Compositions and methods for targeting the dystrophin gene

Abstract

Described herein are methods, compositions, and systems for targeting the dystrophin gene.

Description

Attorney Docket No. 00010.032.1801 COMPOSITIONS AND METHODS FOR TARGETING THE DYSTROPHIN GENECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Application No.63 / 753,418, filed on February 3, 2025, the entire content of which is hereby incorporated by reference herein in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 2, 2026, is named 00010_032_1801_SL.xml and is 2,159,990 bytes in size.SUMMARY

[0003] Described herein, in certain embodiments, are engineered nuclease systems, comprising: (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217. 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0004] Described herein, in certain embodiments, are engineered nuclease system, comprising: (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478. 480-484, and 485-545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 588, 785. 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid - 1 - #593201Attomey Docket No. 00010.032.1801 sequence within a dystrophin gene, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368. 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

[0005] In certain embodiments, the base editor is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

[0006] In certain embodiments, the base editor is encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 216, 276. 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588. 785, 1-4. and 582-587.

[0007] In certain embodiments, the base editor is encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

[0008] In certain embodiments, the endonuclease comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172. 179, 183, 104-123. 125-157. 159-165. 167-171. 173-175. 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity’ to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 100% sequence identity’ to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, 183, 104-123, 125-157. 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8. and 886-891.- 2 - #593201Attorney Docket No. 00010.032.1801

[0009] In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

[0010] In certain embodiments, the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632. 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625. 626-631. 633-636. 643-652, 637-642, and 786-788.

[0011] Described herein, in certain embodiments, are engineered nuclease systems, comprising: an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: T33R, E49R, Q58R, N72R, S89R, E103R, Q132R, E139R, N153R, S164R, N195R, T217R, Q227R, Q228R, N232R, G247R, E267R. T277R, S281R. N307R, L348R, D357R, N401R, Q435R, A524R, D527R, E542N, D556Q, N564R, E575Q, E577R, A583R, E588R, D598K, I602T, I602E, N604R, E624Q, E624R, M627R, P638R, H654Q, N655K, E672R, L673R, G700R, T733R, and S737T as compared to SEQ ID NO: 789; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0012] In certain embodiments, the endonuclease comprises the amino acid modifications El 39R, D357R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E575Q. E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E575Q, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E575Q, E577R, and E624R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E575Q. E577R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R,- 3 - #593201Attorney Docket No. 00010.032.1801 E575Q, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R. E577R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modification A583R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modification E577R as compared to SEQ ID NO: 789.

[0013] In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 365-368 and 375.

[0014] Described herein, in certain embodiments, are engineered nuclease systems, comprising: an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 194; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0015] In certain embodiments, the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 194. In certain embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E255K, and N329E as compared to SEQ ID NO: 194. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 156-191. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 156-191. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 156-191. In certain embodiments, the endonuclease comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 156-191.

[0016] In certain embodiments, the endonuclease comprises one or more of the following sets of amino acid modifications selected from the group consisting of: i) E236K and E255K; ii) M204K and E236K; iii) K93R, E236K, and E255K; iv) K93R, E202K, and E236K; v) M204K. E236K, and N329E; and vi) K93R, M204K, and E236K as compared to SEQ ID NO: 194.

[0017] Described herein, in certain embodiments, are engineered nuclease systems, comprising: an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 176. 124, 158, 166. 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632. 9-23. 85-103, 192, 193, 626-636, 643-652, and 786-788.- 4 - #593201Attorney Docket No. 00010.032.1801

[0018] In certain embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178. and 180-191. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191. In certain embodiments, the endonuclease comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 176, 124, 158. 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191.

[0019] In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103. 192, 193, 626-636, 643-652, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 626-636, 643-652, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632. 9-23. 85-103, 192, 193, 626-636, 643-652, and 786-788.

[0020] Described herein, in certain embodiments, are engineered nuclease systems, comprising: an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 104-191; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-23, 94, and 101-103.

[0021] In some embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 104-191.

[0022] In some embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 104-191.

[0023] In some embodiments, the endonuclease comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 104-191.

[0024] In some embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135.- 5 - #593201Attomey Docket No. 00010.032.1801

[0025] In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38.

[0026] In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38.

[0027] In some embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38.

[0028] In certain embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is RNA. In certain embodiments, the engineered endonuclease binds non-covalently to the engineered guide polynucleotide. In certain embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In certain embodiments, the endonuclease is fused to the engineered guide polynucleotide.

[0029] Described herein, in certain embodiments, is a method for modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with the engineered nuclease system of any one disclosed herein.

[0030] Described herein, in certain embodiments, is a method for modifying a target nucleic acid sequence within a dystrophin gene of a mammalian cell comprising contacting the mammalian cell with the engineered nuclease system disclosed herein.

[0031] In certain embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence.

[0032] In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

[0033] In some embodiments, the modification is in vitro. In some embodiments, the modification is in vivo. In some embodiments, the modification is ex vivo.

[0034] In some embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

[0035] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581. 593-625. 626-631, 633-636, 643-652, 637-642, and 786-788.

[0036] Described herein, in certain embodiments, is a method of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence - 6 - #593201Attorney Docket No. 00010.032.1801 identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 588, 785. 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence.

[0037] Described herein, in certain embodiments, is a method of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence identity’ to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity’ to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194. 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23. 85-103, 192. 193. 365-368. 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

[0038] In some embodiments, the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587. In certain embodiments, the endonuclease is encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587. In certain embodiments, the endonuclease is encoded by a sequence having at least 100% sequence identity’ to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the endonuclease comprises an amino acid sequence having at least 100% sequence identity to any - 7 - #593201Attomey Docket No. 00010.032.1801 one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891. In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885. In certain embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

[0039] In certain embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is RNA. In certain embodiments, the engineered endonuclease binds non-covalently to the engineered guide polynucleotide. In certain embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In certain embodiments, the endonuclease is fused to the engineered guide polynucleotide. In certain embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632. 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

[0040] Described herein, in certain embodiments, are methods of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: T33R, E49R, Q58R, N72R, S89R, E103R, Q132R, E139R, N153R, S164R, N195R, T217R, Q227R, Q228R, N232R, G247R, E267R, T277R, S281R, N307R, L348R, D357R. N401R, Q435R, A524R. D527R, E542N, D556Q, N564R, E575Q, E577R, A583R, E588R, D598K, I602T, I602E, N604R, E624Q, E624R, M627R, P638R, H654Q, N655K, E672R, L673R, G700R, T733R, and S737T as compared to SEQ ID NO: 789; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.- 8 - #593201Attomey Docket No. 00010.032.1801

[0041] In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E575Q. E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E577R, E624R, and E672R as compared to SEQ ID NO: 789.

[0042] In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E575Q, E624R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications E139R, D357R, E575Q, E577R, and E624R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E575Q. E577R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E575Q, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modifications D357R, E577R, and E672R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modification A583R as compared to SEQ ID NO: 789. In certain embodiments, the endonuclease comprises the amino acid modification E577R as compared to SEQ ID NO: 789. In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 365-368 and 375.

[0043] Described herein, in certain embodiments are methods of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 194; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0044] Described herein, in certain embodiments are methods of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: an endonuclease comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 156-191; and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having- 9 - #593201Attomey Docket No. 00010.032.1801 at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581. 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

[0045] In certain embodiments, the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 194.

[0046] In certain embodiments, the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 156-191.

[0047] In certain embodiments, the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 156-191.

[0048] In certain embodiments, the endonuclease comprises an amino acid sequence having at least 100% sequence identity to any one of SEQ ID NOs: 156-191.

[0049] In certain embodiments, the endonuclease comprises one or more of the following sets of amino acid modifications selected from the group consisting of: i) E236K and E255K; ii) M204K and E236K; iii) K93R, E236K, and E255K; iv) K93R, E202K, and E236K; v) M204K, E236K, and N329E; and vi) K93R, M204K, and E236K as compared to SEQ ID NO: 194.

[0050] In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578. and 838-885.

[0051] In certain embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

[0052] In certain embodiments, the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

[0053] In certain embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In certain embodiments, the engineered guide polynucleotide is RNA. In certain embodiments, the engineered endonuclease binds non-covalently to the engineered guide polynucleotide. In certain embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In certain embodiments, the endonuclease is fused to the engineered guide polynucleotide. In certain embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625. 626-631, 633-636, 643-652, 637-642, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788. In certain embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs:- 10 - #593201Attorney Docket No. 00010.032.1801 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

[0054] Described herein, in certain embodiments, is a cell comprising the engineered nuclease system of any one of disclosed herein.

[0055] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.

[0056] In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5. Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa. Saos, C2C12, L cell, HT1080, HepG2, Huh7, K.562, primary cell, or a derivative thereof.

[0057] In some embodiments, the cell is an engineered cell.

[0058] In some embodiments, the cell is a stable cell.

[0059] Described herein, in certain embodiments, is a viral vector comprising the engineered nuclease system of any one of disclosed herein.

[0060] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0061] 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-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l. AAV-hu37, AAV- Anc80, AAV-Anc80L65. AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16. or a derivative thereof.

[0062] Additional aspects and advantages of the present disclosure w ill become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.- 11 - #593201Attorney Docket No. 00010.032.1801 BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also " Figure” and “FIG.” herein), of which:

[0064] FIG. 1 depicts a plot showing total productive editing (%) by MG 3-6 (SEQ ID NO: 582), MG3-6 / 3-4 (SEQ ID NO: 583). MG3-6 / 3-8 (SEQ ID NO: 584), MG21-1 (SEQ ID NO: 585), MG29-1 (SEQ ID NO: 586). MG71-43 (SEQ ID NO: 587). MG119-28 (SEQ ID NO: 588) nucleases and ABE-103 (SEQ ID NO: 589), ABE-106 (SEQ ID NO: 590), ABE-180 (SEQ ID NO: 591), ABE-158 (SEQ ID NO: 592) editors in human skeletal muscle cells for their ability to cause restorative exon skipping or reframing. 49 guide RNAs showed productive editing ranging from 5%-30% targeting different exons. % predictive editing is the editing predicted to cause restorative exon skipping or reframing.

