell, a Cry ptococcus 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.,
  • 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.
  • 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.
  • Systems of the present disclosure may be used for various applications, such as, for example, nucleic acid editing (e.g., gene editing) or binding to a nucleic acid molecule (e.g., sequence-specific binding). Such systems may be used, for example, for remediating (e.g..
  • a genetically inherited mutation that may cause a disease in a subject; inactivating a gene in order to ascertain its function in a cell; as a diagnostic tool to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or an amplified DNA sequence encoding a disease-causing mutation); as deactivated enzymes in combination with a probe to target and detect a specific nucleotide sequence (e.g., sequence encoding antibiotic resistance int bacteria); to render viruses inactive or incapable of infecting host cells by targeting viral genomes; to add genes or amend metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites; to establish a gene drive element for evolutionary selection, and/or to detect cell perturbations by foreign small molecules and nucleotides as a biosensor.
  • disease-causing genetic elements e.g., via cleavage of reverse-transcribed viral RNA or an amplified DNA sequence encoding a disease-causing mutation
  • the method comprises delivering to the target nucleic acid locus the engineered nuclease system described herein.
  • the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure.
  • the complex is configured such that upon binding of the complex to the target nucleic acid locus, the complex modifies the target nucleic locus.
  • modifying the target nucleic acid locus comprises binding, nicking, cleaving, or marking the target nucleic acid locus.
  • the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the target nucleic acid comprises genomic eukaryotic DNA, viral DNA. or bacterial DNA.
  • the target nucleic acid comprises bacterial DNA.
  • the bacterial DNA is derived from a bacterial species different to a species from which the endonuclease was derived.
  • the target nucleic acid locus is in vitro.
  • the nucleic acid locus is within a cell.
  • the endonuclease and the engineered guide nucleic acid structure are provided encoded on separate nucleic acid molecules.
  • the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.
  • the cell is derived from a species different to a species from which the endonuclease is derived.
  • delivering the engineered nuclease system to the target nucleic acid locus comprises delivering the nucleic acid described herein or the vector described herein. In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a nucleic acid comprising an open reading frame encoding the endonuclease. In some embodiments, the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked. In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a capped mRNA containing the open reading frame encoding said endonuclease. In some embodiments, delivering the engineered nuclease system to said target nucleic acid locus comprises delivering a translated polypeptide.
  • delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a deoxyribonucleic acid (DNA) encoding the engineered guide ribonucleic acid structure operably linked to a ribonucleic acid (RNA) pol III promoter.
  • the endonuclease induces a single-stranded break or a double-stranded break at or proximal to the target locus.
  • kits comprising one or more nucleic acid constructs encoding the various components of the engineered nuclease system.
  • the nucleotide sequence comprises a heterologous promoter that drives expression of the engineered nuclease system components.
  • the engineered nuclease system disclosed herein is assembled into a pharmaceutical, diagnostic, or research kit to facilitate its use in therapeutic, diagnostic, or research applications.
  • a kit may include one or more containers housing any of the vectors disclosed herein and instructions for use.
  • the kit may be designed to facilitate use of the methods described herein by researchers and can take many forms.
  • Each of the compositions of the kit may be provided in liquid form (e.g. in solution), or in solid form, (e.g., a dry' powder).
  • some of the compositions may be constitutable or otherwise processable (e g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g, videotape, DVD, etc.), Internet, and/or web-based communications, etc.
  • the written instructions in some embodiments, are in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.
  • SMART nuclease homologs were discovered by mining a large assembly-driven metagenomic database of microbial, viral, and eukaryotic genomes. HMM profiles of previously described SMART nucleases were built and searched against all proteins in the database using a software package for sequence analysis. CRISPR arrays were predicted on assembled contigs with a program to find CRISPRs in full genomes or environmental datasets such as assembled contigs from metagenomes. Proteins were filtered by e-value ( ⁇ 1x1 O' 5 ) and size (> 550 amino acids and ⁇ 1000 amino acids), and partial ORFs were removed.
