Endonuclease system

Abstract

This disclosure provides endonuclease enzymes and methods for using such enzymes or variants thereof.

Description

Background Art

[0001] Cross-reference This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 503,927, filed May 23, 2023, and U.S. Provisional Patent Application No. 63 / 520,864, filed Aug. 21, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML copy created on May 23, 2024, is named MTG-027WO_SL.xml and is 3,611,069 bytes in size.

Summary of the Invention

[0003] Many class 2 Cas effectors are large (over 1200 amino acids (aa)), making delivery in therapeutic applications difficult. Accordingly, methods, compositions, and systems related to putative guide dsDNA nucleases called SMART (SMall ARchaeal-associaTed) nuclease systems are described herein. These endonuclease effectors are characterized by being small (from about 400 aa to about 1050 aa) and the presence of RuvC and HNH catalytic domains.

[0004] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0005] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise sequences having at least 90% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 90% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0006] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and a modified guide polynucleotide which forms a complex with the endonucleases and hybridizes to a target nucleic acid sequence. In some embodiments, the endonucleases comprise sequences having at least 95% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 95% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0007] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 99% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise sequences having at least 99% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 99% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0008] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases containing a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323, 1324, and 1329-1346.

[0009] In some embodiments, the modified guide polynucleotide is a single guide nucleic acid. In some embodiments, the modified guide polynucleotide is a dual guide nucleic acid. In some embodiments, the modified guide polynucleotide is RNA. In some embodiments, the endonuclease is non-covalently bonded to the modified guide polynucleotide. In some embodiments, the endonuclease is covalently bonded to the modified guide polynucleotide. In some embodiments, the endonuclease is fused to the modified guide polynucleotide.

[0010] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

[0011] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 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. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with sequence number 1410 or 1960.

[0012] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1410, 1412, 1953, 1956, 1960, 1961, 1966, 1970, and 1478.

[0013] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 2157, 2159, and 2160.

[0014] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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.

[0015] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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.

[0016] In some embodiments, the modified guide polynucleotide includes a sequence having 100% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

[0017] In certain embodiments, methods for modifying a target nucleic acid sequence are described herein, comprising contacting the target nucleic acid sequence with a modified nuclease system described herein. In some embodiments, modifying a target nucleic acid sequence includes binding, nicking, or cleaving the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence includes genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the modification is in vitro. In some embodiments, the modification is in vivo. In some embodiments, the modification is ex vivo.

[0018] In certain embodiments, a method for modifying a target nucleic acid sequence in mammalian cells is described herein, comprising contacting the mammalian cells with a modified nuclease system described herein. In some embodiments, the method further includes selecting cells containing the modification.

[0019] In certain embodiments, cells comprising the modified nuclease system described herein are described herein. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cells are modified cells. In some embodiments, the cells are stable cells.

[0020] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be recognized, other different embodiments of the present disclosure are possible, and some of its details can be modified in various obvious ways without departing from the present disclosure. Accordingly, the drawings and specification should be considered illustrative and not restrictive. [Brief explanation of the drawing]

[0021] Novel features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are utilized, and to the appended drawings (also referred to herein as "Figure" and "FIG.").