[0065] FIG. 2 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis) in K562 cells. The scaffold length is represented by diagonal hatched (134 nt) and open (119 nt) bars (n = 2 or 3). The spacer length for the DMD_H1 guide (18-25 nt; SEQ ID NOs: 85-100) is shown on the X axis.

[0066] FIG. 3 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis) in K562 cells of each protein variant. Editing efficiency obtained with the WT MG119-28 nuclease and a sgRNA comprising the 119 nt scaffold and a 22 nt spacer targeting hDMD (SEQ ID NO: 89) is shown as a dotted line. 52 single point mutant (SPM) proteins were tested (protein variants).

[0067] FIG. 4 depicts editing efficiency of each protein as percent NGS reads with insertions or deletions (InDeis; bars in K562 cells). The percent out of frame (% OOF) total obtained as % OOF InDeis is shown as circles within the bars. The level of editing efficiency by the WT protein is shown as a dotted line and a bar. This experiment tested sgRNAs with the 134 nt scaffold and 19 nt spacer (hDl 19-28L-P4Hlb; SEQ ID NO: 626; spacer sequence of SEQ ID NO: 858). 34 double and triple mutants and a hexamutant were screened and are shown on the X axis as protein variants.

[0068] FIGs. 5A-5D depict the editing efficiency of the indicated proteins as percent NGS reads with insertions or deletions (InDeis; bars). The MG119-28 WT, six combinatorial mutants, and one SPM were tested at four different mRNA doses, 400, 200, 100, and 50 ng shown on the x-axis in SkM cells. The sgRNA comprised the 134 nt scaffold and a 19 nt spacer targeting the - 12 - #593201Attorney Docket No. 00010.032.1801 hD119-28L_P4Hlb (SEQ ID NO: 94) (FIG. 5A), hD119-28L_P4A2b (SEQ ID NO: 101) (FIG.5B), hD119-28L_P4E2b (SEQ ID NO: 102) (FIG. 5C) and hDl 19-28L_P4C4b (SEQ ID NO: 103) (FIG. 5D) sites.

[0069] FIGs. 6A-6B depict a cell fluorescence screen to identify ABE variants. FIG. 6A shows a schematic of HEK293T cell fluorescence screen. FIG. 6B shows a molecular basis of the cell fluorescence screen. An ABE converts the target adenine in an in-frame stop codon of the target (DMD) to a TGG sense codon, which enables expression of the mCherry protein. FIG. 6B discloses SEQ ID NOs: 575-580, respectively, in order of appearance.

[0070] FIGs.7A-7D depict a domain-walking of monomeric ADA MG68-4 variants and arginine scanning in MG102-71 nickase. FIG. 7A shows activity of MG102-71 ABE domain- walking variants at the human DMD exon 45 splice acceptor site. FIG. 7B shows activity of MG102-71 ABE domain- walking variants at the human DMD exon 51 splice acceptor site. FIG. 7C shows activity of MG102-71 ABE arginine-scanning variants (SEQ ID NOs. 207-229) at the human DMD exon 45 splice acceptor site. Threshold for improvement determination (dotted line) was one of ABE-192 (denoted by SEQ ID NO: 476). FIG. 7D shows activity of MG34-29 ABE domain-walking variants at the human DMD exon 45 splice acceptor site.

[0071] FIGs. 8A-8B depict an activity plot of improved SMART ABE domain-walking variants.FIG. 8A shows an activity plot of MG102-71 ABE domain-walking variants (SEQ ID NOs. 350-369) that performed above the guide-free apo control at human AAVS1 and human DMD exon 45. FIG. 8B shows an activity plot of MG34-29 ABE domain-walking variants (SEQ ID NOs.316-349) that performed above the guide-free apo control at human AAVS1 and human DMD exon 45.

[0072] FIG. 9 depicts a max A to G conversion by a small base editors ABE- 163 (denoted by SEQ ID NO: 447) targeting a nGG PAM at DMD locus in replicates of two. FIG.9 discloses SEQ ID NO: 581.

[0073] FIGs. 10A-10D depict base editing data in SkM cells for rationally engineered small base editors variants. Total on-target A-G conversion and total base substitutions are represented. FIG.10A: MG34-29 Variants E.\45_gl2; FIG. 10B: MG34-29 Variants Ex45_gl3; FIG. 10C:MG102-71 Variants Ex45_gE and FIG. 10D: MG102-71 Variants Ex51_gl.

[0074] FIGs. 11A-11C depict dystrophin protein restoration with MG119-28 candidates. FIG.11A shows a Western blot of the indicated amounts of WT myocyte input amounts, and mock treated AEx52 myocytes or AEx52 myocytes edited with hD119-28-P4C4 (SEQ ID NO: 786), hD119-28-P4B4 (SEQ ID NO: 787), hDl 19-28-g09b (SEQ ID NO: 788). The blots were probed with dystrophin antibody and vinculin (loading control) antibody. FIG. 11B shows WT input that - 13 - #593201Attorney Docket No. 00010.032.1801 was quantified and the relative intensity plotted against the %WT input. FIG. 11C shows the dystrophin signal from MG119-28 edited AEx52 myocytes that was then quantified and normalized against vinculin to identify %dystrophin expression relative to the WT myocytes.

[0075] FIGs. 12A-12C depict dystrophin expression restoration in human AEx44 myocytes with MG29-1, MG3-6 / 3-8, MG21-1, MG3-6x ABE candidates. FIG. 12A is a plot showing mRNA expression measured using ddPCR. FIG. 12B are representative Western blot images demonstrating absence of dystrophin protein in mock-treated AEx44 myocytes and varying levels of dystrophin restoration following genome editing with MG system guide RNA candidates: hD29-P3C8 (SEQ ID NO: 616), hD368-Gl (SEQ ID NO: 597), hD368_Pl A5 (SEQ ID NO: 599), hD211_P1B9 (SEQ ID NO: 607), hDABE103_Ex45_g4 (SEQ ID NO: 637), and hDABE106_Ex45_g5 (SEQ ID NO: 638). FIG. 12C are immunofluorescence images showing robust dystrophin expression in WT myocytes, absence of dystrophin signal in mock-treated AEx44 myocytes, and restored dystrophin expression in AEx44 myocytes edited with guide RNAs hD368_P1A5 (SEQ ID NO: 599), hD211_P1B9 (SEQ ID NO: 607), and hDABE106_Ex45_g5 (SEQ ID NO: 638).

[0076] FIGs. 13A-13B depict dystrophin expression restoration in human AEx44 myocytes with MG21-1 and MG119-28 candidates. FIG. 13A is a plot showing mRNA expression measured using ddPCR. FIG. 13B are representative Western blot images demonstrating absence of dystrophin protein in mock-treated AEx44 myocytes and vary ing levels of dystrophin restoration following genome editing with MG system guide RNA candidates: hD211_P1B9 (SEQ ID NO: 607). hD119-28L-P4G1b (SEQ ID NO: 629), hD119-28L-A9b (SEQ ID NO: 632), mhD119-28L-B9b (SEQ ID NO: 634) in AEx44 myocytes.

[0077] FIGs. 14A-14B depict dystrophin expression restoration in human AEx44 myocytes with SMART ABEs and MG119-28 and MG21-1 candidates. FIG. 14A is a plot showing mRNA expression measured using ddPCR. FIG. 14B are representative Western blot images demonstrating absence of dystrophin protein in mock -treated AEx44 myocytes and varying levels of dystrophin restoration following genome editing with MG system guide RNA candidates: hDABE180_Ex45_gl (SEQ ID NO: 639), hDABE180_Ex45_g2 (SEQ ID NO: 640), hD119-28L-A9b (SEQ ID NO: 632), hD211_P1B9 (SEQ ID NO: 607) in AEx44 myocytes.

[0078] FIGs. 15A-15D depict dystrophin expression restoration in human AEx52 myocytes with MG3-6 / 3-4, MG3-6 / 3-8, MG29-1 and MG119-28 candidates. FIG. 15A is a plot showing mRNA expression measured using ddPCR. FIG. 15B are representative Western blot images demonstrating absence of dystrophin protein in mock-treated AEx52 myocytes and varying levels of dystrophin restoration following genome editing with MG system guide RNA candidates:- 14 - #593201Attorney Docket No. 00010.032.1801 hD364_B9 (SEQ ID NO: 594), hD368_E3 (SEQ ID NO: 605), hD29_A9 (SEQ ID NO: 621), hD119-28L-P4C4b (SEQ ID NO: 635), hD119-28L-P4B4b (SEQ ID NO: 631), hD119-28L-g09b (SEQ ID NO: 636) in AEx52 myocytes. FIG. 15C are immunofluorescence images showing robust dystrophin expression in WT myocytes, absence of dystrophin signal in mock-treated AEx52 myocytes, and restored dystrophin expression in AEx52 myocytes edited with guide RNAs hD29_A9 (SEQ ID NO: 621), hD119-28L-P4C4b (SEQ ID NO: 635). FIG. 15D is showing results from long read sequencing analysis.

[0079] FIGs. 16A-16B relate to MG119-28 guide screen for human guideRNA candidates in human skeletal muscle cells and editing efficiency of lead candidates in mouse myoblasts. FIG.16A is a guide screen of human DMD sgRNAs in human skeletal muscle cells (SkM). The SkM cells were transfected with 200 ng of MG119-28 mRNA with 1:5 molar ratio of synthetic guideRNA (SEQ ID NOs: 626-635. 643- 649). Editing efficiency of hDMD spacers as percent insertions or deletions (%InDels) reads was measured by NGS, three-days post transfection. FIG.16B is a plot showing editing efficiency of mDMD spacers as percent insertions or deletions (%InDels) measured by next generation sequencing (NGS).