  • MG34 ancestor variants were generated using two different methodologies.
  • first method previously-generated ancestors were modified by removing amino acid positions to approximate the sequence length of MG34-1 (SEQ ID NO: 2).
  • second method chimeras were designed from previously -generated ancestors by fusing the recognition lobe from active candidates with the protein backbone of active ancestors to determine if sequence divergence in the recognition lobe is at least partially responsible for the lack of activity of particular ancestral sequences.
  • Candidate MG34 nuclease effector proteins were codon optimized for E. coli and cloned into a vector with a T7 promoter and C-terminal His tag. The gene was PCR amplified with primer binding sites 150 bp upstream and downstream from the T7 promoter and terminator sequences, respectively. This PCR product was added to a reconstituted protein synthesis system where all components needed for in vitro transcription and translation are purified from E. coli at 5 nM minimum final concentration and expressed for 2 hr at 37 °CC.
  • a cleavage reaction was assembled in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgC12 with a 5-fold dilution of the protein synthesis system. 5 nM of an 8N PAM plasmid library', and 50 nM of sgRNA targeting the PAM library.
  • the sgRNA sequences used were some or all of those identified from four active MG34 homologs: MG34-1 (sgl) (SEQ ID NO: 613), MG34-9 (sg9) (SEQ ID NO: 615), MG34-16 (sgl 6) (SEQ ID NO: 616), and MG34-25 (sg25) (SEQ ID NO: 1369).
  • the cleavage products from the protein synthesis system reactions were recovered via clean up with beads.
  • the DNA was blunted via addition of Klenow fragments and dNTPs.
  • Blunt-end products were ligated with a 100-fold excess of double stranded adapter sequences and used as template for the preparation of an NGS library, from which PAM requirements were determined from sequence analysis.
  • Raw NGS reads were filtered by a quality score >20.
  • the 14-24 bp representing the known DNA sequence from the backbone adjacent to the PAM was used as a reference to find the P AM-proximal region and the 8 bp adjacent were identified as the putative PAM.
  • the distance between the PAM and the ligated adapter was also measured for each read.
  • PAM sequences were filtered by cut site frequency such that only PAMs with the most frequent cut site ⁇ 2 bp were included in the analysis.
  • MG34-35 (SEQ ID NO: 1347) is a novel system identified from metagenomics data
  • MG34-38 (SEQ ID NO: 1352) is a reconstructed MG34 ancestor
  • MG34-47 (SEQ ID NO: 1361) is a chimeric ancestor consisting of the recognition lobe of one ancestor inserted into the backbone of another ancestor (Table 5).
  • nucleases were active with all four tested sgRNAs, as shown by the expected cleavage band at approximately 180 bp (FIGs. 2A - 2B, 3A - 3B, and 4A - 4B).
  • the 3’ PAM sequence nGG appears to be the most commonly recognized by these nucleases, with subtle differences between them, and they exhibit a preference for cleavage at positions 6-8 from the PAM (FIGs. 2A - 2B, 3A - 3B, and 4A - 4B)
  • Example 2 - SMART I nucleases recognize diverse PAM sequences and are active in RNP complexes
  • Novel SMART I effectors were quantitatively assayed for cleavage activity via an in vitro cleavage assay.
  • the effectors were expressed in in vitro transcription/translation (IVTT) reactions from a PCR template as described in Example 1, either in the absence or presence of 0.4 pM of the single guide RNA from other active MG34 nucleases.
  • the IVTT mixture containing the RNP was incubated in a reaction consisting of 30% v/v IVTT and 5 nM of a plasmid DNA target with nRR PAMs (nAA, nAG, nGA, or nGG P AMs) in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgC12 for 1 hour.
  • Cleavage products were analyzed by nucleic acid electrophoresis and peak area for uncleaved (-3500 bp supercoiled) and cleavage products (-2200 bp linearized) were plotted as a percent of RNA-guided cleavage.