[0022] [Figure 1A]Figures 1A–1D show phylogenetic trees of SMART I nucleases for ancestral reconstruction of the MG34 (Figures 1A–1C) and MG102 (Figure 1D) clades. The phylogenetic trees were estimated using FastTree or RAxML from multi-sequence alignments of MAFFT global (G-INS-i) or local (L-INS-i). In each phylogenetic tree, the individual ancestors shown as circles represent different ancestral ages and are based on different phylogenetic trees. [Figure 1B] Figures 1A–1D show phylogenetic trees of SMART I nucleases for ancestral reconstruction of the MG34 (Figures 1A–1C) and MG102 (Figure 1D) clades. The phylogenetic trees were estimated using FastTree or RAxML from multi-sequence alignments of MAFFT global (G-INS-i) or local (L-INS-i). In each phylogenetic tree, the individual ancestors shown as circles represent different ancestral ages and are based on different phylogenetic trees. [Figure 1C] Figures 1A–1D show phylogenetic trees of SMART I nucleases for ancestral reconstruction of the MG34 (Figures 1A–1C) and MG102 (Figure 1D) clades. The phylogenetic trees were estimated using FastTree or RAxML from multi-sequence alignments of MAFFT global (G-INS-i) or local (L-INS-i). In each phylogenetic tree, the individual ancestors shown as circles represent different ancestral ages and are based on different phylogenetic trees. [Figure 1D] Figures 1A–1D show phylogenetic trees of SMART I nucleases for ancestral reconstruction of the MG34 (Figures 1A–1C) and MG102 (Figure 1D) clades. The phylogenetic trees were estimated using FastTree or RAxML from MAFFT global (G-INS-i) or local (L-INS-i) multisequence alignments. Each individual ancestor, indicated by a circle in each phylogenetic tree, represents a different ancestral age and is based on a different phylogenetic tree. Figure 1D shows the previously characterized effectors MG102-2, MG102-39, and MG102-42 within the phylogenetic tree. [Figure 2A]Figures 2A-2B show that the novel SMART I effector is an active nuclease with a non-natural guide. The SMART I effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 2A shows the PCR results. The effector was expressed in an in vitro transcription / translation (IVTT) reaction in the presence of a single guide RNA from another active MG34 nuclease and added to a PAM library (dsDNA target). Cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. When RNA-guided cleavage by the nuclease was successful, a band of the expected size of 180 bp was obtained. [Figure 2B-1] Figures 2A-2B show that the novel SMART I effector is an active nuclease with a non-natural guide. The SMART I effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 2B shows the NGS sequencing results. Using the NGS sequencing of the bands confirmed in Figure 2A, the PAM sequence and the cleavage position relative to the PAM sequence were determined. [Figure 2B-2] Figures 2A-2B show that the novel SMART I effector is an active nuclease with a non-natural guide. The SMART I effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 2B shows the NGS sequencing results. Using the NGS sequencing of the bands confirmed in Figure 2A, the PAM sequence and the cleavage position relative to the PAM sequence were determined. [Figure 2B-3] Figures 2A-2B show that the novel SMART I effector is an active nuclease with a non-natural guide. The SMART I effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 2B shows the NGS sequencing results. Using the NGS sequencing of the bands confirmed in Figure 2A, the PAM sequence and the cleavage position relative to the PAM sequence were determined. [Figure 2B-4]Figures 2A-2B show that the novel SMART I effector is an active nuclease with a non-natural guide. The SMART I effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 2B shows the NGS sequencing results. The NGS sequencing of the bands confirmed in Figure 2A was used to determine the PAM sequence and the cleavage positions relative to the PAM sequence. [Figure 3A] Figures 3A-3B show that the novel SMART I effector is an active nuclease. The SMART I ancestral effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 3A shows the PCR results. The effector was expressed in an in vitro transcription / translation (IVTT) reaction in the presence of a single guide RNA from another active MG34 nuclease and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. When RNA-guided cleavage by the nuclease was successful, a band of the expected size of 180 bp was obtained. [Figure 3B-1] Figures 3A-3B show that the novel SMART I effector is an active nuclease. The SMART I ancestral effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 3B shows the NGS sequencing results. The NGS sequencing of the bands confirmed in Figure 3A was used to determine the PAM sequence and the cleavage positions relative to the PAM sequence. [Figure 3B-2] Figures 3A-3B show that the novel SMART I effector is an active nuclease. The SMART I ancestral effector was assayed for cleavage activity using a PAM enrichment protocol. Figure 3B shows the NGS sequencing results. The NGS sequencing of the bands confirmed in Figure 3A was used to determine the PAM sequence and the cleavage positions relative to the PAM sequence. [Figure 3B-3]Figures 3A and 3B show that the novel SMART I effector is an active nuclease. The SMART I ancestral effector was assayed for cleavage activity using the PAM enrichment protocol. Figure 3B shows the NGS sequencing results. The PAM sequence and the cleavage site relative to the PAM sequence were determined using the NGS sequencing of the bands confirmed in Figure 3A. [Figure 3B-4] Figures 3A and 3B show that the novel SMART I effector is an active nuclease. The SMART I ancestral effector was assayed for cleavage activity using the PAM enrichment protocol. Figure 3B shows the NGS sequencing results. The PAM sequence and the cleavage site relative to the PAM sequence were determined using the NGS sequencing of the bands confirmed in Figure 3A. [Figure 4A] Figures 4A and 4B show that the novel chimeric ancestral SMART I effector is an active nuclease. The chimeric ancestral SMART I effector was assayed for cleavage activity using the PAM enrichment protocol. Figure 4A shows the PCR results. The effector was expressed in vitro by transcription / translation (IVTT) in the presence of a single guide RNA from another active MG34 nuclease and added to a PAM library (dsDNA target). The cleavage product was amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 4B] Figures 4A and 4B show that the novel chimeric ancestral SMART I effector is an active nuclease. The chimeric ancestral SMART I effector was assayed for cleavage activity using the PAM enrichment protocol. Figure 4B shows the NGS sequencing results. For MG34-47, the PAM sequence and the cleavage site relative to the PAM sequence were determined using the NGS sequencing of the bands confirmed in Figure 4A. [Figure 5]Figure 5 shows a bar graph illustrating the recognition of diverse PAM sequences by the SMART I ancestral effector. MG34 nucleases were quantitatively assayed for cleavage activity by in vitro cleavage assays. Effectors were expressed in in vitro transcription / translation (IVTT) reactions in the absence (apo) or presence of a single guide RNA from another active MG34 nuclease, and cleavage was initiated by incubation of a dsDNA target with an nRR PAM (nAA, nAG, nGA, or nGG PAM) with a ribonucleoprotein complex. The cleavage products were analyzed by nucleic acid electrophoresis, and the peak areas of uncleaved (approximately 3500 bp superhelical) and cleavage products (approximately 2200 bp linear) were plotted as the percentage of RNA-guided cleavage (y-axis). [Figure 6A] Figures 6A and 6B show the results of SDS-PAGE gel analysis demonstrating the solubility of the SMART I ancestral effector. Figure 6A shows the purification of MG34-27. The sample was treated with 2x concentration Laemmli buffer, separated on a Stain-Free 4-20% gradient SDS-PAGE gel, and visualized by fluorescence imaging. The "Sonication" lane shows the whole cell sample after lysis, and the "Load" lane shows the contents of the soluble fraction of the cell lysate. In Figure 6A, lanes A11-B10 show the eluates obtained by gradient elution with 20-300 mM imidazole. [Figure 6B] Figures 6A and 6B show the results of SDS-PAGE gel analysis demonstrating the solubility of the SMART I ancestral effector. Figure 6B shows the purification of MG34-29. The sample was treated with 2x concentration Laemmli buffer, separated on a Stain-Free 4-20% gradient SDS-PAGE gel, and visualized by fluorescence imaging. The "Sonication" lane shows the whole cell sample after lysis, and the "Load" lane shows the contents of the soluble fraction of the cell lysate. Figure 6B shows the results of stepwise elution at three different pH levels. [Figure 7A]Figures 7A and 7B show the results of activity assays demonstrating RNA-induced cleavage of plasmids using MG34-27 (Figure 7A) and MG34-29 (Figure 7B) with sgRNAs derived from existing nucleases. MG34-29 (50 nM) was complexed with excess MG34-1 sgRNA (sg1) and used to cleave a superhelical plasmid containing nGG PAM. MG34-27 was complexed with sgRNAs (sg1 and sg25) derived from MG34-1 and MG34-25 and used to cleave four separate PAM-containing plasmids. The expected size of the cleaved plasmids was 2200 bp, and the products are shown as red ovals. [Figure 7B] Figures 7A and 7B show the results of activity assays demonstrating RNA-induced cleavage of plasmids using MG34-27 (Figure 7A) and MG34-29 (Figure 7B) with sgRNAs derived from existing nucleases. MG34-29 (50 nM) was complexed with excess MG34-1 sgRNA (sg1) and used to cleave a superhelical plasmid containing nGG PAM. MG34-27 was complexed with sgRNAs (sg1 and sg25) derived from MG34-1 and MG34-25 and used to cleave four separate PAM-containing plasmids. The expected size of the cleaved plasmids was 2200 bp, and the products are shown as red ovals. [Figure 8] Figure 8 shows the results of the nuclease activity assay. Nuclease activity was evaluated by nucleofection of K562 cells with mRNA (500 ng) of MG34-27 or MG34-29 and sgRNA (200 pmol) targeting each target site within the AAVS1 locus. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of two replicates. [Figure 9]Figure 9 shows the results of the nuclease activity assay. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-29 mRNA (500 ng) and sequentially increased doses of sgRNA (100, 200, 300, or 400 pmol) targeting each target site within the AAVS1 locus. After 72 hours, cells were harvested, and an NGS library was prepared to evaluate editing efficiency. Each bar represents the editing efficiency at a site targeted by a specific spacer (C7, E7, F7, or G7) and is the mean of two replicates. [Figure 10A] Figures 10A–10C show the identification of ancestral sequences. Ancestral intermediate sequences were generated based on phylogenetic trees containing 441 sequences (Figure 10A) or 190 sequences (Figure 10B). Ancestral intermediate sequences were generated by randomly introducing amino acids from one ancestor (First) into another ancestor, using the weights shown in Figure 10C and the PAML probabilities obtained from the original ancestral sequence reconstruction. For the two phylogenetic trees, up to four different ancestral groups were generated based on ancestral nodes 1 through 4, according to the relative weights in the random sampling of each ancestor shown in the "Sampling weight" column of Figure 10C. [Figure 10B] Figures 10A–10C show the identification of ancestral sequences. Ancestral intermediate sequences were generated based on phylogenetic trees containing 441 sequences (Figure 10A) or 190 sequences (Figure 10B). Ancestral intermediate sequences were generated by randomly introducing amino acids from one ancestor (First) into another ancestor, using the weights shown in Figure 10C and the PAML probabilities obtained from the original ancestral sequence reconstruction. For the two phylogenetic trees, up to four different ancestral groups were generated based on ancestral nodes 1 through 4, according to the relative weights in the random sampling of each ancestor shown in the "Sampling weight" column of Figure 10C. [Figure 10C]Figures 10A–10C show the identification of ancestral sequences. Ancestral intermediate sequences were generated based on phylogenetic trees containing 441 sequences (Figure 10A) or 190 sequences (Figure 10B). Ancestral intermediate sequences were generated by randomly introducing amino acids from one ancestor (First) into another ancestor, using the weights shown in Figure 10C and the PAML probabilities obtained from the original ancestral sequence reconstruction. For the two phylogenetic trees, up to four different ancestral groups were generated based on ancestral nodes 1 through 4, according to the relative weights in the random sampling of each ancestor shown in the "Sampling weight" column of Figure 10C. [Figure 11A] Figure 11A shows the cleavage activity assay. The MG34 SMART I effector was assayed for cleavage activity using the PAM enrichment protocol. The effector was expressed in vitro by transcription / translation (IVTT) in the presence of a single guide RNA from the active nuclease MG34-1 and added to a PAM library (dsDNA target). The cleavage product was amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp (arrow). The positive control shows the MG34-1 nuclease and its natural guide RNA, and the lane number indicates the tested MG34 candidate (e.g., lane 55 is MG34-55). [Figure 11B] Figure 11B shows the cleavage activity assay. The SMART I effector was assayed for cleavage activity using the PAM enrichment protocol. The effector was expressed in vitro in transcription / translation (IVTT) in the presence of a single guide RNA from the active nuclease MG34-25 and added to a PAM library (dsDNA target). The cleavage product was amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. The lane numbers represent the tested MG34 candidate (e.g., lane 55 is MG34-55). [Figure 12-1]Figure 12 shows the NGS sequences of the bands identified in Figures 11A-11B, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-71, MG34-72, MG34-73, and MG34-74. [Figure 12-2] Figure 12 shows the NGS sequences of the bands identified in Figures 11A-11B, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-71, MG34-72, MG34-73, and MG34-74. [Figure 12-3] Figure 12 shows the NGS sequences of the bands identified in Figures 11A-11B, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-71, MG34-72, MG34-73, and MG34-74. [Figure 12-4] Figure 12 shows the NGS sequences of the bands identified in Figures 11A-11B, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-71, MG34-72, MG34-73, and MG34-74. [Figure 13-1] Figure 13 shows the NGS sequences of the bands identified in Figure 11, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79. [Figure 13-2] Figure 13 shows the NGS sequences of the bands identified in Figure 11, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79. [Figure 13-3] Figure 13 shows the NGS sequences of the bands identified in Figure 11, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79. [Figure 13-4] Figure 13 shows the NGS sequences of the bands identified in Figure 11, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79. [Figure 13-5] Figure 13 shows the NGS sequences of the bands identified in Figure 11, which were used to generate preferred PAM sequences and cleavage sites for the PAM sequences of the active nucleases MG34-75, MG34-76, MG34-77, MG34-78, and MG34-79. [Figure 14-1] Figure 14 shows the cleavage activity assay. MG34-35 effectors were assayed for cleavage activity using the PAM enrichment protocol. The effectors were expressed in vitro by transcription / translation (IVTT) in the presence of their single guide RNAs (SEQ ID NOs. 1392-1399) and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 14-2] Figure 14 shows the cleavage activity assay. MG34-35 effectors were assayed for cleavage activity using the PAM enrichment protocol. The effectors were expressed in vitro by transcription / translation (IVTT) in the presence of their single guide RNAs (SEQ ID NOs. 1392-1399) and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 14-3]Figure 14 shows the cleavage activity assay. MG34-35 effectors were assayed for cleavage activity using the PAM enrichment protocol. The effectors were expressed in vitro by transcription / translation (IVTT) in the presence of their single guide RNAs (SEQ ID NOs. 1392-1399) and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 14-4] Figure 14 shows the cleavage activity assay. MG34-35 effectors were assayed for cleavage activity using the PAM enrichment protocol. The effectors were expressed in vitro by transcription / translation (IVTT) in the presence of their single guide RNAs (SEQ ID NOs. 1392-1399) and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 14-5] Figure 14 shows the cleavage activity assay. MG34-35 effectors were assayed for cleavage activity using the PAM enrichment protocol. The effectors were expressed in vitro by transcription / translation (IVTT) in the presence of their single guide RNAs (SEQ ID NOs. 1392-1399) and added to a PAM library (dsDNA target). The cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by the nuclease yielded a band of the expected size of 180 bp. [Figure 15-1]Figure 15 shows the cleavage activity assay. MG102 SMART I effectors were assayed for cleavage activity using the PAM enrichment protocol. Effectors were expressed in vitro in transcription / translation (IVTT) in the presence of their single guide RNAs and added to a PAM library (dsDNA target). For native effectors (51, 53, 55, 63), four single guide designs were tested, while for ancestral effectors (64-81), guides derived from the active candidate RNAs MG102-2, MG102-39, and MG102-42 were used. Cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by nuclease yielded a band of the expected size of 180 bp. [Figure 15-2] Figure 15 shows the cleavage activity assay. MG102 SMART I effectors were assayed for cleavage activity using the PAM enrichment protocol. Effectors were expressed in vitro in transcription / translation (IVTT) in the presence of their single guide RNAs and added to a PAM library (dsDNA target). For native effectors (51, 53, 55, 63), four single guide designs were tested, while for ancestral effectors (64-81), guides derived from the active candidate RNAs MG102-2, MG102-39, and MG102-42 were used. Cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by nuclease yielded a band of the expected size of 180 bp. [Figure 15-3]Figure 15 shows the cleavage activity assay. MG102 SMART I effectors were assayed for cleavage activity using the PAM enrichment protocol. Effectors were expressed in vitro in transcription / translation (IVTT) in the presence of their single guide RNAs and added to a PAM library (dsDNA target). For native effectors (51, 53, 55, 63), four single guide designs were tested, while for ancestral effectors (64-81), guides derived from the active candidate RNAs MG102-2, MG102-39, and MG102-42 were used. Cleavage products were amplified by ligation to the cleavage site and subsequent PCR amplification. Successful RNA-guided cleavage by nuclease yielded a band of the expected size of 180 bp. [Figure 16A-1] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-2] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-3]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-4] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-5] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-6]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16A-7] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16B-1] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16B-2]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16B-3] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16B-4] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16C-1]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16C-2] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16C-3] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16C-4]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16D-1] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16D-2] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16D-3]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16D-4] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16E-1] Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 16E-2]Figures 16A-16E show the NGS sequences of the bands identified in Figure 15, which allowed us to determine the preferred PAM sequence (Figure 16A) and cleavage sites relative to the PAM sequence (Figures 16B-16E) 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, and MG102-80. [Figure 17] Figure 17 shows the mammalian activity of MG34-29 at the β2 microglobulin (B2M) locus using the MG34-1 nGG PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-29 mRNA (500 ng) and sgRNAs (200 pmol) targeting each target site within the B2M locus using the MG34-1 guide scaffold. 192 B2M guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of three replicates. [Figure 18] Figure 18 shows the mammalian activity of MG34-29 at the hRosa26 locus using the MG34-1 nGG PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-29 mRNA (500 ng) and sgRNAs (200 pmol) targeting each target site within the hRosa26 locus using the MG34-1 guide scaffold. 72 hRosa26 guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of three replicates. [Figure 19]Figure 19 shows the mammalian activity of ASR MG34-29 at the AAVS1 locus using the MG34-1 nGG PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-29 mRNA (500 ng) and sgRNAs (200 pmol) targeting each target site within the AAVS1 locus using the MG34-1 guide scaffold. In this study, 192 B2M guides and 96 AAVS1 guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of three replicates. [Figure 20] Figure 20 shows the mammalian activity of ASR MG34-29 using the MG34-1 ABE active guide targeting EMX1. Nuclease activity was evaluated by nucleofection of K562 cells using the MG34-1 guide scaffold with MG34-29 mRNA (500 ng) and sgRNAs (100, 200, 300, and 400 pmol) targeting each target site within AAVS1 (pPE guide). Four different locus guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar shows the editing efficiency at the site targeted by a specific spacer (X-axis). [Figure 21] Figure 21 shows the mammalian activity of ASR MG34-29 in AAVS1 using the MG34-25 scaffold nGG PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-29 mRNA (500 ng) and sgRNAs (200 pmol) targeting each target site within the AAVS1 locus using the MG34-25 guide scaffold. 96 AAVS1 guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of two replicates. [Figure 22]Figure 22 shows the mammalian activity of ASR MG34 using four AAVS1 locus MG34-1 and four MG34-25 scaffold activity spacers. Nuclease activity was evaluated by nucleofection of K562 cells with various MG34 nuclease mRNAs (500 ng) and sgRNAs (200 pmol) targeting each of the four sites within the AAVS1 locus, using either the MG34-1 (left) or MG34-25 (right) guide scaffold. Four AAVS1 guides were tested. After 72 hours, cells were harvested, and NGS libraries were prepared to evaluate editing efficiency. Each bar represents the editing efficiency at the targeted site by a specific spacer (C7, E7, F7, or G7) and guide scaffold combination. [Figure 23] Figure 23 shows the mammalian activity of ASR MG34-72 in AAVS1 using the MG34-25 scaffold nGG PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG34-72 mRNA (500 ng) and sgRNA (200 pmol) targeting various sites within the AAVS1 locus using the MG34-25 guide scaffold. 96 AAVS1 guides were tested. Cells were harvested after 72 hours and prepared for NGS to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (X-axis) and is the mean of two replicates. [Figure 24] Figure 24 shows the mammalian activity of MG102 using the MG102-53 nAR AAVS1 guide. Nuclease activity was evaluated by nucleofection of K562 cells targeting various sites within the AAVS1 locus using MG102-53 (500 ng) and its own nAR PAM sgRNA (450 pmol). 96 AAVS1 guides were tested. After 72 hours, cells were harvested, an NGS library was prepared, and editing efficiency was evaluated. Each bar represents the editing efficiency at the site targeted by a specific spacer (x-axis). [Figure 25]Figure 25 shows the mammalian activity of ASR MG102-68 using the MG102-39 nRC PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG102-68 mRNA (500 ng) and sgRNAs (450 pmol) targeting various sites within the AAVS1 locus using the MG102-39 guide scaffold at the nRC PAM site. 96 AAVS1 guides were tested. After 72 hours, cells were harvested, NGS libraries were prepared, and editing efficiency was evaluated. Each bar represents the editing efficiency at the site targeted by a specific spacer (x-axis). [Figure 26] Figure 26 shows the mammalian activity of ASR MG102 using the MG102-2 nRC PAM activity spacer and various MG102 guide scaffolds. Nuclease activity was evaluated by nucleofection of K562 cells with MG102 mRNA (500 ng) and sgRNA (450 pmol) targeting various sites within the AAVS1 locus. The guide scaffold used was either the MG102-2, MG102-39, or MG102-53 guide, and the spacer was pre-validated with MG102-2 nuclease. Twenty-four AAVS1 guides were tested. After 72 hours, cells were harvested, and NGS libraries were prepared to evaluate editing efficiency. Each bar represents the editing efficiency at the site targeted by a specific spacer (x-axis). [Figure 27] Figure 27 shows the mammalian activity of ASR MG102-71 using the MG102-39 nRC PAM guide. Nuclease activity was evaluated by nucleofection of K562 cells with MG102-71 mRNA (500 ng) and sgRNAs (450 pmol) targeting various sites within the AAVS1 locus using the MG102-39 guide scaffold at the nRC PAM site. 96 AAVS1 guides were tested. After 72 hours, cells were harvested, NGS libraries were prepared, and editing efficiency was evaluated. Each bar represents the editing efficiency at the site targeted by a specific spacer (x-axis).

[0023] A brief explanation of sequence listings The sequence listings submitted with this specification provide exemplary polynucleotide and polypeptide sequences for use in the methods, compositions, and systems described herein. The following is an exemplary description of some of these sequences.

[0024] MG33 Nuclease Sequence IDs 1, 463-486, 981-988, and 1289-1312 show the full-length peptide sequences of the MG33 nuclease.

[0025] Sequence IDs 199 and 669-670 show the nucleotide sequences of tracrRNAs predicted to function with the MG33 nuclease.

[0026] Sequence IDs 201 and 1003–1005 show the nucleotide sequences of the predicted single guide RNA (sgRNA) sequence that is expected to function with the MG33 nuclease. "N" indicates a variable residue, and non-N residues represent the scaffold sequence.