[0080] FIGs. 17A-17D relate to in-vivo editing at DMD locus by MG119-28 mouse guideRNA candidates delivered via AAV. FIG. 17A is an exemplary AAV cargo construct designed to express MG119-28 and a guide RNA from a single AAV. The payload is flanked by two, 141 bp inverted terminal repeats (ITRs). A U6 polymerase III promoter is placed upstream of the guide RNA sequence in the reverse orientation at the 5' end of the cargo followed by a CMV promoter driving the MG119-28-2a-NLS-EGFP open reading frame. MG119-28 is N-terminally tagged with a V5 epitope. A synthetic polyadenylation sequence (SpA) follows MG119-28. FIG. 17B is a schematic outlining systemic delivery via tail vein injection of myotropic AAVs carrying MG119-28 with guideRNA targeting mouse DMD or mouse ROSA26 (SEQ ID NOs: 653- 657).FIG. 17C is a plot showing editing efficiency of mDMD and mROSA26 spacers as percent insertions or deletions (%InDels) reads measured by NGS from diaphragm, quadriceps and gastrocnemius muscle tissues. FIG. 17D is a plot showing editing efficiency of mDMD and mROSA26 spacers as percent insertions or deletions (%InDels) reads measured by NGS from heart tissue samples pre and post FACS enrichment of EGFP+.

[0081] FIGs. 18A-18B relate to in vivo editing at albumin locus by MG119-28 mouse guideRNA candidates delivered via AAV. FIG. 18A is a schematic outlining systemic delivery via tail vein injection of AAV8 carrying MG119-28 with guideRNA targeting mouse albumin or mouse ROSA26 (SEQ ID NOs: 653, 658, 659). Two weeks post injection, liver tissues were harvested and editing was measured by NGS. FIG. 18B is a plot showing editing efficiency of mAlbumin - 15 - #593201Attorney Docket No. 00010.032.1801 and mROSA26 spacers as percent insertions or deletions (%InDels) reads measured by NGS from left liver lobe (LL), medial liver lobe (ML) and right liver lobe (RL).BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0082] The listing of sequences filed herewith provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions and systems according to the disclosure. Below are exemplar)’ descriptions of sequences therein.

[0083] SEQ ID NOs: 1-4 and 582-585 show the nucleic acid sequences (also include codon-optimized variants) encoding the nucleases, MG3-6, MG3-6 / 3-4, MG3-6 / 3-8, and MG21-1 nucleases, respectively.

[0084] SEQ ID NOs: 5-8 show the protein sequences of nucleases MG3-6, MG3-6 / 3-4, MG3-6 / 3-8, and MG21-1 nucleases, respectively. SEQ ID NOs: 886 and 887 show the protein sequences of the MG3-6 / 3-8, and MG21-1 nucleases, respectively. SEQ ID NOs: 888-891 show the protein sequences of nucleases MG3-6, MG3-6 / 3-4, MG3-6 / 3-8, and MG21-1 nucleases, respectively without a nuclear localization sequence.

[0085] SEQ ID NOs: 9-23 and 85-103 show the nucleic acid sequences encoding guide RNA targeting human DMD.

[0086] SEQ ID NOs: 24-38 and 838-885 show the DNA sequences of DMD target sites.

[0087] SEQ ID NOs: 39-84 show the amino acid sequence of exemplary nuclear localization sequences (NLSs).

[0088] SEQ ID NOs: 104-191 show the protein sequences of MG119 effectors. SEQ ID NOs: 104-155 are single point mutants as compared to WT (SEQ ID NO: 194). SEQ ID NOs: 156-190 are double and triple point mutants as compared to WT. SEQ ID NO: 176 is a MG119-28 variant with K93R-M204K-E236K. SEQ ID NO: 191 is a hexamutant as compared to WT. SEQ ID NO: 194 shows the protein sequence of MG119-28 effector.

[0089] SEQ ID NOs: 192-193 show the nucleotide sequences of MG119-28 effector sgRNA.

[0090] SEQ ID NO: 785 shows the DNA sequence encoding the MG119-28 nuclease variant (DNA sequence encoding MG119-350 (MG119-28 with K93R-M204K-E236K)), includes NLS sequence. This sequence is identical to SEQ ID NO: 588.

[0091] SEQ ID NOs: 786-788 show the nucleic acid sequences for guide RNAs targeting human DMD.

[0092] SEQ ID NOs: 195-217 show the nucleic acid sequences of SMART arginine-scanning (MG102-71 ABE arginine-scanning variants); and 376-398 show the protein sequences of SMART arginine-scanning (MG102-71 ABE arginine-scanning variants).- 16 - #593201Attorney Docket No. 00010.032.1801

[0093] SEQ ID NOs: 218-303 show the nucleic acid sequences of SMART ABE 2nd-generation and 399-484 show the protein sequences of SMART ABE 2nd-generatation. SEQ ID NOs: 589-592 show the nucleic acid sequences encoding the ABE- 103, ABE- 106. ABE- 180, and ABE- 158, respectively. SEQ ID NO: 591 is identical to SEQ ID NO: 298. SEQ ID NO: 592 is identical to SEQ ID NO: 276.

[0094] SEQ ID NOs: 304-364 show the nucleic acid sequences of SMART ABE 3rd-generation and 485-545 show the protein sequences of SMART ABE 3rd-generation.

[0095] SEQ ID NOs: 365-368 shows the nucleic acid sequences of MG34-29 sgRNA targeting human DMD Exon 45 splice acceptor.

[0096] SEQ ID NOs: 369-371 show the nucleic acid sequences of MG102-71 sgRNA targeting human DMD Exon 45 splice acceptor.

[0097] SEQ ID NOs: 372-374 show the nucleic acid sequences of MG102-71 sgRNA targeting human DMD Exon 51 splice acceptor

[0098] SEQ ID NO: 375 shows the nucleic acid sequence of MG34-29 sgRNA targeting human DMD Exon 45 splice acceptor.

[0099] SEQ ID NOs: 546-549 and 556 show the nucleic acid sequences of MG34-29 spacer targeting human DMD Exon 45 splice acceptor.

[0100] SEQ ID NOs: 550-552 show the nucleic acid sequences of MG102-71 spacer targeting human DMD Exon 45 splice acceptor.

[0101] SEQ ID NOs: 553-555 show the nucleic acid sequences of MG102-71 spacer targeting human DMD Exon 51 splice acceptor.

[0102] SEQ ID NOs: 557-574 and 679-784 show sequences of primers used to amplify human DMD guide target site and for next generation sequencing.

[0103] SEQ ID NOs: 575 shows the nucleotide sequence of exon 45 DMD target site (translated amino acid sequence is shown in SEQ ID NOs: 576 and 577) and SEQ ID NO: 578 shows the nucleotide sequence of exon 51 DMD target site (and translated amino acid sequence is shown in SEQ ID NOs: 579 and 580).

[0104] SEQ ID NO: 581 is nucleotide sequence of DMD exon 45 targeting guide hD_ABE_Ex45-§12.

[0105] SEQ ID NOs: 586-588 show the nucleic acid sequences encoding the nucleases, MG29-1, MG71-43, and MG119-28 nucleases, respectively.

[0106] SEQ ID NOs: 593-652 show the full sequences of guide RNAs targeting human DMD.

[0107] SEQ ID NOs: 653-659 show the AAV cargo plasmid. SEQ ID NO: 653 is the nucleic acid sequence of AAV cargo plasmid expressing V 5 -tagged MG119-28 and guide R26g4. SEQ ID NO:- 17 - #593201Attorney Docket No. 00010.032.1801 654 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mhD119-28L-P4A2b. SEQ ID NO: 655 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mhD119-28L-B9b. SEQ ID NO: 656 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mD119-28L-P4B4b. SEQ ID NO: 657 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mDl 19-28L-g05b. SEQ ID NO: 658 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mAlb3. SEQ ID NO: 659 is the nucleic acid sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mAlb4.

[0108] SEQ ID NOs: 660-665 are nucleic acid sequences for ddPCR primer probe sets.

[0109] SEQ ID NO: 666 is the nucleic acid sequence of a primer targeting Exon 1 of the Dp427m isoform and SEQ ID NO: 667 is the nucleic acid sequence of a primer targeting the 3' UTR to generate the full-length DMD cDNA.

[0110] SEQ ID NOs: 668-678 are placeholder sequences.

[0111] SEQ ID NOs: 786-788 show the nucleic acid sequences for guide RNAs targeting human DMD.

[0112] SEQ ID NO: 789 is the amino acid sequence of MG34-29 (wild-type) and SEQ ID NOs: 790-837 are amino acid sequences of MG34-29 variants.DETAILED DESCRIPTIONDefinitions

[0113] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0114] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor 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 (R. I. Freshney. ed. (2010)).- 18 - #593201Attomey Docket No. 00010.032.1801

[0115] As used herein, the singular forms “a,” "an" and ‘‘the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0116] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art. which will depend in part on how the value is measured or determined, i.e.. the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1 % of a given value.

[0117] As used herein, a “cell” refers to a biological cell. A cell may be the basic structural, functional, or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, fems, clubmosses, homworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa. Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., ayeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc ), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g.. a cell can be a synthetically made, sometimes termed an artificial cell).

[0118] The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the - 19 - #593201Attorney Docket No. 00010.032.1801 nucleic acid molecule containing them. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP. ddGTP, ddITP. and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels of nucleotides include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N, N, N', N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red. Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [Rl lOJdCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA] ddGTP, and [dROX]ddTTP available from Perkin Elmer. Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP. FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein- 12-UTP, and Fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP. BOD1PY-FL-4-UTP. B0DIPY-TMR-14-UTP, BOD1PY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary' chemically-modified nucleotide is biotin-dNTP. Nonlimiting 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).