  • SMART I nucleases MG34-27 (SEQ ID NO: 1314) and MG34-29 (SEQ ID NO: 1316) were most active with targets with nGG and nAG PAM sequences, but also showed significant activity with nGA and more limited activity with nAA PAM sequences (Table 6; FIG. 5).
  • the amount of cleavage of targets with each PAM sequence differs depending on the single guide RNA used.
  • MG34-27 shows the highest activity for the nGG PAM with sg25 (SEQ ID NO: 1369) but shows the highest activity' for the nAG PAM with sg9 (SEQ ID NO: 615; FIG. 5).
  • Candidate MG34 nucleases were cloned into pET21b under T7 RNA polymerase- controlled promoters and expressed in cells. Cultures were grown until induction at 37° C and then temperature was lowered to 18°C and the cells expressed for 18 hours.
  • the lysis buffers contained 500 mM NaCl, 10% Glycerol, 0.5 mM TCEP and 50 mM Buffering agent.
  • CHES pH 9.0 was the buffering agent; for MG34-29 a range of buffers were tried - MES pH 6.0, Tris pH 7.5, CHES pH 9.0. Samples with 2x Laemmli buffer were separated on a Stain-Free 4-20% gradient SDS PAGE gel and visualized by fluorescence imaging.
  • MG34-29 50 nM was complexed with 75 nM MG34-1 sgRNA (sgl) (SEQ ID NO: 613) and used to cleave a supercoiled plasmid containing an nGG PAM.
  • MG34-27 50 nM was complexed with sgRNAs from MG34-1 (sgl) (SEQ ID NO: 613) and MG34-25 (sg25) (SEQ ID NO: 1369) and used to cleave four separate PAM containing plasmids (nAA, nAG, nGA, nGG).
  • Example 3 Ancestral Reconstructions of SMART I nucleases are active gene editors in human cells
  • CRISPR-associated SMART I nucleases of the MG34 family are capable of efficient dsDNA cleavage activity in vitro and in E. coll, but they have not previously shown detectable levels of activity as nucleases in human cells.
  • This family of enzymes has been expanded herein by applying ancestral sequence reconstruction methods for computational protein diversification. Computational reconstruction generated new MG34 effectors that were active for dsDNA cleavage in vitro. Herein, the activity of these enzymes in human cells was evaluated.
  • MG34-27 (SEQ ID NO: 1314) and MG34-29 (SEQ ID NO: 1316) were codon optimized for human expression and cloned into an expression vector with a T7 promoter, 5‘ and 3’ UTRs, and a polyA tail.
  • the coding sequence contained an N-terminal SV40 nuclear localization signal and a C-terminal nucleoplasmin nuclear localization signal.
  • the expression vector was midi-prepped, linearized with SapI, and used for in vitro transcription with Hi-T7.
  • In vitro transcription reactions contained N1 -methylpseudouridine in place of uridine and transcription reagent. The resulting mRNA was checked for product size and purity and diluted to 250 ng/pL in sterile water for use in nucleofection.
  • K562 cells (ATCC CCL-243) were cultured in IMDM media +10% FBS for 1-2 passages prior to nucleofection. On the day of nucleofection, cells were harvested, counted, washed in IX PBS, and resuspended in buffer according to manufacturer instructions. 120,000 cells were distributed per well and nucleofected with 500 ng of mRNA and 200 pmol of sgRNA. For some experiments, the amount of guide added was varied from 100 to 400 pmol. Cells were added to recovery' media and grown for 72 hours before genomic DNA was harvested. Resulting gDNA was diluted 1 :3 and used as a template for NGS PCR. Indels were quantified from resulting NGS reads mapped to the reference amplicon.
  • MG34-27 and MG34-29 were each tested with 96 guides with NGG PAMs targeting the AAVS1 locus.
  • the target sites are listed in Table 7.
  • Table 7 AAVS1 genome editing targets for MG34-27 and MG34-29
  • MG34-27 showed activity over 5% with three guides (FIG. 8, SEQ ID NOs: 1404. 1407, and 1412), while MG34-29 showed activity over 5% with 15 guides (FIG. 8, SEQ ID NOs: 1400-1414).