[0027] Sequence IDs 1023-1028 show PAM sequences that match the MG33 nuclease.

[0028] Sequence IDs 1045-1054 represent the CRISPR repeats of the MG33 nuclease described herein.

[0029] MG34 Nuclease Sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440 show the full-length peptide sequences of the MG34 nuclease.

[0030] Sequence IDs 200 and 1348 show the nucleotide sequences of tracrRNAs predicted to function with the MG34 nuclease.

[0031] Sequence IDs 202, 203, 613–616, and 1369 show the nucleotide sequences of the predicted single guide RNA (sgRNA) sequences that are expected to function with the MG34 nuclease. "N" indicates a variable residue, and non-N residues represent the scaffold sequence.

[0032] Sequence IDs 1023-1028 and 1441-1450 represent PAM sequences that match the MG34 nuclease.

[0033] Sequence IDs 1055-1057 and 1349 represent the CRISPR repeats of the MG34 nuclease described herein.

[0034] Sequence IDs 1392-1414 show the nucleotide sequences of the MG34 single guide RNA.

[0035] Sequence IDs 1470-2242 show the nucleotide sequences of chemically synthesized / modified MG34 sgRNAs. Sequence IDs 1470-1485 show the nucleotide sequences of MG34-35 sgRNAs targeting AAVS1. Sequence IDs 1486-1489 show the nucleotide sequences of MG34-25 sgRNAs targeting AAVS1. Sequence IDs 1490-1493 show the nucleotide sequences of MG34-1 sgRNAs targeting AAVS1. Sequence IDs 1494-1685 show the nucleotide sequences of MG34-1 sgRNAs targeting B2M. Sequence IDs 1686-1757 show the nucleotide sequences of MG34-1 sgRNAs targeting hRosa26. Sequence IDs 1758-1806 show the nucleotide sequences of MG34-1 sgRNAs targeting TRAC. Sequence ID 1807 shows the nucleotide sequence of MG34-1 sgRNA targeting the VISTA enhancer hs267 regulatory region pPE633. Sequence ID 1808 shows the nucleotide sequence of MG34-1 sgRNA targeting the Sharpr-MPRA regulatory region 15312 pPE634. Sequence ID 1809 shows the nucleotide sequence of MG34-1 sgRNA targeting the EMX1 intron pPE641. Sequence ID 1810 shows the nucleotide sequence of MG34-1 sgRNA targeting the HSPA12A gene (Hsp70 member 12A) pPE635. Sequence IDs 1811-1906 show the nucleotide sequences of MG34-1 sgRNA targeting AAVS1. Sequence IDs 1907-2002 show the nucleotide sequences of MG34-25 sgRNA targeting AAVS1. Sequence IDs 2003-2098 show the nucleotide sequences of MG102-39 sgRNA targeting AAVS1. Sequence IDs 2099-2194 show the nucleotide sequences of MG102-53 sgRNA targeting AAVS1. Sequence IDs 2195-2202 show the nucleotide sequences of MG102-2 sgRNA targeting AAVS1. Sequence IDs 2203-2210 show the nucleotide sequences of MG102-39 sgRNA targeting AAVS1. Sequence IDs 2211-2218 show the nucleotide sequences of MG102-53 sgRNA targeting AAVS1.Sequence IDs 2219-2226 show the nucleotide sequences of MG102-2 sgRNA that targets TRAC. Sequence IDs 2227-2234 show the nucleotide sequences of MG102-39 sgRNA that targets TRAC. Sequence IDs 2235-2242 show the nucleotide sequences of MG102-53 sgRNA that targets TRAC.

[0036] MG35 Nuclease Sequence IDs 25-198, 221-459, 489-580, 617-668, and 674-675 show the full-length peptide sequences of the MG35 nuclease.

[0037] Sequence IDs 460-461 show the nucleotide sequences of MG35 tracrRNA, which originates from the same gene locus as the MG35 nuclease.

[0038] Sequence IDs 462, 676, and 1229-1230 represent the CRISPR repeats of the MG35 nuclease described herein.

[0039] Sequence IDs 677-686, 1006-1012, and 1231-1259 show the nucleotide sequences of the MG35 single guide RNA.

[0040] Sequence IDs 687-974 show the nucleotide sequences of the MG35 single guide RNA coding sequence.

[0041] Sequence IDs 1029-1034 represent PAM sequences that match the MG35 nuclease.

[0042] Sequence IDs 1172-1228 show the nucleotide sequences of the gene loci encoding the MG35 nuclease described herein.

[0043] MG102 Nuclease Sequence IDs 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346 show the full-length peptide sequences of the MG102 nuclease.

[0044] Sequence IDs 672-673, 1327-1328, and 1370-1372 show the nucleotide sequences of MG102 tracrRNA, which originate from the same locus as the MG102 nuclease.

[0045] Sequence IDs 205-220 show exemplary nuclear localization sequences (NLS) that are added to the nucleases according to the present disclosure in some embodiments.

[0046] Sequence IDs 1013-1022 and 1376-1391 show the nucleotide sequences of the MG102 single guide RNA.

[0047] Sequence IDs 1035-1044 and 1451-1469 represent PAM sequences that match the MG102 nuclease.

[0048] Sequence IDs 1058-1072, 1325-1326, and 1373-1375 represent the CRISPR repeats of the MG102 nuclease described herein.

[0049] Sequence ID 1171 shows the nucleotide sequence of the gene locus encoding the MG102 nuclease described herein.

[0050] MG143 Nuclease Sequence ID 975 shows the full-length peptide sequence of MG143 nuclease.

[0051] Sequence ID 1073 represents the CRISPR repeat of the MG143 nuclease described herein.

[0052] MG144 Nuclease Sequence IDs 976-979 and 1274-1288 show the full-length peptide sequences of the MG144 nuclease.

[0053] Sequence IDs 1074-1077 represent the CRISPR repeats of the MG144 nuclease described herein.

[0054] MG145 Nuclease Sequence ID 980 shows the full-length peptide sequence of MG145 nuclease.

[0055] Sequence ID 1078 represents the CRISPR repeat of the MG145 nuclease described herein.

[0056] MG102 TRAC targeting Sequence IDs 1079-1082 and 1145-1166 show the DNA sequences of the TRAC target sites.

[0057] Sequence IDs 1083-1086 and 1123-1144 show the nucleotide sequences of sgRNAs modified to function with the MG102 nuclease in order to target TRAC.

[0058] MG33 TRAC targeting Sequence IDs 1167-1168 show the nucleotide sequences of sgRNA modified to function with the MG33 nuclease in order to target TRAC.

[0059] Sequence IDs 1169-1170 show the DNA sequences of the TRAC target sites.

[0060] AAVS1 targeting Sequence IDs 1087-1104 show the nucleotide sequences of sgRNAs modified to function with the MG102 nuclease in order to target AAVS1.

[0061] Sequence IDs 1105-1122 show the DNA sequences of the AAVS1 target site.

[0062] others Sequence IDs 2270-2330 show the nucleotide sequences of target sites in the genome. [Modes for carrying out the invention]

[0063] definition While various embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions can be conceived by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used.

[0064] The practices of some of the methods disclosed herein employ techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise indicated. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMAusubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies: A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RIFreshney, ed. (2010)).

[0065] Where used in this disclosure, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context otherwise explicitly indicates. Furthermore, the terms “including,” “includes,” “having,” “has,” and “with,” or their variations thereof, are intended to be as inclusive as the term “comprising,” to the extent that they are used in either the detailed description and / or the claims.

[0066] The terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined, i.e., the limits of the measuring system. For example, “about” may mean within one or more standard deviations according to the practice of the art. Alternatively, “about” may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0067] As used in this disclosure, the term “nucleotide” refers to a base-sugar-phosphate combination. Nucleotides intended to be nucleotides include naturally occurring and synthetic nucleotides. A nucleotide is the monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates such as adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, e.g., dATP, dCTP, dITP, dUTP, dGTP, dTTP, or their derivatives. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used in this disclosure, the term nucleotide also includes dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Exemplary examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or may be labeled in a detectable manner, such as by using optically detectable moieties (e.g., fluorophores) or moieties containing quantum dots. Examples of detectable labels include radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Examples of fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer (Foster City, Calif), FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer (Mannheim, Indianapolis, India), and Molecular Chromosome-labeled nucleotides available from Probes (Eugene, Oreg) include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically modified nucleotide is biotin-dNTP.Non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0068] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, whether single-stranded, double-stranded, or multi-stranded, that are either deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides as intended include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene fragments, multiple loci (a single locus) defined by binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. When T is referred to in the context of polynucleotides, T means U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., modified backbones, sugars, or nucleic acid bases). Where present, modifications to the nucleotide structure are conferred before or after polymer assembly. Non-limiting examples of modifications include 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein conjugated to sugars), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosin, and waiosin. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0069] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of at least two amino acid residues joined by peptide bonds. These terms do not imply a specific length of the polymer and are not intended to imply or distinguish whether peptides are produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers containing 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 (e.g., domains) with or without secondary or tertiary structures. The terms also encompass amino acid polymers modified by any other operations, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with labeling components. As used in this disclosure, the terms “amino acid” and “multiple 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 chemical moiety that is not naturally present on the amino acid. The term "amino acid" includes both D-amino acids and L-amino acids.

[0070] As used in this disclosure, “non-natural” means a nucleic acid or polypeptide sequence that does not exist in nature. Non-natural means a nucleic acid or polypeptide sequence that does not exist in nature, including modifications such as mutations, insertions, or deletions. The term non-natural encompasses fusion nucleic acids or polypeptides in which the non-natural sequence encodes or exhibits activity (e.g., enzyme activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) of the nucleic acid or polypeptide sequence to which it is fused. Non-natural nucleic acids or polypeptide sequences include those that are genetically engineered to ligate a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to produce a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.

[0071] As used in this disclosure, “operably linked,” “operable linkage,” “operatively linked,” or their grammatical equivalents refer to the arrangement of gene elements, such as promoters, enhancers, polyadenylation sequences, etc., where the operation (e.g., movement or activation) of a first gene element has some effect on a second gene element. The effect on the second gene element may, but does not have to be, the same type as the operation of the first gene element. For example, if the movement of the first element causes the activation of the second element, then the two gene elements are operably linked. For example, if a regulatory element, which may include a promoter sequence and / or an enhancer sequence, helps initiate transcription of a coding sequence, then the regulatory element is operably linked to the coding region. Intervening residues may exist between the regulatory element and the coding region, as long as this functional relationship is maintained.

[0072] A “functional fragment” of a DNA or protein sequence refers to a fragment that possesses biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to influence expression in a manner attributable to the full-length sequence.

[0073] The terms “modified,” “synthetic,” and “artificial” are used interchangeably herein to refer to objects modified by human intervention. For example, these terms refer to polynucleotides or polypeptides that do not exist in nature. Modified peptides have, but do not require, low sequence identity to naturally occurring human proteins (e.g., less than 50%, less than 25%, less than 10%, less than 5%, less than 1%). For example, the VPR domain and VP64 domain are synthetic transactivation domains. Non-limiting examples include: nucleic acids modified by altering their sequence to a sequence that does not occur in nature; nucleic acids modified by ligating them to nucleic acids that are not naturally associated so that the ligated product possesses a function not present in the original nucleic acid; modified nucleic acids synthesized in vitro using sequences that do not exist in nature; proteins modified by altering their amino acid sequence to a sequence that does not exist in nature; and engineered proteins that acquire new functions or properties. A “modified” system contains at least one modified component.

[0074] As used herein, the term “optimally aligned” refers to the alignment of two amino acid sequences that gives the highest identity percentage score or maximizes the number of matched residues.

[0075] The terms "tracrRNA" or "tracr sequence" refer to the transactivation of CRISPR RNA. TracrRNA interacts with CRISPR(cr)RNA to form the guide (g)RNA for type II and subtype VB CRISPR-Cas systems. When tracrRNA is manipulated, it may have approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA derived from S. pyogenes, S. aureus). TracrRNA may refer to modified forms of tracrRNA, which may include nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeric forms. The term tracrRNA encompasses nucleic acids that may be at least approximately 60% identical to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from S. pyogenes, S. aureus, etc.) over a stretch of at least six consecutive nucleotides. For example, a tracrRNA sequence may have at least approximately 60% identity, at least approximately 65% ​​identity, at least approximately 70% identity, at least approximately 75% identity, at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 95% identity, at least approximately 98% identity, at least approximately 99% identity, or 100% identity over a stretch of at least six consecutive nucleotides to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from S. pyogenes, S. aureus, etc.). Type II tracrRNA sequences can be predicted on a genomic sequence by identifying regions that are complementary to a portion of the repetitive sequences in an adjacent CRISPR array.