[0119] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary - 20 - #593201Attorney Docket No. 00010.032.1801 polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA). ribosomal RNA (rRNA), short interfering RNA (siRNA), shorthairpin RNA (shRNA), micro-RNA (miRNA), ribozy mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and / or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g, rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0120] As used herein, the term “base editor” refers to an enzyme that catalyzes the conversion of one target base or base pair into another (e.g., A: T to G: C, C: G to T: A) without requiring the creation and repair of a double-strand break. An exemplary’ base editor is a deaminase. In some embodiments, the base editor comprises a deaminase and a nuclease that is deficient in nuclease activity. In some embodiments, the base editor comprises a deaminase and a catalytically inactive nuclease. In some embodiments, the base editor comprises a fusion of a deaminase and a catalytically inactive nuclease.

[0121] As used herein, the term “deaminase” refers to a protein or enzyme that catalyzes a deamination reaction (i.e., a reaction that removes an amino group). Deaminases include adenosine deaminases, which catalyze the hydrolytic deamination of adenine or adenosine (e.g., an engineered adenosine deaminase that deaminates adenosine in DNA), and cytidine (or cytosine) deaminases, which catalyze the hydrolytic deamination of cytidine (or cytosine) or deoxy cytidine to uridine (or uracil) or deoxyuridine, respectively. The deaminase or deaminase domain can be a naturally-occurring deaminase or deaminase domain from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, mouse, or bacterium (e.g., E. coli), a variant of a naturally-occurring deaminase or deaminase domain, or a non-naturally occurring deaminase or deaminase domain.- 21 - #593201Attomey Docket No. 00010.032.1801

[0122] The terms “transfection” or “transfected” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See. e.g., Sambrook et al.. 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0123] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertian’ structure (e.g.. domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, refer to natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. The term “amino acid” includes both D-amino acids and L-amino acids.

[0124] As used herein, the “non-native” can refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein. Non-native may refer to affinity tags. Non-native may refer to fusions. Non-native may refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions, or deletions. A non-native sequence may exhibit or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) that may also be exhibited by the nucleic acid or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence may be linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.

[0125] The term “promoter”, as used herein, refers to the regulatory DNA region which controls transcription or expression of a polynucleotide (e.g., a gene) and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene - 22 - #593201Attorney Docket No. 00010.032.1801 transcription. A “basal promoter”, also referred to as a “core promoter”, may refer to a promoter that contains all the basic necessary elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically, though not necessarily, contain a TATA-box and / or a CAAT box. The term “expression”, as used herein, refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0126] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring. An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature: a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring a new function or property. An “engineered” system comprises at least one engineered component.

[0127] A “vector” as used herein, refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0128] As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, an expression cassette refers to the combination of regulatory elements and a gene or genes to which the are operably linked for expression.- 23 - #593201Attomey Docket No. 00010.032.1801

[0129] As used herein, “operably linked’’, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g.. movement or activation) of a first genetic element has some effect on the second genetic element. The effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, a regulatory’ element, which may comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0130] As used herein, the terms “gene editing” and “genome editing” can be used interchangeably. Gene editing or genome editing means to change the nucleic acid sequence of a gene or a genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0131] As used herein, a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.” A nucleic acid-targeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”

[0132] The term “binds” or any grammatical variation thereof (e.g., binding, etc.) when made in reference to the binding of two molecules (e.g., extracellular antigen binding domain of a CAR - 24 - #593201Attorney Docket No. 00010.032.1801 and a tumor-associated antigen, etc.) refer to an interaction of the two molecules that is dependent upon the presence of a particular structure on one or both of the molecules.

[0133] The term "tumor antigen7’ or "tumor associated antigen” as used herein refers to a substance, produced by tumor cells, which is recognized by the immune system and which elicits an antigen specific immune response in a host (e.g., which is presented by MHC complexes). In some embodiments, a tumor antigen is on the surface of a tumor cell. In some embodiments, the tumor antigen is presented by an MHC / HLA.

[0134] A “functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence. A biological activity of a DNA sequence includes its ability to influence expression in a manner attributed to the full-length sequence.

[0135] As used herein, the term “optimally aligned” refers to an alignment of two amino acid sequences that give the highest percent identity score or maximizes the number of matched residues.

[0136] The term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA. tracrRNA interacts with the CRISPR (cr) RNA to form guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. If the tracrRNA is engineered, it may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes, S. aureus). tracrRNA may refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. The term tracrRNA encompasses a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a tracrRNA sequence has at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. Type II tracrRNA sequences can be predicted on a genome sequence by identifying regions with complementarity to part of the repeat sequence in an adjacent CRISPR array.

[0137] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g, in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino - 25 - #593201Attomey Docket No. 00010.032.1801 acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); or CLUSTALW with parameters of the Smith-Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0138] As used herein, the term “RuvC III domain” refers to a third discontinuous segment of a RuvC endonuclease domain (the RuvC nuclease domain being comprised of three discontiguous segments, RuvC_I, RuvC_II, and RuvC_III). A RuvC domain or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g.. Pfam HMM PF18541 for RuvC III).

[0139] As used herein, the term “HNH domain” refers to an endonuclease domain having characteristic histidine and asparagine residues. An HNH domain can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0140] As used herein, the term '‘recognition domain” or ‘'REC domain” refers to a domain thought to interact with the repeat: anti-repeat duplex of the gRNA and to mediate the formation of a Cas endonuclease / gRNA complex.

[0141] As used herein, the term ‘'PAM interacting domain” or “PI domain” refers to a domain interacting with the protospacer- adjacent motif (PAM) external to the seed sequence in a region targeted by a Cas protein. Examples of P AM-interacting domains include, but are not limited to. Topoisomerase-homology (TOPO) domains and C-terminal domains (CTD) present in Cas proteins. A PAM interacting domain or segments thereof can generally be identified by alignment- 26 - #593201Attomey Docket No. 00010.032.1801 to documented domain sequences, structural alignment to proteins with annotated domains, or by comparison to Hidden Markov Models (HMMs) built based on documented domain sequences

[0142] As used herein, the term '‘complex’7refers to a joining of at least two components. The two components may each retain the properties / activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0143] Included in the current disclosure are variants of any of the enzy mes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three- dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%. at least about 50%. at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%. at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity any one of the systems described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease is not disrupted. In some embodiments, a functional variant of any of the systems described herein lack substitution of at least one of the conserved or functional residues described herein. In some embodiments, a functional variant of any of the systems described herein lacks substitution of all of the conserved or functional residues described herein

[0144] In accordance with IUPAC conventions, the following abbreviations are used throughout the examples:A = adenineC = cytosineG = guanine- 27 - #593201Attomey Docket No. 00010.032.1801 T = thymineR = adenine or guanineY = cytosine or thymineS = guanine or cytosineW = adenine or thymineK = guanine or thymineM = adenine or cvtosineB = C, G. or TD = A, G, or TH = A, C. or TV = A, C, or G.

[0145] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groupseach contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F). Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)Overview

[0146] Duchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disorder caused by mutations in the DMD gene on the X chromosome. This gene encodes dystrophin, a protein essential for maintaining muscle cell integrity by linking the internal cytoskeleton to the dystroglycan complex at the cell membrane. The absence or malfunction of dystrophin destabilizes muscle membranes, leading to excessive calcium influx, muscle degeneration, and necrosis. Individuals with DMD typically exhibit symptoms in early childhood, such as difficulty walking, frequent falls, and trouble climbing stairs. As the disease progresses, it affects the respiratory and cardiac muscles, leading to severe complications like cardiomyopathy and heart failure. The spectrum of mutations in the DMD gene also results in Becker Muscular Dystrophy (BMD), a milder form of the disease where some functional dystrophin is produced, leading to a slower progression of muscle weakness. Modifying the DMD gene utilizing CRISPR-based editing systems is one strategy for precisely correcting the genetic defects responsible for DMD.

[0147] DMD is caused by frameshifting or nonsense mutations in the DMD gene that reduce or abolish dystrophin expression, leading to progressive muscle degeneration and early mortality. The wild-type exon DMD open reading frame can be disrupted by frameshifting due to the - 28 - #593201Attomey Docket No. 00010.032.1801 deletion of exon 44, which causes a frameshift and premature stop codon in some DMD patients. A > G conversion of the conserved splice acceptor adenine of exon 45 can skip exon 45 and recover the reading frame despite the deletion of exon 44. Skipping of exon 45 is predicted to benefit subjects carrying common upstream deletions that disrupt the reading frame, most frequently deletions involving exon 44, by re-establishing an in-frame transcript through removal of exon 45. This strategy is estimated to be applicable to approximately 8.1% of individuals with Duchenne muscular dystrophy.

[0148] The wild-ti pe exon DMD open reading frame can also be disrupted by frameshifting due to the deletion of exons 49 and / or 50 which causes a frameshift and premature stop codon in some DMD patients. A> G conversion of the conserved splice acceptor adenine of exon 51 can skip exon 45 and recover the reading frame despite the deletion of exon 44. Skipping of exon 51 targets a complementary patient population harboring frequent downstream multi-exon deletions within the hotspot locus, and is predicted to benefit approximately 13.1% of patients with DMD.CRISPR / Cas Enzymes

[0149] The discovery of new Cas enzymes with unique functionality and structure offers the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzy mes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species offers the potential to drastically increase the number of new CRISPR / Cas systems documented and speed the discovery of new oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX / CasY CRISPR systems from metagenomic analysis of natural microbial communities.

[0150] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins / enzymes. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8- 29 - #593201Attomey Docket No. 00010.032.1801 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer- adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.

[0151] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes, and comprise Types I, III, and IV.

[0152] Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nucleases function primarily as DNA nucleases.