  • Example 4 Diversification of SMART I effector sequences generates active nucleases
  • ASR ancestral sequence reconstruction
  • Candidate MG34 and MG102 effectors were tested in vitro for nuclease activity and PAM preference, as described previously (Example 1).
  • the sgRNA sequences used belong to two active MG34 homologs: MG34-1 (sgl) (SEQ ID NO: 613) and MG34-25 (sg25) (SEQ ID NO: 1369).
  • the sgRNA from three active homologs were used: MG102-2 (sgl) (SEQ ID NO: 1013), MG102-39 (sg39) (SEQ ID NO: 1017), and MG102-42 (sg42) (SEQ ID NO: 1018).
  • MG102 native sequences recovered here four single guide designs were tested per protein (SEQ ID NOs: 1376-1391), and for MG34 native sequences, eight single guide designs were tested (SEQ ID NOs: 1392- 1399).
  • the different sgRNA designs included different spacer lengths, spacers, and trim positions in the CRISPR repeat and predicted tracrRNA.
  • nGG appears to be the most commonly recognized by ancestral nucleases of the MG34 family (FIGs. 12-13), while nGG or nRG is recognized by MG34-35 (FIG. 14).
  • Some nucleases show weak preferences in the 4th base position, for example NGGT over NGGN where a T is more enriched in cleavage products but not strictly required. All exhibit a preference for cleavage at positions 5-8 from the PAM (FIGs. 12-14).
  • MG102-51 SEQ ID NO: 1262
  • MG102- 53 SEQ ID NO: 1264
  • MG102-55 SEQ ID NO: 1266
  • MG102-63 SEQ ID NO: 1324
  • the ancestral nucleases MG102-65 through MG102-68 SEQ ID NOs: 1330-1333
  • MG102- 71 SEQ ID NO: 1336
  • MG102-73 SEQ ID NO: 1338
  • MG102-74 SEQ ID NO: 1339
  • MG102-77 SEQ ID NOs: 1342-1345
  • Example 5 - SMART I nucleases are active gene editors in human cells
  • Sequences for nucleases were codon optimized for human expression and cloned into an expression vector with a CleanCap T7 promoter, 5’ and 3’ UTRs, and a poly A tail. Additionally, some plasmids containing nucleases codon optimized for human expression were ordered in an expression vector and a CleanCap T7 promoter and 5’-UTR, and separately a 3'-UTR and polyA tail, were added via PCR primers. The coding sequences contained an N-terminal SV40 nuclear localization signal and a C-terminal nucleoplasmin nuclear localization signal. Cloned expression vectors for nucleases were midi-prepped), linearized with SapI, and used for in vitro transcription with Hi-T7.
  • PCR amplified nuclease coding sequences were directly used for in vitro transcription following the same Hi-T7 methods.
  • In vitro transcription reactions contained N1 -methylpseudouridine in place of uridine and had added CleanCap reagent.
  • the resulting mRNA was checked for product size and purity via Tapestation and diluted to 250 ng/pL in sterile water for use in nucleofection.
  • K.562 cells were cultured in IMDM media + 10% FBS for 1-2 passages prior to nucleofection. On the day of nucleofection, cells were harvested, counted, washed in IX PBS, and resuspended in SF buffer . 120,000 cells were distributed per well and nucleofected with 500 ng of mRNA and 200 (MG34s) or 450 (MG102s) pmol of sgRNA using a SF Cell Line 96- well NucleofectorTM Kit. Cells were added to recovery media and grown for 72 hours before genomic DNA was harvested with QuickExtract. Resulting gDNA was diluted 1 :3 and used as a template for NGS PCR. Indels were quantified from resulting NGS reads mapped to the reference amplicon.
  • the ancestral nuclease MG34-29 (SEQ ID No. 1316) was found to be active with guides with the MG34-1 guide backbone and NGG PAMs targeting three new genetic loci: Beta-2 microglobulin (B2M) (FIG. 17), hRosa2 (FIG. 18), and TRAC.