[0076] As used herein, “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that can hybridize to a target nucleic acid, thereby directing the relevant nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid may include crRNA or tracrRNA, or a combination of both. The term guide nucleic acid encompasses engineered guide nucleic acids and programmable guide nucleic acids that specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. A double-stranded target polynucleotide chain that is complementary to the guide nucleic acid and hybridizes with it is called the complementary chain. A double-stranded target polynucleotide chain that is complementary to the complementary chain and therefore not complementary to the guide nucleic acid is called the non-complementary chain. A guide nucleic acid having a polynucleotide chain is called a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is called a “double guide nucleic acid.” Unless otherwise specified, the term “guide nucleic acid” is inclusive and refers to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may include a segment referred to as a “nucleic acid targeting segment,” “nucleic acid targeting sequence,” or “spacer.” A nucleic acid targeting segment may include a subsegment referred to as a “protein-binding segment,” “protein-binding sequence,” or “Cas protein-binding segment.”

[0077] The terms “sequence identity” or “identity rate” in relation to two or more nucleic acid or polypeptide sequences refer to two or more sequences (e.g., in pairwise alignment) or (e.g., in multiple sequence alignment) that are identical or have a specific proportion of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence across a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP using a BLOSUM62 scoring matrix with a word length (W) parameter of 3, an expected value (E) parameter of 10, and gap costs set by 11 presence and 1 extension, and with conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using a PAM30 scoring set gap cost of 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); or CLUSTALW using parameters of the Smith-Waterman homology search algorithm with a match parameter of 2, a mismatch parameter of -1, and a gap parameter of -1; MUSCLE using default parameters; MAFFT using parameter retrieval of 2 and a maximum of 1000 iterations; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0078] As used herein, the term “RuvC_III domain” refers to the third discontinuous segment of the RuvC endonuclease domain (the RuvC nuclease domain consists of three discontinuous segments: RuvC_I, RuvC_II, and RuvC_III). The RuvC domain or its segments can generally be identified by alignment to a documented domain sequence, structural alignment to a protein with an annotated domain, or comparison with a hidden Markov model (HMM) constructed based on a documented domain sequence (e.g., Pfam HMM PF18541 of RuvC_III).

[0079] As used in this disclosure, the term “HNH domain” refers to an endonuclease domain having characteristic histidine and asparagine residues. HNH domains can generally be identified by alignment to a documented domain sequence, structural alignment to a protein having an annotated domain, or comparison to a hidden Markov model (HMM) constructed based on a documented domain sequence (e.g., Pfam HMM PF01844 of the HNH domain).

[0080] As used herein, the term “bridge helix domain” or “BH domain” refers to the arginine-rich helix domain present in the Cas enzyme, which plays a crucial role in initiating cleavage activity upon binding to target DNA.

[0081] As used herein, the term “recognition domain” or “REC domain” refers to a domain that is thought to interact with the repeat:antirepeat double helix of gRNA and mediate the formation of the Cas endonuclease / gRNA complex.

[0082] As used herein, the term “wedge” (WED) domain primarily refers to domains that interact with sgRNA and PAM double repeats:anti-repeat doubles (e.g., those present in Cas proteins). WED domains can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison with hidden Markov models (HMMs) constructed based on documented domain sequences.

[0083] As used herein, the terms “PAM interaction domain” or “PI domain” refer to a domain found in a Cas enzyme that is positioned in an endonuclease DNA complex to recognize a PAM sequence on the non-complementary DNA strand of a guide RNA.

[0084] As used in this disclosure, the term “complex” refers to the conjugation of at least two components. Each of the two components may retain properties / activities it had before forming the complex, or may acquire properties as a result of forming the complex. The conjugation includes, but is not limited to, covalent bonds, non-covalent bonds (i.e., hydrogen bonds, ionic interactions, van der Waals interactions, and hydrophobic bonds), the use of linkers, fusion, or any other preferred method. The components intended for the complex include polynucleotides, polypeptides, or combinations thereof. For example, the complex includes an endonuclease and a guide polynucleotide.

[0085] In accordance with IUPAC convention, the following abbreviations will be used throughout the examples. A = Adenine C = Cytosine G = Guanine T = Chimin R = adenine or guanine Y = Cytosine or Thymine S = Guanine or Cytosine W = Adenine or Thymine K = guanine or thymine M = adenine or cytosine B = C, G, or T D = A, G, or T H = A, C, or T V = A, C, or G.

[0086] overview The discovery of novel Cas enzymes with unique functionalities and structures has the potential to further transform deoxyribonucleic acid (DNA) editing technology, improving speed, specificity, functionality, and ease of use. Relatively few functionally characterized CRISPR / Cas enzymes exist in the literature compared to the predicted prevalence of clustered and regularly arranged short palindromic repeat (CRISPR) systems in microorganisms and the complete diversity of microbial species. This is partly because a vast number of microbial species may not readily culture under laboratory conditions. Metagenomic sequencing from natural environmental niches representing a large number of microbial species dramatically increases the number of documented novel CRISPR / Cas systems, potentially accelerating the discovery of new oligonucleotide editing functions. A fruitful recent example of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0087] The CRISPR / Cas system is an RNA-directed nuclease complex described as functioning as an adaptive immune system in microorganisms. In their natural context, the CRISPR / Cas system arises in a CRISPR (clustered, regularly spaced short palindromic repeat sequence) operon or locus, which generally consists of two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences encoding an RNA-based targeting element; and (ii) an ORF encoding a Cas that encodes a nuclease polypeptide directed by the RNA-based targeting element, aligned with an accessory protein / enzyme. 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 (target seed) and a crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined neighborhood of the target seed (PAMs are typically sequences not commonly represented in the host genome). Depending on the exact function and structure of the system, CRISPR-Cas systems are typically organized into two classes, five types, and sixteen subtypes based on shared functional characteristics and evolutionary similarities.

[0088] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes and include types I, III, and IV.

[0089] The type I CRISPR-Cas system is considered to have moderate complexity in terms of its components. In the type I CRISPR-Cas system, an array of RNA targeting elements is transcribed as a long precursor crRNA (precrRNA) processed by repeat elements, and a short mature crRNA that orients the nuclease complex to the nucleic acid target is subsequently released, followed by a preferred short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via the endoribonuclease subunit (Cas6) of a large endonuclease complex called a cascade, which also includes the nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nucleases primarily function as DNA nucleases.

[0090] The type III CRISPR system can be characterized by the presence of a central nuclease known as Cas10, along with a repeat-associated mysterious protein (RAMP) containing a Csm or Cmr protein subunit. Similar to the type I system, mature crRNA is processed from pre-crRNA using a Cas6-like enzyme. Unlike the type I and II systems, the type III system appears to target and cleave DNA-RNA double strands (such as the DNA strand used as a template for RNA polymerase).

[0091] The type IV CRISPR-Cas system harbors an effector complex containing a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins from the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted smaller subunit. Such systems are generally found on endogenous plasmids.

[0092] Class 2 CRISPR-Cas systems have a single polypeptide multi-domain nuclease effector and include types II, V, and VI.

[0093] Type II CRISPR-Cas systems are considered the simplest in terms of their components. In type II CRISPR-Cas systems, processing a CRISPR array with mature crRNA does not require the presence of a special endonuclease subunit, but rather a small transcoding 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 with endogenous RNAse III to produce a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are DNA nucleases. Type II effectors generally exhibit a structure containing a RuvC-like endonuclease domain that fits into an RNase H fold, which has an unrelated HNH nuclease domain inserted into the fold of a RuvC-like nuclease domain. The RuvC-like domain is involved in cleaving target (e.g., crRNA-complementary) DNA strands, while the HNH domain is involved in cleaving substitution DNA strands.

[0094] Type V CRISPR-Cas systems are characterized by a nuclease effector structure (e.g., Cas12) similar to that of type II effectors, containing a RuvC-like domain. Like type II, most (but not all) type V CRISPR systems use tracrRNA to process precrRNA into mature crRNA; however, unlike type II systems which require RNAse III to cleave precrRNA into multiple crRNAs, type V systems can cleave precrRNA using the effector nuclease itself. Similar to type II CRISPR-Cas systems, type V CRISPR-Cas systems are DNA nucleases.

[0095] Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to possess robust single-strand nonspecific deoxyribonuclease activity, activated by primary crRNA-directed cleavage of a double-stranded target sequence.

[0096] Type VI CRISPR-Cas systems possess RNA-inducible RNA endonucleases. Instead of a RuvC-like domain, the single polypeptide effector of type VI systems (e.g., Cas13) contains two HEPN ribonuclease domains. Unlike both type II and type V systems, type VI systems also appear to not require tracrRNA to process precrRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) appear to possess robust single-strand nonspecific nuclease (ribonuclease) activity, which is activated by the first crRNA-directed cleavage of the target RNA.

[0097] Due to their simpler structure, Class 2 CRISPR-Cas have been the most widely adopted for manipulation and development as designer nucleases / genome editing applications.

[0098] One of the initial adaptations of such a system for in vitro use is (i) recombinantly expressed purified full-length Cas9 (e.g., class 2, type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature crRNA of approximately 42 nt carrying a 5' sequence of approximately 20 nt complementary to the target DNA sequence to be cleaved, followed by a 3' tracr binding sequence (total crRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence), (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg 2+ The system was subsequently improved and engineered, and involved a crRNA of (ii) ligated to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) capable of inducing Cas9 to the target by itself (compare the upper and lower panels in Figure 2).

[0099] Such modified systems can be adapted for use in mammalian cells by providing DNA vectors that encode (i) an ORF encoding codon-optimized Cas9 (e.g., class 2, type II Cas enzyme) under a suitable mammalian promoter having a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal), and (ii) an ORF encoding sgRNA (having a 5' sequence beginning with G, followed by a 20nt complementary targeting nucleic acid sequence, linker, and tracrRNA sequence bound to a 3' tracr binding sequence) under a suitable polymerase III promoter (e.g., U6 promoter).

[0100] MG Enzyme In this specification, in some embodiments, modified nuclease systems comprising low molecular weight endonucleases are provided.

[0101] In some embodiments, the endonuclease includes a RuvC-1 domain or a RuvC domain. In some embodiments, the endonuclease includes an HNH domain. In some embodiments, the endonuclease includes a RuvC domain and an HNH domain. In some embodiments, the endonuclease includes an arginine-rich region containing an RRxRR motif or a domain having PF14239 homology. In some embodiments, the endonuclease includes an REC domain. In some embodiments, the endonuclease includes a BH (Bridge Helix) domain. In some embodiments, the endonuclease includes a WED (wedge) domain. In some embodiments, the endonuclease includes a (PAM interaction) domain. In some embodiments, the endonuclease is configured to be selective for a target adjacent motif (TAM) sequence containing one of the following: ANGG (SEQ ID NO: 1029), NARAA (SEQ ID NO: 1030), ATGAAA (SEQ ID NO: 1031), ATGA (SEQ ID NO: 1032), or WTGG (SEQ ID NO: 1033).

[0102] In some embodiments, the endonuclease is derived from an uncultured 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.

[0103] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0104] In some embodiments, the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease.

[0105] In some embodiments, the endonuclease has 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 with Cas9 endonuclease. In some embodiments, the endonuclease has less than 80% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 75% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 70% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 65% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 60% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 55% identity with Cas9 endonuclease. In some embodiments, the endonuclease has less than 50% identity with the Cas9 endonuclease.

[0106] In some embodiments, the endonuclease contains a sequence having 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 with 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 contains a sequence having at least 70% sequence identity with 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 contains a sequence having at least 75% sequence identity with 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 contains a sequence having at least 75% sequence identity with 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 contains a sequence having at least 80% sequence identity with 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 contains a sequence having at least 81% sequence identity with 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 contains a sequence having at least 82% sequence identity with 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 contains a sequence having at least 83% sequence identity with 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 contains a sequence having at least 84% sequence identity with 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 contains a sequence having at least 85% sequence identity with 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 contains a sequence having at least 86% sequence identity with 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 contains a sequence having at least 87% sequence identity with 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 contains a sequence having at least 88% sequence identity with 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 contains a sequence having at least 89% sequence identity with 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 contains a sequence having at least 90% sequence identity with 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 contains a sequence having at least 91% sequence identity with 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 contains a sequence having at least 92% sequence identity with 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 contains a sequence having at least 93% sequence identity with 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 contains a sequence having at least 94% sequence identity with 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 contains a sequence having at least 95% sequence identity with 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 contains a sequence having at least 96% sequence identity with 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 contains a sequence having at least 97% sequence identity with 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 contains a sequence having at least 98% sequence identity with 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 contains a sequence having at least 99% sequence identity with any one of sequence numbers 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 contains a sequence having at least 100% sequence identity with any one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440.

[0107] In some embodiments, the endonuclease contains a sequence having 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 with any one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 80% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 81% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 82% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 83% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440.In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 86% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 87% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 88% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 89% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 91% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 92% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440.In some embodiments, the endonuclease contains a sequence having at least 93% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 94% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 95% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 98% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 99% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 100% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440.

[0108] In some embodiments, the endonuclease is an MG33 nuclease. In some embodiments, the endonuclease contains a sequence having 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 with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 70% sequence identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 81% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 82% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 83% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312.In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 86% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 87% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 88% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 89% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 91% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 92% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 93% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 94% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312.In some embodiments, the endonuclease contains a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 98% sequence identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 99% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312. In some embodiments, the endonuclease contains a sequence having at least 100% sequence identity with one of sequence numbers 1, 463-486, 981-988, and 1289-1312.