[0153] Type III CRISPR systems may be characterized by the presence of a central nuclease, known as CaslO, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre-crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0154] Type IV CRISPR-Cas systems possess an effector complex that comprises a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.

[0155] Class 2 CRISPR-Cas systems have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.

[0156] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g, Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature - 30 - #593201Attorney Docket No. 00010.032.1801 effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are DNA nucleases. Type II effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0157] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g, Cas 12) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are DNA nucleases.

[0158] Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., Casl2a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.

[0159] Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (e.g., Casl3) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to not need a tracrRNA for processing of pre-crRNA into crRNA. Similar to ty pe V systems, however, some Type VI systems (e.g. C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.

[0160] Because of their simpler architecture, class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease / genome editing applications.

[0161] One of the early adaptations of such a system for in vitro use involved (i) recombinantly-expressed, purified full-length Cas9 (e.g.. a class 2, Type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature ~42 nt crRNA bearing a ~20 nt 5' sequence complementary to the target DNA sequence to be cleaved followed by a 3' tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg2+. A later improved, engineered system involved the crRNA of (ii) joined to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.- 31 - #593201Attomey Docket No. 00010.032.1801

[0162] Such engineered systems can be adapted for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g., a class 2, Type II Cas enzy me) under a suitable mammalian promoter with a C -terminal nuclear localization sequence (e.g.. SV40 NLS) and a suitable polyadenylation signal (e.g, TK pA signal); and (ii) an ORF encoding an sgRNA (having a 5' sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence joined to a 3' tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g, the U6 promoter).Engineered EndonucleasesMG119

[0163] Described herein, in certain embodiments, are engineered endonucleases. In some embodiments, the endonuclease is a Class 2, Type V endonuclease. In some embodiments, the endonuclease is a MG119 endonuclease. In some embodiments, the endonuclease is a MG119-28 endonuclease. In some embodiments, the endonuclease is modified. In some embodiments, the endonuclease comprises point mutations. In some embodiments, the endonuclease comprises a single point mutation. In some embodiments, the endonuclease comprises two point mutations. In some embodiments, the endonuclease comprises three point mutations. In some embodiments, the endonuclease comprises four point mutations. In some embodiments, the endonuclease comprises five point mutations. In some embodiments, the endonuclease comprises six point mutations.

[0164] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 5-8, 104-191, and 194.

[0165] In some embodiments, the endonuclease is a MG119 endonuclease (i.e., SEQ ID NOs: 104-191 and 194). In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%. at least about 98%. or at least about 99% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease - 32 - #593201Attorney Docket No. 00010.032.1801 comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity' to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity' to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 104-191 and 194.

[0166] In some embodiments, the endonuclease comprises one or more amino acid modifications compared to a MG119 endonuclease described herein. In some embodiments, the endonuclease comprises one or more amino acid modifications as compared to SEQ ID NO: 194 (i.e., MG119-28). In some embodiments, the endonuclease comprises one or more amino acid modifications at aposition selected from the group consisting of: K93, E202, M204, E236, E236, E255, SI 74, and N329 as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236. E255, and N329 as compared to SEQ ID NO: 194.

[0167] In some embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E236R, E255K, S174R, andN329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K. E255K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification K93R as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification E202K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification M204K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification E236R as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification S174R as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modification N329E as compared to SEQ ID NO:- 33 - #593201Attomey Docket No. 00010.032.1801 194. In some embodiments, the endonuclease comprises the modifications K93R and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K.93R, M204K, and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E236K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K, E236K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K, E236K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K, M204K. and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K and E236K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E236K and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E236K, and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E236K and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, M204K, andN329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R and E202K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K. E236K, and E255K, as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E236K, E255K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R and M204K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E255K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K, M204K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E255K and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K.93R,- 34 - #593201Attorney Docket No. 00010.032.1801 E202K, and M204K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K and M204K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K, E236K, and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K, E255K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R, M204K, and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications M204K, E255K, and N329E as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications E202K, M204K, and E255K as compared to SEQ ID NO: 194. In some embodiments, the endonuclease comprises the modifications K93R. E202K, M204K. E236K, E255K, and N329E as compared to SEQ ID NO: 194.

[0168] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%. at least about 65%. at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises asequence having at least about 96% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least - 35 - #593201Attomey Docket No. 00010.032.1801 about 98% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 104-191.

[0169] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%. at least about 65%. at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises asequence having at least about 96% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 156-191.

[0170] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%. at least about 90%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at - 36 - #593201Attorney Docket No. 00010.032.1801 least about 80% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 110, 117, 119. 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 110, 117. 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135.

[0171] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises asequence having at least about 96% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 104-191. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 104-191. In - 37 - #593201Attorney Docket No. 00010.032.1801 some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 104-191.

[0172] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises asequence having at least about 96% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 156-191. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 156-191.

[0173] In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%. at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 104-191 and 194. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 110, 117. 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any - 38 - #593201Attomey Docket No. 00010.032.1801 one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 110, 117, 119, 124. 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 110, 117, 119, 124, 133, and 135.MG34-29

[0174] Described herein, in certain embodiments, are engineered systems comprising: (a) an endonuclease; and (b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0175] In some embodiments, the endonuclease comprises a RuvC-1 domain or a RuvC domain. In some embodiments, the endonuclease comprises an HNH domain. In some embodiments, the endonuclease comprises a RuvC domain and an HNH domain. In some embodiments, the endonuclease comprises an arginine rich region comprising an RRxRR motif or a domain with PF14239 homology. In some embodiments, the endonuclease comprises a REC domain. In some embodiments, the endonuclease comprises a BH (Bridge Helix) domain. In some embodiments, the endonuclease comprises a WED (wedge) domain. In some embodiments, the endonuclease comprises a PI (PAM interacting) domain. In some embodiments, the endonuclease is configured to be selective for a target adjacent motif (TAM) sequence comprising any one of ANGG, NARAA, ATGAAA, ATGA, or WTGG.

[0176] In some embodiments, the endonuclease is modified. In some embodiments, the endonuclease comprises point mutations. In some embodiments, the endonuclease comprises a single point mutation. In some embodiments, the endonuclease comprises two point mutations. In some embodiments, the endonuclease comprises three point mutations. In some embodiments, the endonuclease comprises four point mutations. In some embodiments, the endonuclease comprises five point mutations. In some embodiments, the endonuclease comprises six point mutations.- 39 - #593201Attomey Docket No. 00010.032.1801

[0177] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% identity to any one of SEQ ID NOs: 789-837.

[0178] In some embodiments, the endonuclease is a MG-34 endonuclease (i.e., SEQ ID NOs: 789-837). In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%. at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity' to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 789-837. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 789-837.

[0179] In some embodiments, the endonuclease comprises one or more amino acid modifications compared to a MG-34 endonuclease described herein. In some embodiments, the endonuclease comprises one or more amino acid modifications as compared to SEQ ID NO: 789 (i.e., MG34-29). In some embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: T33R, E49R, Q58R, N72R, S89R, E103R, Q132R, E139R, N153R, S164R, N195R, T217R, Q227R, Q228R, N232R, G247R, E267R, T277R, S281R, N307R, L348R, D357R, N401R. Q435R, A524R, D527R, E542N, D556Q, N564R, E575Q, E577R, A583R, E588R, D598K. I602T. I602E, N604R. E624Q. E624R, M627R. P638R. H654Q,- 40 - #593201Attorney Docket No. 00010.032.1801 N655K, E672R, L673R, G700R, T733R, S737T as compared to SEQ ID NO: 789 (i.e., MG34-29).

[0180] In some embodiments, the endonuclease comprises the modification T33R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E49R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification Q58R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N72R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification S89R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E103R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification Q132R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E139R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N153R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification S164R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N195R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification T217R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification Q227R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification Q228R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N232R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification G247R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E267R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification T277R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification S281R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N307R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification L348R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification D357R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N401R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification Q435R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification A524R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification D527R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E542N as compared to SEQ ID NO: 789. In some embodiments, the endonuclease - 41 - #593201Attomey Docket No. 00010.032.1801 comprises the modification D556Q as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N564R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E575Q as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E577R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification A583R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E588R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification D598K as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification I602T as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification I602E as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N604R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E624Q as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E624R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification M627R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification P638R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification H654Q as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification N655K as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification L673R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification G700R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification T733R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modification S737T as compared to SEQ ID NO: 789.

[0181] In some embodiments, the endonuclease comprises the modifications E139R, D357R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications D357R, E575Q, E577R. E624R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, E575Q, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R, E577R, E624R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R, E575Q, E624R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R,- 42 - #593201Attorney Docket No. 00010.032.1801 E575Q, E577R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R, E575Q, E577R, and E624R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications D357R, E575Q, E577R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications D357R, E575Q, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications D357R, E577R, and E672R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R, E575Q, E624R, E672R and A583R as compared to SEQ ID NO: 789. In some embodiments, the endonuclease comprises the modifications E139R, D357R, E575Q, E577R, E624R, and A583R as compared to SEQ ID NO: 789.

[0182] In some embodiments, the endonuclease comprises a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 70% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 75% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 80% sequence identity' to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 81% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 82% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 83% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 84% sequence identity' to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 85% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 86% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 87% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 88% sequence identity’ to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 89% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 90% sequence identity' to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 91% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 92% sequence identity - 43 - #593201Attomey Docket No. 00010.032.1801 to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 93% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 94% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 95% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 96% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 97% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 98% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence with at least 99% sequence identity to SEQ ID NO: 789. In some embodiments, the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 789.Base editing

[0183] Base editing is the conversion of one target base or base pair into another (e.g., A: T to G: C, C: G to T: A) without requiring the creation and repair of a double-strand break. The base editing may be achieved with the help of DNA and RNA base editors that allow the introduction of point mutations at specific sites, in either DNA or RNA. Generally, DNA base editors may comprise a fusion of a catalytically inactive nuclease and a catalytically active base-modification enzyme that acts on single-stranded DNAs (ssDNAs). RNA base editors may comprise of similar, RNA-specific enzymes. Base editing may increase the efficiency of gene modification, while reducing the off-target and random mutations in the DNA.