  • B2M Beta-2 microglobulin
  • 192 guides were tested and eight (8) showed an activity of >5% indel formation, with maximum editing of 47% (FIG. 17, Table 10)
  • For hRosa2 72 guides were tested and five (5) showed an activity of >5% indel formation, with maximum editing of 13% (FIG. 18, Table 10).
  • TRAC 49 guides were tested and one (1) showed an activity of >5% indel formation, with maximum editing of 8% (Table 10)
  • MG34-29 was also found to be active with additional MG34-1 guide backbone NGG PAM guides at AAVS1 (FIG. 19). An additional 100 guides were tested and 14 showed activity >5% indel formation, with maximum editing of 48% (FIG. 19, Table 10). MG34-29 was also tested with 4 guides targeting diverse loci designed to test MG34-1 and guide pPE641 targeting the EMX1 intron demonstrated high in-cell editing >80% indels, but did not demonstrate a dosedependent response when increasing sgRNA concentrations up to 400 pmol (FIG. 20, Table 10). MG34-29 was also found to be active with MG34-25 guide backbone NGG PAM guides at AAVS1, with maximum editing of 29% (FIG. 21, Table 10).
  • MG34-29 guides with >5% indel formation at B2M, hRosa26, TRAC, or AAVS1
  • the ancestral nuclease MG34-71 (SEQ ID No. 1431) was found to be active with guides with the MG34-1 and MG34-25 guide backbones and NGG PAMs targeting AAVS1. 24 guides were tested and two (2) showed activity >5% indel formation, with maximum editing of 10%
  • the ancestral nuclease MG34-72 (SEQ ID NO: 1432) was found to be active with guides with the MG34-1, MG34-25, and MG34-35 guide backbones and NGG PAMs targeting AAVS 1. 120 guides were tested and nine (9) showed an activity of >5% indel formation, with an editing (% indels) of up to 52% (FIGs. 22 and 23, Table 12).
  • the ancestral nuclease MG34-79 (SEQ ID No. 1439) was found to be active with guides with the MG34-25 guide backbone and NGG PAMs targeting AAVS1. 24 guides were tested and only one (1) showed an activity of >5% indel formation, with an editing (% indels) of up to 11% (FIG. 22, Table 13).
  • the native nuclease MG102-53 (SEQ ID No. 1264) was found to be active with guides with the MG102-53 guide backbone and NAR PAMs targeting AAVS1. 96 guides were tested and three (3) showed an activity of >5% indel formation, with an editing (% indels) of >20% (FIG. 24, Table 14)
  • the ancestral nuclease MG102-68 (SEQ ID No. 1333) was found to be active with guides with the MG102-39 guide backbone (FIG. 25), MG102-2 guide backbone (Table 15), and MG102-53 guide backbone (FIG. 26) with NRC PAMs targeting AAVS1. 120 guides were tested and twenty-five (25) showed activity >5% indel formation, with maximum editing of 50% (FIGs. 25 and 26, Table 15)
  • the ancestral nuclease MG102-71 (SEQ ID No. 1336) was found to be active with guides with the MG102-2, MG102-39, MG102-53 guide backbone and NRC PAMs targeting AAVS 1. 120 guides were tested and twenty -five (25) showed an activity of >5% indel formation, with an editing (% indels) of up to 60% (FIGs. 26 and 27, Table 16).
  • CRISPR Recognition Tool a tool for automatic detection of clustered regularly interspaced palindromic repeats
  • MAFFT multiple sequence alignment software version 7 improvements in performance and usability. Mol Biol Evol. 2013;30(4):772-780. doi: 10. 1093/molbev/mst010.

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Abstract

La présente invention concerne des enzymes endonucléases ainsi que des procédés d'utilisation de telles enzymes ou variants de celles-ci.
EP24811938.0A 2023-05-23 2024-05-23 Systèmes d'endonucléases Pending EP4716752A2 (fr)

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