[0109] In some embodiments, the nuclease is the MG34 nuclease. In some embodiments, the endonuclease contains a sequence having 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 with any one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 70% sequence identity with any one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 80% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 81% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 82% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440.In some embodiments, the endonuclease contains a sequence having at least 83% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 86% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 87% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 88% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 89% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440.In some embodiments, the endonuclease contains a sequence having at least 91% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 92% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 93% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 94% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 95% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 98% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440.In some embodiments, the endonuclease contains a sequence having at least 99% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 100% sequence identity with one of sequence numbers 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440.

[0110] In some embodiments, the endonuclease is an MG35 nuclease. In some embodiments, the endonuclease contains a sequence having 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 with any one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 70% sequence identity with any one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 80% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 81% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 82% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 83% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675.In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 86% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 87% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 88% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 89% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 91% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 92% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675.In some embodiments, the endonuclease contains a sequence having at least 93% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 94% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 95% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 98% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 99% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675. In some embodiments, the endonuclease contains a sequence having at least 100% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675.

[0111] In some embodiments, the endonuclease is the MG102 nuclease. In some embodiments, the endonuclease contains a sequence having 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 with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 70% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 80% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 81% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 82% sequence identity with any one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.In some embodiments, the endonuclease contains a sequence having at least 83% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 86% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 87% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 88% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 89% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 90% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 91% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.In some embodiments, the endonuclease contains a sequence having at least 92% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 93% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 94% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 95% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease includes a sequence having at least 98% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease includes a sequence having at least 99% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346. In some embodiments, the endonuclease includes a sequence having at least 100% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1346.

[0112] In some embodiments, the endonuclease is the MG144 nuclease. In some embodiments, the endonuclease contains a sequence having 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 with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 70% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 75% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 75% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 80% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 81% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 82% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 83% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 84% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 85% sequence identity with one of sequence numbers 976-979 and 1274-1288.In some embodiments, the endonuclease includes a sequence having at least 86% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 87% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 88% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 89% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 90% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 91% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 92% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 93% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 94% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease includes a sequence having at least 95% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 96% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 97% sequence identity with one of sequence numbers 976-979 and 1274-1288.In some embodiments, the endonuclease contains a sequence having at least 98% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 99% sequence identity with one of sequence numbers 976-979 and 1274-1288. In some embodiments, the endonuclease contains a sequence having at least 100% sequence identity with one of sequence numbers 976-979 and 1274-1288.

[0113] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1324 and 1329-1346. In some embodiments, the endonuclease contains a sequence having 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 with any one of sequence numbers 1350-1368 and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 80% identity with any one of sequence numbers 1350-1368 and 1415-1440. In some embodiments, the endonuclease contains a sequence having at least 80% identity with any one of sequence numbers 1324 and 1329-1346.

[0114] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to a target nucleic acid sequence. In some embodiments, the endonucleases comprise a sequence having 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 with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440. In some embodiments, the endonuclease includes a sequence having at least 90% identity with SEQ ID NOs. 1350-1368 and 1415-1440. In some embodiments, the endonuclease includes a sequence having at least 90% identity with SEQ ID NOs. 1324 and any one of SEQ ID NOs. 1329-1346.

[0115] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and a modified guide polynucleotide which forms a complex with the endonucleases and hybridizes to a target nucleic acid sequence. In some embodiments, the endonucleases comprise sequences having at least 95% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 95% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0116] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 99% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise sequences having at least 99% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 99% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0117] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases containing a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323, 1324, and 1329-1346.

[0118] In some embodiments, the disclosure provides endonucleases described herein that are configured to induce double-strand breaks proximal to the target locus on the 5' side of a protospacer-adjacent motif (PAM). In some embodiments, the endonucleases induce double-strand breaks of 6 to 8 nucleotides or 7 nucleotides from the PAM. In some embodiments, the endonucleases described herein are configured to induce single-strand breaks on the 5' side of a protospacer-adjacent motif (PAM) in the vicinity of the aforementioned target locus. In some embodiments, the endonucleases induce single-strand breaks of 6 to 8 nucleotides or 7 nucleotides from the PAM. In some embodiments, the endonucleases configured to induce single-strand breaks include inactivating mutations in one or more catalytic residues of the endonucleases described herein.

[0119] In some embodiments, the endonuclease includes one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS includes sequences selected from SEQ ID NOs. 205-220 and 2243-2268. In some embodiments, the NLS includes a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of the sequences 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 85% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 90% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 91% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 92% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 93% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 94% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 95% identity with sequence numbers 205-220 and 2243-2268.In some embodiments, the NLS includes sequences having at least about 96% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 97% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 98% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having at least about 99% identity with sequence numbers 205-220 and 2243-2268. In some embodiments, the NLS includes sequences having 100% identity with sequence numbers 205-220 and 2243-2268.

[0120] [Table 1-1]

[0121] [Table 1-2]

[0122] In some embodiments, the endonucleases described herein are variants thereof having sequence identity with a particular domain. In some embodiments, the domain may be an arginine-rich domain (e.g., a PF14239 homology domain), a REC (recognition) domain, a BH (bridge helix) domain, a WED (wedge) domain, a PI (PAM interaction) domain, a PF14239 homology domain, or any other domain described herein. In some embodiments, residues containing one or more of these domains are identified in the protein by alignment with one of the following proteins (e.g., when the following proteins and one of the proteins of interest are optimally aligned), and residue boundaries, e.g., domains are described.

[0123] [Table 2]

[0124] Guide polynucleotides In certain embodiments, an endonuclease system is disclosed herein, comprising (a) an endonuclease disclosed herein and (b) a modified guide polynucleotide, such as a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA. When T is referred to in polynucleotides, T means U (uracil) in RNA and T (thymine) in DNA.

[0125] In some embodiments, the modified guide polynucleotide is configured to form a complex with an endonuclease. In some cases, the modified guide polynucleotide includes a spacer sequence. In some cases, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some cases, the endonuclease is configured to bind to a protospacer-adjacent motif (PAM) sequence.

[0126] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, the mammalian cell is a pig, cattle, goat, sheep, rodent, rat, mouse, non-human primate, or human cell. In some embodiments, the target gene or locus is TRAC. In some embodiments, the target gene or locus is AAVS1.

[0127] In some embodiments, the guide polynucleotide is 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 SEQ ID NOs: 199-203 It is coded by an array that has at least 90%, 95%, 97%, 98%, or 99% array identity with one of the following: 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. In some embodiments, the guide polynucleotide is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% of 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. , comprising a sequence containing at least 46 to 80 consecutive nucleotides having identity of at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with any one of the following sequence numbers: 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.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having at least approximately 99% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with any one of the following sequence numbers: 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.

[0128] In some embodiments, the guide polynucleotide is 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 SEQ ID NOs: 199-203 It is coded by an array that has at least 90%, 95%, 97%, 98%, or 99% array identity with any one of the following: 460-461, 613-616, 669-670, 672-673, 677-974, 1003-1022, 1231-1259, 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-224. In some embodiments, the guide polynucleotide is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% of 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. , comprising a sequence containing at least 46 to 80 consecutive nucleotides having identity of at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of the following sequences: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of the following sequences: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of the following sequences: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of the following sequences: 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.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 97% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 98% identity with any one of the following sequence numbers: 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. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with any one of the following sequence numbers: 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.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with any one of the following sequence numbers: 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.

[0129] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the guide polynucleotide is encoded by any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242, or by a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

[0130] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0131] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with SEQ ID NO: 1410 or 1960. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with either SEQ ID NO: 1410 or 1960.

[0132] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1410, 1412, 1953, 1956, 1960, 1961, 1966, 1970, and 1478. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1410, 1412, 1953, 1956, 1960, 1961, 1966, 1970, and 1478.

[0133] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with one of SEQ ID NOs: 2157, 2159, and 2160. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NOs: 2157, 2159, and 2160.

[0134] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0135] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with 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.

[0136] In some embodiments, the modified guide polynucleotide includes a sequence having 100% sequence identity with any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

[0137] In some embodiments, the guide polynucleotide (e.g., SEQ ID NOs: 199, 201, 669-670, and 1003-1005) is configured to form a complex with the MG33 nuclease. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with any one of sequence numbers 199, 201, 669-670, and 1003-1005.

[0138] In some embodiments, the guide polynucleotide is encoded by one of sequence numbers 199, 201, 669-670, and 1003-1005, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of sequence numbers 199, 201, 669-670, and 1003-1005.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 97% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 98% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.

[0139] In some embodiments, 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 the MG34 nuclease. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 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 with one of sequence numbers 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 90% identity with one of sequence numbers 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 with one of sequence numbers 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 with one of sequence numbers 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 97% identity with one of sequence numbers 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 with one of sequence numbers 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 with one of sequence numbers 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of sequence numbers 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.

[0140] In some embodiments, the guide polynucleotide is encoded by one of sequence numbers 1348 and 1392-1414, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with one of sequence numbers 1348 and 1392 to 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 1348 and 1392 to 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with one of sequence numbers 1348 and 1392-1414.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity with one of sequence numbers 1348 and 1392-1414. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of sequence numbers 1348 and 1392-1414.

[0141] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with 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 complementary sequence having at least about 85% identity with 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 complementary sequence having at least about 90% identity with 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 complementary sequence having at least about 95% identity with 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 complementary sequence having at least about 96% identity with 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 complementary sequence having at least about 97% identity with 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 complementary sequence having at least about 98% identity with 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 complementary sequence having at least about 99% identity with 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 complementary sequence having 100% identity with one of the sequence numbers 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.

[0142] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 97% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 98% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with one of SEQ ID NOs: 1348 and 1392-1414. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with one of sequence numbers 1348 and 1392-1414.

[0143] In some embodiments, the guide polynucleotides (e.g., SEQ ID NOs. 460-461, 677-974, 1006-1012, and 1231-1259) are configured to form a complex with the MG35 nuclease. In some embodiments, the guide polynucleotides are encoded by any one of SEQ ID NOs. 460-461, 677-974, 1006-1012, and 1231-1259, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 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 with one of sequence numbers 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 with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 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 with one of sequence numbers 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 with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of sequence numbers 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 with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259.

[0144] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 97% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 98% identity with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with one of sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with one of the sequence numbers 460-461, 677-974, 1006-1012, and 1231-1259.

[0145] In some embodiments, the guide polynucleotide targets a sequence having at least about 80% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity with one of sequence numbers 2270-2330. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity with one of sequence numbers 2270-2330.

[0146] In some embodiments, the guide polynucleotides (e.g., SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391) are configured to form a complex with the MG102 nuclease. In some embodiments, the guide polynucleotides are encoded by any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 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 with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 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 85% identity with one of sequence numbers 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 with one of sequence numbers 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 95% identity with one of sequence numbers 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 with one of sequence numbers 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 with one of the sequence numbers 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 98% identity with one of the sequence numbers 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 with one of the sequence numbers 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 with any one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.

[0147] In some embodiments, the guide polynucleotide is encoded by one of sequence numbers 1376-1391, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 1376-1391. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of sequence numbers 1376 to 1391. In some embodiments, the guide polynucleotide is encoded by a sequence that has at least about 80% identity with any one of sequence numbers 1376 to 1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with one of sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with one of sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with one of sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with one of sequence numbers 1376-1391.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of the sequence numbers 1376-1391.

[0148] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with 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 complementary sequence having at least about 97% identity with 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 complementary sequence having at least about 98% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with one of the sequence numbers 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.

[0149] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 85% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 90% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 95% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 96% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 97% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 98% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 99% identity with one of the sequence numbers 1376-1391. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having 100% identity with one of the sequence numbers 1376-1391.

[0150] In some embodiments, the target gene is a TRAC. In some embodiments, the guide polynucleotide is encoded by one of sequence numbers 1083-1086, 1123-1144, and 1167-1168, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of the sequence numbers 1083-1086, 1123-1144, and 1167-1168.

[0151] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a target nucleic acid sequence within a TRAC gene or an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence having at least about 80% identity with any one of SEQ ID NOs: 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 85% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 90% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 95% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 96% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 97% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 98% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 99% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having 100% identity with one of sequence numbers 1083-1086, 1123-1144, and 1167-1168.

[0152] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TRAC gene or within the intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets one of the sequences 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 with one of the sequences 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 80% identity with one of the sequences 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 with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170.In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity with one of sequence numbers 1079-1082, 1145-1166, and 1169-1170.

[0153] In some embodiments, the target gene is AAVS1. In some embodiments, the guide polynucleotide is encoded by one of sequence numbers 1087-1104, or by a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide includes a sequence comprising at least 46 to 80 consecutive nucleotides that have at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any one of the SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence that has at least about 80% identity with any one of the SEQ ID NOs. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity with one of sequence numbers 1087-1104.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity with one of the sequence numbers 1087-1104.

[0154] In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence 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 one of sequence numbers 1087-1104, or a complementary sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 80% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 85% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 90% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 95% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 96% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 97% identity with one of the sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 98% identity with one of the sequence numbers 1087-1104.In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having at least about 99% identity with one of sequence numbers 1087-1104. In some embodiments, the guide polynucleotide hybridizes or targets a complementary sequence to a sequence having 100% identity with one of sequence numbers 1087-1104.