[0184] DNA base editors are engineered ribonucleoprotein complexes that act as tools for single base substitution in cells and organism. They may be created by fusing an engineered basemodification enzyme and a catalytically deficient CRISPR endonuclease variant that cannot cut dsDNA, but it is able to unfold the dsDNA in a protospacer adjacent motif (PAM) sequencedependent manner, such that a guide RNA can find its complementary target to indicate a ssDNA scission site. The guide RNA anneals to the complementary DNA, displacing a fragment of ssDNA and directing the CRISPR ‘scissors’ to the base modification site. The cellular repair machinery will repair the nicked non-edited strand using information from the complementary edited template.

[0185] So far, two types of DNA editors, cytosine base (CBEs) and adenine base editors (ABEs) have been developed. However, recent findings indicate that off-target modifications are present in DNA, and that many off-target modifications are also introduced into RNA by DNA base editors.- 44 - #593201Attorney Docket No. 00010.032.1801 MG Base Editors

[0186] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor; (b) an endonuclease configured to bind the base editor and is deficient in nuclease activity; and (c) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene. Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor encoded by a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 195-217; and (b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0187] In some embodiments, the base editor encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 70% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 75% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 195-217. In some embodiments, the base editor is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 195-217.

[0188] In certain embodiments, the engineered systems comprises: (a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 376-398; and (b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence- 45 - #593201Attomey Docket No. 00010.032.1801

[0189] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%. at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 95% identify to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 96% identify to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 97% identify to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 98% identify’ to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having at least about 99% identify to any one of SEQ ID NOs: 376-398. In some embodiments, the base editor comprises a sequence having 100% identify to any one of SEQ ID NOs: 376-398.

[0190] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor; (b) an endonuclease configured to bind the base editor and is deficient in nuclease activity; and (c) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene. Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor encoded by a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identify to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592; and (b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0191] In some embodiments, the base editor encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identify to any one of SEQ ID NOs: 276, 298. 218-275. 277-297. 299-303. 591, and 592. In some embodiments, the - 46 - #593201Attomey Docket No. 00010.032.1801 base editor is encoded by a sequence having at least about 70% identity to any one of SEQ ID NOs: 276, 298, 218-275. 277-297. 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 75% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 276. 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 90% identity’ to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592. In some embodiments, the base editor is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 276, 298, 218-275, 277-297, 299-303, 591, and 592.

[0192] In certain embodiments, the engineered systems comprises: (a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 457, 479. 399-456, 458-478, and 480-484; and (b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence

[0193] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%. at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and - 47 - #593201Attomey Docket No. 00010.032.1801 480-484. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 96% identity' to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484.

[0194] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor encoded by a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 304-364; and (b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0195] In some embodiments, the base editor encoded by a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 70% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 75% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 304-364. In some - 48 - #593201Attomey Docket No. 00010.032.1801 embodiments, the base editor is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 304-364. In some embodiments, the base editor is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 304-364.

[0196] In certain embodiments, the engineered systems comprises: (a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 485-545; and (b) an engineered guide polynucleotide configured to form a complex with an endonuclease of the base editor and comprises a spacer sequence that hybridizes to a target nucleic acid sequence

[0197] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 485-545. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 485-545.

[0198] Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor; (b) an endonuclease configured to bind the base editor and is deficient in nuclease activity;- 49 - #593201Attomey Docket No. 00010.032.1801 and (c) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to a target nucleic acid sequence. Described herein, in certain embodiments, are engineered systems comprising: (a) a base editor comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 399-545; and (b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

[0199] In some embodiments, the base editor comprises an amino acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 70% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 75% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 80% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 85% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 95% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 96% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 97% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 98% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having at least about 99% identity to any one of SEQ ID NOs: 399-545. In some embodiments, the base editor comprises an amino acid sequence having 100% identity to any one of SEQ ID NOs: 399-545.

[0200] In some embodiments, the base editor comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 376-398, 459. 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 376-398,- 50 - #593201Attorney Docket No. 00010.032.1801 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 376-398. 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 376-398, 459, 468. 476, 519, and 520-538. In some embodiments, the base editor comprises a sequence having 100% identity to any one of SEQ ID NOs: 376-398, 459, 468, 476, 519, and 520-538.

[0201] In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least 70%, at least 75%, at least 80%, at least 81%, at least 82%. at least 83%. at least 84%. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid sequence having at least about 70% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor comprises a sequence having at least about 75% identity' to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base is encoded by a nucleic acid having a sequence with at least about 80% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 85% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 90% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 95% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 96% identity to - 51 - #593201Attomey Docket No. 00010.032.1801 any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 97% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 98% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence with at least about 99% identity to any one of SEQ ID NOs: 195-217 and 591. In some embodiments, the base editor is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 195-217 and 591.

[0202] In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of the endonuclease. In some embodiments, the base editor comprises one or more nuclear localization sequences (NLSs) proximal to an N-or C-terminus of the endonuclease. The NLS can comprise any of the sequences in Table 1 below, or a combination thereof.

[0203] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 39-84, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%. at least about 40%. at least about 45%. at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS- 52 - #593201Attorney Docket No. 00010.032.1801 comprises a sequence having at least about 99% identity to SEQ ID NOs: 39-84. In some embodiments, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 39-84. Table 1. Example NLS SequencesSource NLS amino acid sequence SEQ ID NO: SV40 PKKKRKV 39 nucleoplasmin bipartite NLS KRPAATKKAGQAKKKK 40c-myc NLS PAAKRVKLD 41c-myc NLS RQRRNELKRSP 42hRNPAl M9NLS NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRN 43QGGYImportin-alpha IBB domain RMRIZFKNKGKDT AELRRRRVE V S VELRK AKKDE 44QILKRRNVMyoma T protein VSRKRPRP 45Myoma T protein PPKKARED 46p53 PQPKKKPL 47mouse c-abl IV SALIKKKKKMAP 48influenza virus NS1 DRLRR 49influenza virus N SI PKQKKRK 50Hepatitis virus delta antigen RKLKKKIKKL 51mouse Mxl protein REKKKFLKRR 52human poly(ADP-ribose) KRKGDE VDG VDE V AKKK SKK 53 polymerasesteroid hormone receptor (human) RKCLQAGMNLEARKTKK 54 glucocorticoid5' SV40 NLS MAPKKKRKVGGGGS 553' SV 40 1 NLS SGGAPKKKRKV 563' SV402 NLS SGGAPKKKRKV 573' SV403 NLS SGGAPKKKRKVSGGAPKKKRKVSGGAPKKKRKV 585' Nucleoplasmin NLS MKRPAATKKAGQAKKKKGGGGS 593' Nucleoplasmin 1 NLS SGGKRPAATKKAGQAKKKK 60SGGKRPAATKKAGQAKKKKSGGKRPAATKKAGQ3' Nucleoplasmin 2 NLSAKKKK 61 SGGKRPAATKKAGQAKKKKSGGKRPAATKKAGQ3' Nucleoplasmin 3 NLSAKKKKSGGKRPAATKKAGQAKKKK 625' c-Myc NLS MPA AKRVKLDGGGG S 633' c-Myc 1 NLS SGGPAAKRVKLD 643' c-Myc 2 NLS SGGPAAKRVKLDSGGPAAKRVKLD 65SGGPAAKRVKLDSGGPAAKRVKLDSGGPAAKRV3' c-Myc 3 NLSKLD 66Synthetic 5' Class 2-1 NLS MQ AAKRPRTTGGGG S 67Synthetic 3' Class 2-1 1 NLS SGGQAAKRPRTT 68- 53 - #593201Attorney Docket No. 00010.032.1801 Source NLS amino acid sequence SEQ ID NO: Synthetic 3' Class 2-1 2 NLS SGGQAAKRPRTTSGGQAAKRPRTT 69Synthetic 5' Class 2-2 NLS MRAAKRPRTTGGGG S 70Synthetic 3' Class 2-2 1 NLS SGGRAAKRPRTT 71Synthetic 3' Class 2-22 NLS SGGRAAKRPRTT SGGRA AKRPRTT 72Synthetic 5' Class 2-2 NLS MRAAKRPRTTGGGG S 73Synthetic 3' Class 2-2 1 NLS SGGRAAKRPRTT 74Synthetic 3' Class 2-22 NLS SGGRAAKRPRTT SGGRAAKRPRTT 75Synthetic 5' Class 3 NLS MAAAKRSWSMAFGGGGS 76Synthetic 3' Class 3 1 NLS MSGGAAAKRSWSMAF 77Synthetic 3' Class 32 NLS MSGGAAAKRSWSMAFMSGGAAAKRSWSMAF 78Synthetic 5' Class 4 NLS MRAAKRKYFAVGGGGS 79Synthetic 3' Class 4 1 NLS MSGGRAAKRKYFAV 80Synthetic 3' Class 42 NLS MSGGRAAKRKYFAVMSGGRAAKRKYFAV 815' HCVNLS MPKPQRKTKRGGGG S 823' HCV NLS SGGPPRKKRTVV 83 3' hRNPAl NLS SGGFGNYNNQSSNFGPMKGGNFGGRSSGPY 84Guide Polynucleotides

[0204] The systems and methods for restoring dystrophin protein expression, described herein, may comprise guide polynucleotides e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA for restoring protein expression of DMD. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0205] In some embodiments, the guide polynucleotide targeting DMD is encoded by a nucleic acid having a sequence of any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788 or a nucleic acid having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-23, 85-103. 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide comprises at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%. at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%. at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371. 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 80% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652,#593201Attorney Docket No. 00010.032.1801 and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 85% identity to any one of SEQ ID NOs: 9-23. 85-103, 192-193, 365-368. 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 90% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 95% identity to any one of SEQ ID NOs: 9-23. 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 97% identity to any one of SEQ ID NOs: 9-23. 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 98% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having at least about 99% identity to any one ofSEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide is encoded by a nucleic acid having a sequence having 100% identity to any one of SEQ ID NOs: 9-23, 85-103. 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788.