[0155] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the AAVS1 gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets any one of sequence numbers 1105-1122, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity with any one of sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity with any one of sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity with any one of sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity with one of the sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity with one of the sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity with one of the sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity with one of the sequence numbers 1105-1122. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity with one of the sequence numbers 1105-1122.In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity with any one of sequence numbers 1105-1122.

[0156] In some embodiments, the system provided herein includes one or more guide polynucleotides. In some embodiments, the guide polynucleotide includes a sense sequence. In some embodiments, the guide polynucleotide includes an antisense sequence. In some embodiments, the guide polynucleotide includes a nucleotide sequence other than a region complementary or substantially complementary to the region of the target sequence. For example, crRNA is part of the guide polynucleotide, is considered part of the guide polynucleotide, or is included in the guide polynucleotide, e.g., a crRNA:tracrRNA chimera.

[0157] In some embodiments, the guide polynucleotide comprises a synthetic or modified nucleotide. In some embodiments, the guide polynucleotide comprises one or more nucleoside linkers modified from natural phosphodiesters. In some embodiments, all or the entire sequence of nucleoside linkers of the guide polynucleotide is modified. For example, in some embodiments, the nucleoside linkages include sulfur (S), such as phosphorothioate nucleoside linkages. In some embodiments, the guide polynucleotide comprises about 10%, 25%, 50%, 75%, or more than 90% modified nucleoside linkers. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified nucleoside linkers (e.g., phosphorothioate nucleoside linkages).

[0158] In some embodiments, the guide polynucleotide comprises modifications to a ribose sugar or nucleic acid base. In some embodiments, the guide polynucleotide comprises one or more nucleosides containing a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acid (LNA)), or an unbound ribose ring typically lacking a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, e.g., a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0159] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modification involves altering a substituent on the ribose ring to a group other than hydrogen, or to a 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', 5' positions, or a combination thereof. In some embodiments, the nucleoside having a modified sugar moiety comprises a 2'-modified nucleoside, e.g., a 2'-substituted nucleoside. In some embodiments, the 2'-sugar-modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (a 2'-substituted nucleoside), or comprises a 2'-linked biradical, and includes 2'-substituted nucleosides and LNA (2'-4' biradical-bridged) nucleosides. Examples of 2'-substituted nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification in the ribose group involves a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0160] In some embodiments, the guide polynucleotide contains one or more modified sugars. In some embodiments, the guide polynucleotide contains only modified sugars. In some embodiments, the guide polynucleotide contains about 10%, 25%, 50%, 75%, or more than 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar contains 2'-O-methyl. In some embodiments, the modified sugar contains 2'-fluoro. In some embodiments, the modified sugar contains a 2'-O-methoxyethyl group. In some embodiments, the guide polynucleotide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., containing 2'-O-methyl or 2'-fluoro).

[0161] In some embodiments, the guide polynucleotide includes both nucleoside linker modifications and nucleoside modifications. In some embodiments, the guide polynucleotide includes about 10%, 25%, 50%, 75%, or more than 90% modified nucleoside linkers and about 10%, 25%, 50%, 75%, or more than 90% modified sugars. In some embodiments, the guide polynucleotide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified nucleoside linkers (e.g., phosphorothioate nucleoside linkers) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., including 2'-O-methyl or 2'-fluoro).

[0162] In some cases, the guide polynucleotide includes a complementary sequence to a eukaryotic, fungal, plant, mammalian, or human genome polynucleotide sequence.

[0163] In some embodiments, the guide polynucleotide has a length of 30 to 250 nucleotides. In some embodiments, the guide polynucleotide has a length of more than 90 nucleotides. In some embodiments, the guide polynucleotide has a length of less than 245 nucleotides. In some embodiments, the guide polynucleotide has a length of 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides. In the embodiment where the guide polynucleotides are added, the number of guide polynucleotides is approximately 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-120, 30-140, 30-160, 30-180, 30-200, 30-220, 30-240, 50-60, 50-70, 50-80, 50-90, 50-100, and The lengths of the nucleotides are approximately 50 to 120, 50 to 140, 50 to 160, 50 to 180, 50 to 200, 50 to 220, 50 to 240, 100 to 120, 100 to 140, 100 to 160, 100 to 180, 100 to 200, 100 to 220, 100 to 240, 160 to 180, 160 to 200, 160 to 220, or 160 to 240.

[0164] In some embodiments, the guide RNA structure includes an RNA sequence that is predicted to contain a hairpin. In some embodiments, the hairpin includes a stem and a loop.

[0165] In some embodiments, the stem contains at least 12 pairs, at least 14 pairs, at least 16 pairs, or at least 18 pairs of ribonucleotides.

[0166] In some embodiments, the guide RNA structure further includes a second stem and a second loop.

[0167] In some embodiments, the second stem contains 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.

[0168] In some embodiments, the guide RNA structure further comprises an RNA structure, which comprises at least two hairpins.

[0169] In some embodiments, the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a prokaryotic genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a bacterial genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to an archaeal genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a eukaryotic genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a fungal genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a plant genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a mammalian genome sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a human genome sequence.

[0170] MG Endonuclease System In certain embodiments, modified nuclease systems comprising an endonuclease and a modified guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence are described herein.

[0171] In some embodiments, the modified nuclease system includes an endonuclease having at least 70% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 75% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 75% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 80% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 81% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonuclease having at least 82% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 83% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 84% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 85% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 86% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonuclease having at least 87% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 88% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 89% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 90% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 91% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonuclease having at least 92% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 93% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 94% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 95% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 96% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 97% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 98% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 99% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having a sequence having at least 100% sequence identity with 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, as well as a modified guide polynucleotide that forms a complex with the endonuclease.

[0172] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise a sequence having at least 80% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0173] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise sequences having at least 90% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 90% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0174] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and a modified guide polynucleotide which forms a complex with the endonucleases and hybridizes to a target nucleic acid sequence. In some embodiments, the endonucleases comprise sequences having at least 95% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 95% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0175] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases comprising a sequence having at least 99% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases comprise sequences having at least 99% identity with SEQ ID NOs: 1350-1368 and 1415-1440. In some embodiments, the endonucleases comprise sequences having at least 99% identity with one of SEQ ID NOs: 1324 and 1329-1346.

[0176] As described herein, in certain embodiments, a modified nuclease system is provided which includes: an endonucleases containing a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323-1324, 1329-1347, 1350-1368, and 1415-1440; and modified guide polynucleotides which form a complex with the endonucleases and hybridize to target nucleic acid sequences. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1347, 1350-1368, and 1415-1440. In some embodiments, the endonucleases contain a sequence having 100% sequence identity to any one of SEQ ID NOs: 1323, 1324, and 1329-1346.

[0177] In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having 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 with any one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as a modified guide polynucleotide configured to form a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 70% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 81% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 82% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 83% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 84% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 85% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 86% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 87% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 88% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 89% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 91% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 92% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 93% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 94% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 96% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 97% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 98% sequence identity with one of SEQ ID NOs: 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 99% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 100% sequence identity with one of sequence numbers 1322-1324, 1329-1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.

[0178] In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having 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 with any one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as a modified guide polynucleotide configured to form a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 70% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 80% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 81% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 82% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 83% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 84% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 85% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 86% sequence identity with one of SEQ ID NOs: 21324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 87% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 88% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 89% sequence identity with one of SEQ ID NOs: 21324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 90% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 91% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 92% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 93% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 94% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 95% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 96% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 97% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 98% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 99% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 100% sequence identity with one of SEQ ID NOs: 1324, 1329-1346, 1347, 1350-1368, and 1415-1440, as well as modified guide polynucleotides that form a complex with the endonucleases.

[0179] In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having 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 with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide configured to form a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having at least 75% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease having at least 80% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 81% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 82% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having at least 83% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having at least 84% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 85% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 86% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 87% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 88% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 89% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 90% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 91% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 92% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 93% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 94% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 95% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 96% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having at least 97% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having at least 98% sequence identity with one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, as well as a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 99% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 100% sequence identity with one of sequence numbers 25-198, 221-459, 489-580, 617-668, and 674-675, as well as modified guide polynucleotides that form a complex with the endonucleases.

[0180] In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having 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 with any one of SEQ ID NOs: 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as a modified guide polynucleotide configured to form a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 70% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 75% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 75% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 80% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 81% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 82% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 83% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 84% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 85% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 86% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 87% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 88% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 89% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 90% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 91% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 92% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 93% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 94% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 95% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 96% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 97% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 98% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases.In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 99% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing sequences having at least 100% sequence identity with one of sequence numbers 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, as well as modified guide polynucleotides that form a complex with the endonucleases.

[0181] In some embodiments, the modified nuclease system includes an endonuclease containing a sequence having 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 with any one of SEQ ID NOs: 976-979 and 1274-1288, and a modified guide polynucleotide configured to form a complex with the endonuclease. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 75% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 80% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonuclease having at least 81% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 82% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 83% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 84% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 85% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 86% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 87% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonuclease having at least 88% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 89% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 90% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 91% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 92% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 93% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 94% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonuclease having at least 95% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonuclease.In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 96% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 97% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 98% sequence identity with one of sequence numbers 976-979 and 1274-1288, and a modified guide polynucleotide that forms a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 99% sequence identity with one of sequence numbers 976-979 and 1274-1288, and modified guide polynucleotides that form a complex with the endonucleases. In some embodiments, the modified nuclease system includes an endonucleases containing a sequence having at least 100% sequence identity with one of sequence numbers 976-979 and 1274-1288, and modified guide polynucleotides that form a complex with the endonucleases.

[0182] In some embodiments, the modified guide polynucleotide is a single guide nucleic acid. In some embodiments, the modified guide polynucleotide is a dual guide nucleic acid. In some embodiments, the modified guide polynucleotide is RNA. In some embodiments, the endonuclease is non-covalently bonded to the modified guide polynucleotide. In some embodiments, the endonuclease is covalently bonded to the modified guide polynucleotide.

[0183] In some embodiments, the modified nuclease system includes an endonuclease having at least about 70% identity with 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 is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 70% identity with any one of the following sequence numbers: 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. In some embodiments, the modified nuclease system includes an endonuclease having at least about 75% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 75% identity with any one of the following sequence numbers: 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.In some embodiments, the modified nuclease system includes an endonuclease having at least about 80% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 80% identity with any one of the following sequence numbers: 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. In some embodiments, the modified nuclease system includes an endonuclease having at least about 85% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 85% identity with any one of the following sequence numbers: 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.In some embodiments, the modified nuclease system includes an endonuclease having at least about 90% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 90% identity with any one of the following sequence numbers: 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. In some embodiments, the modified nuclease system includes an endonuclease having at least about 95% identity with 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 is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 95% identity with any one of the following sequence numbers: 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.In some embodiments, the modified nuclease system includes an endonuclease having at least about 96% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 96% identity with any one of sequence numbers 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. In some embodiments, the modified nuclease system includes an endonuclease having at least about 97% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 97% identity with any one of the following sequence numbers: 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.In some embodiments, the modified nuclease system includes an endonuclease having at least about 98% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 98% identity with any one of the following sequence numbers: 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. In some embodiments, the modified nuclease system includes an endonuclease having at least about 99% identity with one of sequence numbers 1-198, 221-459, 463-612, 617-668, 674-675, 975-1002, 1322-1324, 1329-1347, 1350-1368, and 1415-1440, and is configured to form a complex with said endonuclease. The modified guide polynucleotide comprises a sequence having at least approximately 99% identity with any one of the following sequence numbers: 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.In some embodiments, the modified nuclease system includes an endonuclease having 100% identity with 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 is configured to form a complex with said endonuclease. The modified guide polynucleotide includes a modified guide polynucleotide that has 100% identity with any one of the following sequence numbers: 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.

[0184] In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 70% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 70% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 80% identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 80% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 85% identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 85% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 95% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 95% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 96% identity with one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 96% identity with one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005. In some embodiments, the modified nuclease system includes an endonuclease having 100% identity with any one of SEQ ID NOs: 1, 463-486, 981-988, and 1289-1312, and a modified guide polynucleotide configured to form a complex with the endonuclease, the modified guide polynucleotide having 100% identity with any one of SEQ ID NOs: 199, 201, 669-670, and 1003-1005.

[0185] In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 70% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 70% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 75% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 80% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 80% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 85% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 85% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 90% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 95% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 95% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 96% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 96% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 97% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 98% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 99% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414. In some embodiments, the modified nuclease system includes an endonuclease having 100% identity with any one of SEQ ID NOs: 2-24, 487-488, 1313-1321, 1347, 1350-1368, and 1415-1440, and a modified guide polynucleotide configured to form a complex with the endonuclease, the modified guide polynucleotide having 100% identity with any one of SEQ ID NOs: 200, 202, 203, 613-616, 1348, 1369, and 1392-1414.

[0186] In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 70% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 70% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 75% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 80% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 80% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 85% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 85% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 90% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 95% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 95% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 96% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 96% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 97% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 97% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 98% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide having at least about 98% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a modified guide polynucleotide comprising a sequence having at least about 99% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259. In some embodiments, the modified nuclease system includes an endonuclease having 100% identity with any one of SEQ ID NOs: 25-198, 221-459, 489-580, 617-668, and 674-675, and a modified guide polynucleotide configured to form a complex with the endonuclease, the modified guide polynucleotide having 100% identity with any one of SEQ ID NOs: 460-461, 677-974, 1006-1012, and 1231-1259.