[0206] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the DMD gene (e g., SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 9-23, 85-103, 192-193. 365-368, 369-371, 372-374, 375, 593-652, and 786-788 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 9-23. 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one ofSEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID - 55 - #593201Attomey Docket No. 00010.032.1801 NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 9-23. 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193. 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 9-23, 85-103, 192-193, 365-368, 369-371, 372-374, 375, 593-652, and 786-788.

[0207] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the DMD gene (e g., SEQ ID NOs: 24-38, 575, or 578). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 24-38, 575, or 578 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 24-38, 575. or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polymucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 24-38. 575. or 578. In - 56 - #593201Attorney Docket No. 00010.032.1801 some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 24-38, 575, or 578. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 24-38, 575, or 578.MG Systems

[0208] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NOs: 9-23. 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786- - 57 - #593201Attorney Docket No. 00010.032.1801 788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NOs: 9-23. 85-103, 593-652, and 786-788.

[0209] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 9-23. 593-595, 596-606. 607-613, 614, 615-621. and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613. 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614. 615-621. and - 58 - #593201Attomey Docket No. 00010.032.1801 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 1-4, 582-588, and 886-891 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NOs: 9-23, 593-595, 596-606, 607-613, 614, 615-621, and 622-625.MG 119-28 Targeting of Dystrophin

[0210] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 192. 193, 85-92, 101-103, 626-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 632, 192, 193, 85-92. 94. 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 632. 192, 193, 85-92. 94. 101-103, 626-631, 633-636, 643-652, and 786-788. In some - 59 - #593201Attomey Docket No. 00010.032.1801 embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 194 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 194 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 194 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788.

[0211] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system - 60 - #593201Attorney Docket No. 00010.032.1801 comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 632, 192. 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 632. 192, 193. 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631. 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 98% identity’ to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 632, 192, 193. 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 104-155 and 156-191 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%. 97%, 98%,- 61 - #593201Attomey Docket No. 00010.032.1801 99% or 100% sequence identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788.

[0212] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 632, 192. 193, 85-92, 94, 101-103, 626-631, 633-636,- 62 - #593201Attorney Docket No. 00010.032.1801 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, and 183 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 632, 192. 193. 85-92. 94. 101-103, 626-631, 633-636, 643-652, and 786-788. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158. 166, 172, 179, and 183 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity to any one of SEQ ID NO: 632, 192, 193, 85-92, 94, 101-103, 626-631, 633-636, 643-652, and 786-788.MG34-29 Targeting of Dystrophin

[0213] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 789-837 and the engineered polynucleotide comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. at least about 95%, at least about 96%. at least about 97%. at least about 98%, or at least about 99% identity' to any one of SEQ ID NOs: 365-368 and 375.

[0214] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 789-837 and - 63 - #593201Attomey Docket No. 00010.032.1801 an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 85% identity’ to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 96% identity’ to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having at least about 99% identity’ to any one of SEQ ID NOs: 365-368 and 375. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having 100% identity to any one of SEQ ID NOs: 789-837 and an engineered polynucleotide having 100% identity to any one of SEQ ID NOs: 365-368 and 375.ABEs Targeting of Dystrophin

[0215] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID - 64 - #593201Attorney Docket No. 00010.032.1801 NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 376-398 and the engineered polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103. 593-652. and 786-788.

[0216] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least - 65 - #593201Attomey Docket No. 00010.032.1801 about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 457. 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 9-23, 85-103. 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 457. 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 457, 479, 399-456. 458-478. and 480-484 and the engineered polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any - 66 - #593201Attorney Docket No. 00010.032.1801 one of SEQ ID NOs: 457, 479, 399-456, 458-478, and 480-484 and the engineered polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788.

[0217] In some embodiments, the endonuclease comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 9-23. 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652. and 786-788. In some embodiments,- 67 - #593201Attorney Docket No. 00010.032.1801 the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 9-23. 85-103, 593-652. and 786-788. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 485-545 and the engineered polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 9-23, 85-103, 593-652, and 786-788.

[0218] In some embodiments, the present disclosure provides gRNAs that are position the conserved splice acceptor adenines of exon 45. In some embodiments, the present disclosure provides gRNAs that are position the conserved splice acceptor adenines of exon 51.

[0219] In some embodiments, the nucleases and ABEs disclosed herein target the DMD gene region in which deletions frequently disrupt the dystrophin reading frame to produce a functional dystrophin protein.

[0220] In some embodiments, the guide polynucleotide is configured to form a complex with the endonuclease. In some embodiments, the guide polynucleotide binds to the endonuclease to form a complex. In some embodiments, the guide polynucleotide binds (e.g., non-covalently through electrostatic interactions or hydrogen bonds) to the endonuclease to form a complex. In some embodiments, the guide polynucleotide is fused to the endonuclease to form a complex.

[0221] In some embodiments, the guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0222] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’-4’ biradical bridged) nucleosides. Examples of 2’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl- RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, the modification at the - 68 - #593201Attomey Docket No. 00010.032.1801 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).

[0223] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methyl. In some embodiments, the modified sugar comprises a 2’ -fluoro. In some embodiments, the modified sugar comprises a 2’-O-methoxy ethyl group. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2‘-fluoro).

[0224] In some embodiments, the guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter-nucleoside linkers and greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified inter-nucleoside linkers (e.g, phosphorothioate inter-nucleoside linkage) and 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).

[0225] In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0226] In some embodiments, the guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the guide polynucleotide is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the guide polynucleotide is about 30 to about 40, about 30 to about 50. about 30 to about 60, about 30 to about 70. about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to - 69 - #593201Attomey Docket No. 00010.032.1801 about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90. about 50 to about 100. about 50 to about 120. about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length.Methods of Use

[0227] Provided herein are methods for restoring expression of dystrophin protein using the engineered nuclease systems or base editing systems described herein. Methods for restoring expression of dystrophin protein comprise contacting a target nucleic acid sequence with an engineered nuclease system or a base editing system disclosed herein and an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.Delivery and Vectors

[0228] Disclosed herein, in some embodiments, are nucleic acid sequences encoding an engineered nuclease system comprising an endonuclease and an engineered guide polynucleotide or components of the engineered nuclease system.

[0229] In some embodiments, the nucleic acid encoding the endonuclease system or components thereof is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.

[0230] In some embodiments, the nucleic acid encoding the endonuclease system or components thereof is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl -derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC. pSF-OXB20-BetaGal, pSF- - 70 - #593201Attorney Docket No. 00010.032.1801 OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0231] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac. araBad, trp, Ptac, p5, p19. p40, Synapsin. CaMKII, GRK1, and derivatives thereof. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0232] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.

[0233] 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-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3. AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8. AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8

[0234] 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 - 71 - #593201Attomey Docket No. 00010.032.1801 thereof. In some embodiments, the virus is AAV 10 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 AAV 14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the 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- 72 - #593201Attorney Docket No. 00010.032.1801 AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0235] 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.

[0236] In some embodiments, the nucleic acid encoding the engineered nuclease system or components thereof is delivered by a non-nucleic acid-based delivery system (e.g, a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0237] In some embodiments, the engineered nuclease system or components thereof is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the engineered nuclease system or components thereof is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the engineered nuclease system or components thereof. For example, a cell is transduced (e.g., with a virus encoding the engineered nuclease system or components thereof), or transfected (e.g, with a plasmid encoding the engineered nuclease system or components thereof) with a nucleic acid that encodes the engineered nuclease system or components thereof, or the translated the engineered nuclease system or components thereof. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing the engineered nuclease system or components thereof or containing the engineered nuclease system or components thereof are transduced or transfected with one or more gRNA molecules, for example, when the engineered nuclease system or components thereof comprises a CRISPR nuclease. In some embodiments, a plasmid expressing the engineered nuclease system or components thereof is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g. piggybac) and viral transduction (for example lentivirus or AAV) or other - 73 - #593201Attomey Docket No. 00010.032.1801 methods known to those of skill in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0238] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac). microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g.. U. S. Pat. Nos.5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91 / 17424 and WO 91 / 16024. In some embodiments, the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0239] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0240] In some embodiments, the present disclosure provides a cell comprising a vector or a nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or parts thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.Cells

[0241] Described herein, in certain embodiments, is a cell comprising the systems or vectors described herein.

[0242] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NS0), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, aHuh7 cell. aHepG2 cell, aK562 cell. aN2acell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia rit cell, a Drosophila melanogaster cell, a S2 cell, or aHeliothis virescens cell), ayeast cell (e.g., aSaccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., aE. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria - 74 - #593201Attorney Docket No. 00010.032.1801 cell, or an archaea cell). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.

[0243] In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5. Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa. Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or derivative thereof.Kits

[0244] In some embodiments, this disclosure provides kits comprising one or more nucleic acid constructs encoding the various components of the engineered nuclease system. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the engineered nuclease system components.

[0245] In some embodiments, the engineered nuclease system disclosed herein is assembled into a pharmaceutical, diagnostic, or research kit to facilitate its use in therapeutic, diagnostic, or research applications. A kit may include one or more containers housing any of the vectors disclosed herein and instructions for use.