[0187] In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 70% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 70% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 75% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 75% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 80% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 80% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 85% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 85% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 90% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 90% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 95% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 95% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 96% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 96% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 97% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 97% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 98% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 98% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.In some embodiments, the modified nuclease system includes an endonuclease comprising a sequence having at least about 99% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, comprising a sequence having at least about 99% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391. In some embodiments, the modified nuclease system includes an endonuclease having 100% identity with any one of SEQ ID NOs. 581-612, 989-1002, 1260-1273, 1322-1324, and 1329-1347, and a modified guide polynucleotide configured to form a complex with the endonuclease, the modified guide polynucleotide having 100% identity with any one of SEQ ID NOs. 672-673, 1013-1022, 1327-1328, 1370-1372, and 1376-1391.

[0188] In some embodiments, the modified guide polynucleotide includes a sequence having at least 90% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 92% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 93% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 94% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 95% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 96% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 97% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having at least 98% sequence identity with one of the sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.In some embodiments, the modified guide polynucleotide includes a sequence having at least 99% sequence identity with any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242. In some embodiments, the modified guide polynucleotide includes a sequence having 100% sequence identity with any one of SEQ ID NOs: 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

[0189] In some embodiments, the modified nuclease system further includes a single-stranded DNA repair template. In some embodiments, the modified nuclease system further includes a double-stranded DNA repair template. In some embodiments, the single-stranded or double-stranded DNA repair template includes a first homologous arm in the 5' to 3' direction containing a sequence of at least 20 nucleotides on the 5' side of the target deoxyribonucleic acid sequence. In some embodiments, the single-stranded or double-stranded DNA repair template includes a synthetic DNA sequence of at least 10 nucleotides in the 5' to 3' direction. In some embodiments, the single-stranded or double-stranded DNA repair template includes a second homologous arm in the 5' to 3' direction containing a sequence of at least 20 nucleotides on the 3' side of the target sequence. In some embodiments, the single-stranded or double-stranded DNA repair template includes a first homologous arm in the 5' to 3' direction, comprising a sequence of at least 20 nucleotides on the 5' side relative to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, or a second homologous arm comprising a sequence of at least 20 nucleotides on the 3' side relative to the target sequence.

[0190] In some embodiments, the first homologous arm includes 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. In some embodiments, the first homologous arm includes a sequence of at least 10 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 20 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 30 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 40 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 50 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 60 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 70 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 80 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 90 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 100 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 110 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 120 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 130 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 140 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 150 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 175 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 200 nucleotides.In some embodiments, the first homologous arm includes a sequence of at least 250 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 300 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 400 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 500 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 750 nucleotides. In some embodiments, the first homologous arm includes a sequence of at least 1000 nucleotides.

[0191] In some embodiments, the modified nuclease system is Mg 2+ This further includes the sources of supply.

[0192] In some embodiments, the disclosure provides endonucleases described herein, configured to induce chemical modification of a nucleotide base at or near a target locus targeted by the endonucleases. In this case, chemical modification of a nucleotide base refers to modification of a chemical moiety involved in base pairing, rather than modification of the sugar or phosphate portion of the nucleotide. In some embodiments, the chemical modification includes deamination of an adenosine or cytosine nucleotide. In some embodiments, the endonucleases configured to induce chemical modification include an endonucleases having a base editor conjugated or fused to the endonucleases in a frame. In some embodiments, the endonucleases to which the base editor is fused or conjugated include an inactivating mutation in at least one catalytic residue (e.g., the RuvC domain) of the endonucleases. In some embodiments, the base editor is conjugated to the N-terminus or C-terminus of the endonucleases or linked via chemical conjugation. In some embodiments, the base editor may be, but is not limited to, RNA-specific adenosine deaminase 1 (ADAR1), RNA-specific adenosine deaminase 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), apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B), apolipoprotein B mRNA editing enzyme catalytic subunit 3C (APOBEC3C), apolipoprotein B mRNA editing enzyme catalytic subunit 3D (APOBEC3D), apolipoprotein B mRNA editing enzyme catalytic subunit 3F (APOBEC3F), apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G), apolipoprotein B mRNA editing enzyme catalytic subunit 3H (APOBEC3H), or apolipoprotein B The present invention comprises any adenosine or cytosine deaminase containing MRNA editing enzyme catalytic subunit 4 (APOBEC4) or a functional fragment thereof.In some embodiments, the base editor includes a yeast, eukaryote, mammal, or human base editor.

[0193] In some embodiments, the disclosure provides endonucleases described herein, configured to induce histone chemical modification within or proximal to a target locus targeted by the endonucleases. In some embodiments, the endonucleases configured to induce histone chemical modification include an endonucleases having a histone editor conjugated or fused to the endonucleases in a frame. In some embodiments, the histone editor is conjugated or fused to the endonucleases at the N-terminus or C-terminus. In some embodiments, the chemical modification includes methylation, acetylation, demethylation, or deacetylation. In some embodiments, the endonucleases to which the histone editor is fused or conjugated include an inactivating mutation in at least one catalytic residue (e.g., the RuvC domain) of the endonucleases. In some embodiments, the histone editor is 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, SUV420) The histone editor includes H1 (or SUV420H2), histone demethylases (e.g., KDM1, KDM2, KDM3, KDM4, KDM5, or KDM6 families), histone acetyltransferases (e.g., GNAT or HAT family acetyltransferases), or histone deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7). In some embodiments, the histone editor includes yeast, eukaryote, mammalian, or human histone editors.

[0194] Delivery and vector In this specification, in some embodiments, nucleic acid sequences encoding a modified nuclease system comprising an endonuclease and a modified guide polynucleotide, or components of the modified nuclease system, are disclosed.

[0195] In some embodiments, the nucleic acid encoding the endonuclease system or its components is DNA, such as linear DNA, plasmid DNA, or minicircle DNA. In some embodiments, the nucleic acid encoding the modified nuclease system is RNA, such as mRNA.

[0196] In some embodiments, nucleic acids encoding an endonuclease system or its components are delivered by nucleic acid-based vectors. In some embodiments, the nucleic acid-based vectors are plasmids (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmids (e.g., pWE or sCos vectors), artificial chromosomes, human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1-derived artificial chromosomes (PACs), phagemids, phage derivatives, bacmids, or viruses. In some embodiments, the nucleic acid-based vectors are pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, and pRI 101-AN. The selection is made from a list consisting of DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0197] In some embodiments, the nucleic acid-based vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of mini-promoters, inducible promoters, constitutive promoters, and their derivatives. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synapsin, CaMKII, GRK1, and their derivatives. In some embodiments, the promoter is the U6 promoter. In some embodiments, the promoter is the CAG promoter.

[0198] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anerovirus, bocavirus, vacciniavirus, or retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anerovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vacciniavirus. In some embodiments, the virus is a retrovirus.

[0199] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh1 0, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, A The following are examples of the following: AV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or derivatives thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0200] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof.In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof.In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0201] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0202] In some embodiments, nucleic acids encoding a modified nuclease system or its components are delivered by a non-nucleic acid-based delivery system (e.g., a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with lipids. In some embodiments, the nucleic acid associated with lipids is encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via binding molecules associated with both the liposome and the nucleic acid, confined within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in a micelle, complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is contained in lipid nanoparticles (LNPs).

[0203] In some embodiments, the modified nuclease system or its components are introduced into cells in any preferred manner, either stably or transiently. In some embodiments, the modified nuclease system or its components are transfected into cells. In some embodiments, cells are transfected or transfected with nucleic acid constructs encoding the modified nuclease system or its components. For example, cells are transfected (e.g., with a virus encoding the modified nuclease system or its components) or transfected (e.g., with a plasmid encoding the modified nuclease system or its components) with the modified nuclease system or its components, or with a nucleic acid encoding the translated modified nuclease system or its components. In some embodiments, the transfection is stable or transient. In some embodiments, cells expressing or containing the modified nuclease system or its components are transfected or transfected with one or more gRNA molecules, for example, if the modified nuclease system or its components include a CRISPR nuclease. In some embodiments, the modified nuclease system or plasmids expressing its components are introduced into cells by electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac), and viral transduction (e.g., lentivirus or AAV), or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into cells as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. For example, methods for delivering polypeptides and / or RNPs into cells by electroporation or cell compression are known in the art.

[0204] Exemplary methods for nucleic acid delivery include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced incorporation of DNA. Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam®, Lipofectin®, and SF Cell Line 4D-Nucleofector X Kit® (Lonza)). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include lipids of WO91 / 17424 and WO91 / 16024. In some embodiments, delivery is to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration). In some embodiments, the nucleic acid is contained in liposomes or nanoparticles that specifically target host cells.

[0205] Additional methods for delivering nucleic acids to cells are known to those skilled in the art. See, for example, US2003 / 0087817.

[0206] In some embodiments, this disclosure provides cells containing the vector or nucleic acid described herein. In some embodiments, the cells express a gene editing system or a part thereof. In some embodiments, the cells are human cells. In some embodiments, the cells are genome-edited ex vivo. In some embodiments, the cells are genome-edited in vivo. cell

[0207] In certain embodiments, what is described herein is a cell comprising the system or vector described herein.

[0208] In some embodiments, the cells may be eukaryotic cells (e.g., plant cells, animal cells, protist cells, or fungal cells), mammalian cells (Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK), human fetal kidney (HEK), mouse myeloma (NS0), or human retinal cells), immortalized cells (e.g., HeLa cells, COS cells, HEK-293T cells, MDCK cells, 3T3 cells, PC12 cells, Huh7 cells, HepG2 cells, K562 cells, N2a cells, or SY5Y cells), insect cells (e.g., Spodoptera frugiperda cells, Trichoplusia ni cells, Drosophila melanogaster cells, S2 cells, or Heliothis virescens cells), or yeast cells (e.g., Saccharomyces). These include cerevisiae cells, Cryptococcus cells, or Candida cells, plant cells (e.g., parenchymal cells, plaque cells, or plaque-walled cells), fungal cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells, or Candida cells), or prokaryotic cells (e.g., E. coli cells, Streptococcus bacterial cells, Streptomyces soil bacterial cells, or archaeal cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0209] In some embodiments, the cells are 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 cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0210] How to use The systems of this disclosure may be used for a variety of applications, such as nucleic acid editing (e.g., gene editing) or binding to nucleic acid molecules (e.g., sequence-specific binding). Such systems may be used, for example, to correct (e.g., remove or replace) genetically inherited mutations that may cause disease in a subject; to inactivate genes to confirm their function in cells; as a diagnostic tool to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or amplified DNA sequences encoding disease-causing mutations); as an inactivated enzyme combined with a probe to target and detect specific nucleotide sequences (e.g., sequences encoding antibiotic resistance in bacteria); to inactivate viruses by targeting viral genomes or to prevent them from infecting host cells; to add genes or modify metabolic pathways to manipulate organisms to produce beneficial small molecules, macromolecules or secondary metabolites; to establish gene-driven elements for evolutionary selection; and / or as a biosensor to detect cellular perturbations by foreign small molecules and nucleotides.

[0211] In some embodiments, methods for binding, cleaving, labeling, or modifying double-stranded deoxyribonucleic acid polynucleotides are described herein.

[0212] In certain embodiments described herein, a method for modifying a target nucleic acid locus comprises delivering the modified nuclease system described herein to the target nucleic acid locus, wherein the endonuclease is configured to form a complex with the modified guide ribonucleic acid structure, and the complex is configured to modify the target nucleic acid locus upon binding of the complex to the target nucleic acid locus.

[0213] In some embodiments, the method includes delivering a modified nuclease system described herein to a target nucleic acid locus. In some embodiments, the endonuclease is configured to form a complex with a modified guide ribonucleic acid structure. In some embodiments, the complex is configured to modify the target nucleic acid locus upon binding to it. In some embodiments, modifying the target nucleic acid locus includes binding, nicking, cleaving, or marking the target nucleic acid locus.

[0214] In some embodiments, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the target nucleic acid locus comprises genomic eukaryotic DNA, viral DNA, or bacterial DNA. In some embodiments, the target nucleic acid comprises bacterial DNA. In some embodiments, the bacterial DNA is derived from a bacterial species different from the species from which the endonuclease originates. In some embodiments, the target nucleic acid locus is in vitro. In some embodiments, the nucleic acid locus is intracellular. In some embodiments, an endonuclease encoded on a separate nucleic acid molecule and an engineered guide nucleic acid structure are provided. In some embodiments, the cell is a prokaryotic cell, bacterial cell, eukaryotic cell, fungal cell, plant cell, animal cell, mammalian cell, rodent cell, primate cell, or human cell. In some embodiments, the cell is derived from a species different from the species from which the endonuclease originates.

[0215] In this specification, in some embodiments, a method for disrupting a TRAC locus in a cell is described, the method comprising contacting the aforementioned cell with a composition comprising: an endonuclease having at least 80% sequence identity with any one of SEQ ID NOs: 1324, 1329-1346, 1350-1368, and 1415-1440; and a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence, wherein the modified guide RNA is configured to hybridize to any one of SEQ ID NOs: 1079-1082, 1145-1166, and 1169-1170.

[0216] In this specification, in some embodiments, a method for disrupting the AAVS1 locus in cells is described, the method comprising contacting the aforementioned cells with a composition having at least 80% sequence identity with any one of sequence numbers 1324, 1329-1346, 1350-1368, and 1415-1440, and a modified guide RNA comprising a spacer sequence configured to form a complex with the aforementioned endonuclease, and to hybridize the modified guide RNA to the region of the aforementioned locus, and to hybridize the modified guide RNA to any one of sequence numbers 1105-1122 and 2301-2330.

[0217] In some embodiments, delivery of the modified nuclease system to a target nucleic acid locus includes delivery of a nucleic acid or vector described herein. In some embodiments, delivery of the modified nuclease system to a target nucleic acid locus includes delivery of a nucleic acid containing an open reading frame encoding an endonuclease. In some embodiments, the nucleic acid includes a promoter to which the open reading frame encoding the endonuclease is operably linked. In some embodiments, delivery of the modified nuclease system to a target nucleic acid locus includes delivery of capped mRNA containing the aforementioned open reading frame encoding the endonuclease. In some embodiments, delivery of the modified nuclease system to the aforementioned target nucleic acid locus includes delivery of a translated polypeptide.

[0218] In some embodiments, delivering a modified nuclease system to a target nucleic acid locus involves delivering deoxyribonucleic acid (DNA) encoding a modified guide ribonucleic acid structure operably ligated to a ribonucleic acid (RNA) pol III promoter. In some embodiments, the endonuclease induces a single-strand or double-strand break at or proximal to the target locus.

[0219] kit In some embodiments, the disclosure provides a kit comprising one or more nucleic acid constructs encoding various components of a modified nuclease system. In some embodiments, the nucleotide sequence includes a heterologous promoter that drives the expression of the modified nuclease system component.

[0220] In some embodiments, the modified nuclease systems disclosed herein are incorporated into pharmaceutical, diagnostic, or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kit may comprise one or more containers containing one of the vectors disclosed herein, and instructions for use.

[0221] The kit may be designed to facilitate researchers' use of the methods described herein and may take various forms. Each of the components of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), where applicable. In certain cases, some of the components may be configured to be (e.g., in an active form) or otherwise processable by the addition of a suitable solvent or other kind (e.g., water or cell culture medium), which may or may not be provided with the kit. Where used herein, “instructions” defines the components of the instructions and / or promotional materials and may typically be accompanied by written instructions on or associated with the packaging of the herein. Instructions may also include any oral or electronic instructions provided in any format that clearly indicates to the user that the instructions relate to the kit, such as audiovisual (e.g., videotape, DVD, etc.), the internet, and / or web-based communications. In some embodiments, written instructions may be in the form prescribed by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product, and such instructions may also reflect approval by an agency for manufacture, use, or sale for animal administration. [Examples]

[0222] Example 1 - SMART nuclease Bioinformatics identification of SMART nucleases

[0223] SMART nuclease homologs were discovered by mining large assembly-driven metagenomics databases of microbial, viral, and eukaryotic genomes. HMM profiles of previously reported SMART nucleases were created, and all proteins in the database were searched using sequence analysis software. CRISPR arrays were predicted on assembled contigs using a program for detecting CRISPR in environmental datasets, such as assembled contigs derived from complete genomes and metagenomics. Proteins were identified with E-values ​​(<1 × 10⁻¹⁰). -5 Sequences were filtered by length and size (over 550 amino acids, less than 1000 amino acids), and incomplete ORFs were excluded. Sequences were aligned with reference type II nucleases and SMART nucleases, and phylogenetic trees were constructed. The MG34 and MG102 clades were identified based on their relative positions to known SMART effectors, and further novel candidates were selected for in vitro screening.

[0224] Computational reconstruction of ancestral SMART nuclease sequences

[0225] To further expand the diversity of SMART nucleases, nuclease sequences of the MG34 and MG102 families were generated using a phylogenetic ancestral sequence reconstruction algorithm. In this analysis, 441 SMART I protein sequences were aligned with L-INS-i and G-INS-i parameters to construct a phylogenetic tree. The phylogenetic tree was rooted using SpCas9 and SaCas9. Sequence reconstruction was performed. Insertions and deletions were manually identified for each reconstructed node.

[0226] Furthermore, the ancestral variants of MG34 were generated using two different approaches. In the first method, previously generated ancestral sequences were modified by removing amino acid positions to approximate the sequence length of MG34-1 (SEQ ID NO: 2). In the second method, chimeras were designed based on previously generated ancestral sequences, and the recognition lobe from the activity candidate was fused to the protein backbone of the active ancestor to examine whether the lack of activity of a particular ancestral sequence was at least partially due to the sequence diversity of the recognition lobe.

[0227] Results of bioinformatics

[0228] Based on the search of the metagenome database, one new MG34 sequence, MG34-35 (SEQ ID NO: 1347), and three new MG102 sequences, MG102-63 (SEQ ID NO: 1324), MG102-82 (SEQ ID NO: 1322), and MG102-83 (SEQ ID NO: 1323), were identified. Three new ancestral sequences (Table 3) were generated for the MG34 clade using combinations of different alignment and tree construction algorithms (Figures 1A - 1C). For the MG102 clade, 18 new ancestral sequences were generated (Figure 1D; SEQ ID NOs: 1329 - 1347). Four MG34 ancestral variants (Table 4) were generated from previously identified ancestral sequences by removing positions that are not present in the reference sequence MG34-1 (SEQ ID NO: 2). Twelve MG34 ancestral chimeras (Table 5) were generated by replacing the recognition lobe of the activity candidate with other ancestral sequences.

[0229] [Table 3]

[0230] [Table 4]

[0231] [Table 5]

[0232] In vitro PAM determination assay

[0233] Candidate MG34 nuclease effector proteins were codon-optimized for E. coli and cloned into vectors containing a T7 promoter and a C-terminal His tag. The genes were PCR-amplified at 150 bp primer binding sites upstream and downstream of the T7 promoter sequence and terminator sequence, respectively. The PCR products were added to a protein synthesis system reconstituted at a final concentration of 5 nM or higher, with all components necessary for in vitro transcription and translation purified from E. coli, and expressed at 37°C for 2 hours. The cleavage reaction was assembled by adding a 5-fold dilution of the protein synthesis system in 10 mM Tris (pH 7.5), 100 mM NaCl, and 10 mM MgCl2, along with a 5 nM 8N PAM plasmid library and 50 nM sgRNA targeting the PAM library. The sgRNA sequences used were some or all of the sequences identified from the following four active MG34 homologs: MG34-1(sg1)(SEQ ID NO: 613), MG34-9(sg9)(SEQ ID NO: 615), MG34-16(sg16)(SEQ ID NO: 616), and MG34-25(sg25)(SEQ ID NO: 1369). The cleavage products obtained from the protein synthesis reaction were recovered by purification using beads. The DNA was blunted by the addition of Klenow fragments and dNTPs. The blunt-end products were ligated with a 100-fold excess of double-stranded adapter sequences and used as templates for the preparation of the NGS library, from which the PAM requirements were determined by sequence analysis. Raw NGS reads were filtered by quality score > 20. The proximal region of the PAM was located using 14-24 bp representing known DNA sequences from the backbone adjacent to the PAM as a reference, and the adja...

Claims

1. It is a modified nuclease system, a) Endonucleases containing a sequence having at least 80% sequence identity with any one of sequence numbers 1324, 1329-1346, 1350-1368, and 1415-1440, and b) A modified nuclease system comprising a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

2. The modified nuclease according to claim 1, wherein the endonuclease comprises a sequence having at least 80% identity with any one of sequence numbers 1350-1368 and 1415-1440.

3. The modified nuclease according to claim 1, wherein the endonuclease comprises a sequence having at least 80% identity with any one of sequence numbers 1324 and 1329-1346.

4. It is a modified nuclease system, a) Endonucleases containing a sequence having at least 90% sequence identity with any one of sequence numbers 1324, 1329-1346, 1350-1368, and 1415-1440, and b) A modified nuclease system comprising a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

5. The modified nuclease according to any one of claims 1 to 4, wherein the endonuclease comprises a sequence having at least 90% identity with sequence numbers 1350-1368 and 1415-1440.

6. The modified nuclease according to any one of claims 1 to 4, wherein the endonuclease comprises a sequence having at least 90% identity with any one of sequence numbers 1324 and 1329 to 1346.

7. It is a modified nuclease system, a) Endonucleases containing a sequence having at least 95% sequence identity with any one of sequence numbers 1324, 1329-1346, 1350-1368, and 1415-1440, and b) A modified nuclease system comprising a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

8. The modified nuclease according to any one of claims 1 to 7, wherein the endonuclease comprises a sequence having at least 95% identity with sequence numbers 1350-1368 and 1415-1440.

9. The modified nuclease according to any one of claims 1 to 7, wherein the endonuclease comprises a sequence having at least 95% identity with any one of sequence numbers 1324 and 1329 to 1346.

10. It is a modified nuclease system, a) Endonucleases containing a sequence having at least 99% sequence identity with any one of sequence numbers 1324, 1329-1346, 1350-1368, and 1415-1440, and b) A modified nuclease system comprising a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

11. The modified nuclease according to any one of claims 1 to 10, wherein the endonuclease comprises a sequence having at least 99% identity with sequence numbers 1350-1368 and 1415-1440.

12. The modified nuclease according to any one of claims 1 to 10, wherein the endonuclease comprises a sequence having at least 99% identity with any one of sequence numbers 1324 and 1329 to 1346.

13. It is a modified nuclease system, a) Endonucleases containing sequences having 100% sequence identity with any one of sequence numbers 1323-1324, 1329-1347, 1350-1368, and 1415-1440, and b) A modified nuclease system comprising a modified guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

14. The modified nuclease system according to any one of claims 1 to 13, wherein the endonuclease comprises a sequence having 100% sequence identity with any one of sequence numbers 1347, 1350-1368, and 1415-1440.

15. The modified nuclease according to any one of claims 1 to 13, wherein the endonuclease comprises a sequence having 100% identity with any one of sequence numbers 1323, 1324, and 1329 to 1346.

16. The modified nuclease system according to any one of claims 1 to 15, wherein the modified guide polynucleotide is a single guide nucleic acid.

17. The modified nuclease system according to any one of claims 1 to 15, wherein the modified guide polynucleotide is a dual guide nucleic acid.

18. The modified nuclease system according to any one of claims 1 to 15, wherein the modified guide polynucleotide is RNA.

19. The modified nuclease system according to any one of claims 1 to 18, wherein the endonuclease is non-covalently bonded to the modified guide polynucleotide.

20. The modified nuclease system according to any one of claims 1 to 18, wherein the endonuclease is covalently bonded to the modified guide polynucleotide.

21. The modified nuclease system according to any one of claims 1 to 18, wherein the endonuclease is fused to the modified guide polynucleotide.

22. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

23. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having at least 90% sequence identity with 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.

24. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide comprises a sequence having at least 90% sequence identity with SEQ ID NO: 1410 or 1960.

25. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1410, 1412, 1953, 1956, 1960, 1961, 1966, 1970, and 1478.

26. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 2157, 2159, and 2160.

27. A modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 2017, 2022, 2029, 2031, 2032, 2035, 2044, 2045, 2047, 2048, 2073, 2075, 2090, 2195, 2197, 2198, 2199, 2200, and 2202.

28. A modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having at least 90% sequence identity with any one of sequence numbers 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.

29. The modified nuclease system according to any one of claims 1 to 21, wherein the modified guide polynucleotide includes a sequence having 100% sequence identity with any one of sequence numbers 1327-1328, 1348, 1369-1372, 1376-1391, 1392-1414, and 1470-2242.

30. A method for modifying a target nucleic acid sequence, comprising contacting the target nucleic acid sequence with a modified nuclease system according to any one of claims 1 to 29.

31. The method according to claim 30, wherein modifying the target nucleic acid sequence includes binding, nicking, or cleaving the target nucleic acid sequence.

32. The method according to any one of claims 30 to 31, wherein the target nucleic acid sequence includes genomic DNA, viral DNA, viral RNA, or bacterial DNA.

33. The method according to any one of claims 30 to 32, wherein the modification is in vitro.

34. The method according to any one of claims 30 to 32, wherein the modification is in vivo.

35. The method according to any one of claims 30 to 32, wherein the modification is exvivo.

36. A method for modifying a target nucleic acid sequence in a mammalian cell, comprising contacting the mammalian cell with a modified nuclease system according to any one of claims 1 to 29.

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

38. A cell comprising the modified nuclease system described in any one of claims 1 to 29.

39. The cell according to claim 38, wherein the cell is a eukaryotic cell.

40. The cell according to claim 38, wherein the cell is a mammalian cell.

41. The cell according to claim 38, wherein the cell is an immortalized cell.

42. The cell according to claim 38, wherein the cell is an insect cell.

43. The cell according to claim 38, wherein the cell is a yeast cell.

44. The cell according to claim 38, wherein the cell is a plant cell.

45. The cell according to claim 38, wherein the cell is a fungal cell.

46. The cell according to claim 38, wherein the cell is a prokaryotic cell.

47. The cell according to claim 38, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

48. The cell according to claim 38, wherein the cell is a modified cell.

49. The cell according to claim 38, wherein the cell is a stable cell.

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