[0246] The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit. where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions, in some embodiments, are in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.- 75 - #593201Attomey Docket No. 00010.032.1801EXAMPLES

[0247] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.Example 1 - Guide screen for human DMD gene using mRNA transfection of human skeletal muscle cells (SkMs)

[0248] Guide RNAs for nucleases MG3-6 (SEQ ID NO: 582), MG3-6 / 3-4 (SEQ ID NO: 583), MG 3-6 / 3-8 (SEQ ID NO: 584), MG21-1 (SEQ ID NO: 585), MG29-1 (SEQ ID NO: 586), MG71-43 (SEQ ID NO: 587), MG119-28 (SEQ ID NO: 588), as well as base editors ABE-103 (SEQ ID NO: 589), ABE-106 (SEQ ID NO: 590), ABE-180 (SEQ ID NO: 591, identical to SEQ ID NO: 298), and ABE-158 (SEQ ID NO: 592, identical to SEQ ID NO: 276) targeting the human Dystrophin (DMD) gene were identified by searching for the relevant PAM sequence for each nuclease, as shown in Table 2. The guides were designed and selected to 1) disrupt the 5' splice acceptors of exons 44, 45, 50, 51, and 53 to promote exon skipping during dystrophin mRNA processing, and 2) create small insertions or deletions at the 5' end of exons 45, 46, 48, 51, 52, 53, and 56. and the 3‘ end of each exon from 43 to 51, to shift the reading frame of the out-of-frame exons of DMD patients with exon deletions into the correct open reading frame. A total of 388 guide RNAs for MG3-6, MG 3-6 / 3-4, MG 3-673-8, MG21-1, MG29-1, MG71-43, MG119-28, ABE-103, ABE-106, ABE- 180, and ABE-158 systems were chemically synthesized as single guide RNAs with AltRl / AltR2 end-modifications (IDT). These guides were screened in human skeletal muscle (SkM) cells to evaluate their editing efficiency, as well as their ability to induce the intended exon skipping and / or reframing of the DMD gene.

[0249] Out of 388 guide RNAs screened, 158 demonstrated >2% editing efficiency at the DMD locus across various guide RNA screening experiments. Next-generation sequencing (NGS) of edited loci was used to analyze the INDEL profiles generated by these 158 guides. The resulting data enabled prediction of whether the edits disrupted splice acceptor sites — potentially inducing exon skipping — or introduced frameshifts within coding regions that would restore the DMD reading frame in patients with out-of-frame deletions. From this analysis, 49 guide RNAs were identified as capable of inducing productive exon skipping and / or reframing (Table 2). These guides achieved productive editing rates ranging from 5% to 27%, confirming the functionality of - 76 - #593201Attorney Docket No. 00010.032.1801 the tested nuclease and base editor systems in enabling targeted correction of DMD mutations (FIG. 1). The full sequences of these productive guide RNAs are provided as SEQ ID NOs: 593 to SEQ ID NO: 652. The PAM and spacer sequences are indicated in Table 3.

[0250] Importantly, guideRNA candidates from MG3-6, MG3-6 / 3-4, MG3-6 / 3-8, MG21-1, MG119-28, ABE- 180, and ABE-158 systems offer a significant advantage due to their small size, allowing for packaging into single all-in-one AAV vectors. Systems such as MG21-1, ABE-158, and MG119-28 provide additional space within the AAV packaging limit, enabling incorporation of regulatory elements such as switchable nuclease expression cassettes, tissue-specific promoters, and multiple guide RNA expression units. The sizes of these components and their compatibility with AAV packaging are summarized in Table 4.

[0251] SkM Transfection Protocol

[0252] SkM cells were transfected with mRNA encoding various nucleases or base editors, along with guide RNAs (gRNAs) at a 1:5 mRNA:gRNA molar ratio. For all transfections, the appropriate amount of mRNA and guide RNA were mixed in media and combined with Lipofectamine Messenger Max in a master mix. The mixtures were vortexed briefly, incubated for at least 5-10 minutes at room temperature, and added directly to SkM cells. Editing reagent volumes and cell plating formats were adjusted based on the experiment, as detailed below.

[0253] 24-well plate format transfections:

[0254] For hD368-Gl (SEQ ID NO: 597), hD368-G2 (SEQ ID NO: 598), hD364-B7 (SEQ ID NO: 595), hD368-E2 (SEQ ID NO: 596), and hD29-D6 (SEQ ID NO: 615), SkM cells were trypsinized and seeded into 24-well plates at 75,000 viable cells per well. Each transfection included 2400 ng of nuclease mRNA and 353 ng of guide RNA in a total reaction volume of 50 pL. A master mix of 46.4 pL OptiMEM and 3.6 pL Messenger Max per well was prepared. After reagent incubation, the complexes were added to the cells in 500 pL of pre-equilibrated media. Five days post-transfection, cells were harvested using Cell and Tissue DNA Extraction Buffer and genomic DNA was purified via magnetic bead purification using a genomic DNA isolation kit.

[0255] 96-well plate format transfections

[0256] For all other guide RNAs. SkM cells cultured for fewer than 10 days in Mesenchymal Stem Cell Basal Medium with Primary Skeletal Cell Muscle Growth Kit were seeded at 12,500 viable cells per well in TC-treated 96-well plates, with media adjusted to 200 pL per well.

[0257] For nucleases, 200 ng of mRNA and 29-83 ng of guide RNA were used per transfection. A 5.0 pL RNA reaction was prepared and combined with 4.7 pL OptiMEM and 0.3 pL Messenger Max per well. For base editors, 400 ng of mRNA and corresponding guide RNA were used. The - 77 - #593201Attorney Docket No. 00010.032.1801 RNA was combined in a 5.0 pL reaction volume and mixed with 4.4 pL OptiMEM and 0.6 pL Messenger Max. Transfection complexes were incubated for 10 minutes at room temperature before being added to the cells.

[0258] Post-transfection processing

[0259] For low-dose nuclease screens, cells were harvested three days post-transfection. Media was aspirated, and 50 pL of QuickExtract was added to each well. Plates were incubated at 65 °C for 6 minutes at 300 rpm, followed by 98 °C for 3 minutes. Target regions of the DMD gene were PCR-amplified using a DNA Polymerase with gene-specific primers and adapters for nextgeneration sequencing (NGS). A second PCR using barcoded primers was performed for 10 cycles, and amplicons were analyzed. Data were processed using a custom script to quantify insertion / deletion (INDEL) frequencies at the target sites.Table 2. Guide screens with productive exon skipping and refraining guides for top MG systems within the AAV packaging size limit.Number of Number of guides with Nucleases / Consensus PAM guides Number of guides PRODUCTIVE editing System sequence screened with editing (>2%) (>5%) MG3-6 5' NNRGRYY 3‘ 31 13 4MG3-6 / 3-4 5' NNRAAA 3’ 58 25 3MG3-6 / 3-8 5’ NNRGNNY 3’ 57 37 11MG21-1 5' NNRRRNAR 3' 62 21 7MG29-1 5' TTTN 3' 59 39 7MG71-43 5' NNNRCY 3' 43 6 1MG119-28 5' TTR 3' 47 17 11ABE- 103 5' NNRNYHY 3' 10 NA 1ABE- 106 5' NNNMHTY 3' 10 NA 1ABE- 180 5' NRC 3' 7 NA 2ABE- 158 5' NGG 3' 4 NA 1Total 388 158 49Table 3. PAM and spacer target sequences of productive editing guide RNAs at human DMD gene. _ _ _Human guide ID PAM Spacer Target SequencehD368-Gl GGAGTTC TGCCGCTGCCCAATGCCATCCT (SEQ ID NO: 24) hD368_PlA5 CAAGGGT TAACAAAATGTACAAGGACCGA (SEQ ID NO: 25) hD21-l_PlB9 GTAGGTAA AAATGTACAAGGACCGACAAGG (SEQ ID NO: 26) hD21-l_PlF12 CAGGACAG CAATTTCACCTTGGAGGTCCTA (SEQ ID NO: 27)- 78 - #593201Attomey Docket No. 00010.032.1801 Human guide ID PAM Spacer Target SequencehD368-G2 ACAGTCT CACAGGTTGTGTCACCAGAGTA (SEQ ID NO: 28) hD364-B7 CTAAAA AGAGTAACAGTCTGAGTAGGAG (SEQ ID NO: 29) hD2I-l_PlD9 ACAGAAAA GAGGTCTGCAAACAGCTGTCAG (SEQ ID NO: 30) hD368_P1E5 TAAGTCT AAATACAAATGGTATCTTAAGG (SEQ ID NO: 31) hD36_PlFl CTAGGTC CCAATAGTGGTCAGTCCAGGAG (SEQ ID NO: 32) hD368_P1B8 TTGGAAC CTTACAGGCAACAATGCAGGAT (SEQ ID NO: 33) hD21-1_P1F11 CTGAGAAA TTTCAGGTTTCCAGAGCTTTAC (SEQ ID NO: 34) hD364-F5 CAAAAA TCCTGGAGTTCCTGTAAGATAC (SEQ ID NO: 35) hD368-E2 GAAGATC TTTTCTGTTAAAGAGGAAGTTA (SEQ ID NO: 36) hD21-lJ’lC9 TAAGGTAA ATGCTAAATACAAATGGTATCT (SEQ ID NO: 37) hD21-1_P1D11 AAAGGAAA CAGGTAAAGCTCTGGAAACCTG (SEQ ID NO: 38) hD364_B8 AGGAAA GTGACACAACCTGTGGTTACTA (SEQ ID NO: 838) hD364 B9 ATGAAA CAGAACCGGAGGCAACAGTTGA (SEQ ID NO: 839) hD368 P1F5 GTAGGGC AGCTGTCAGACAGAAAAAAGAG (SEQ ID NO: 840) hD368 Fl CTGGAGT TTTGCCGCTGCCCAATGCCATC (SEQ ID NO: 841) hD368_A2 CAGGAAC GTTTTGCCTTTTTGGTATCTTA (SEQ ID NO: 842) hD368_E3 CTAGAAT CACTGATTCTGAATTCTTTCAA (SEQ ID NO: 843) hD368_H3 GTGGGAT TAGTTGAAAGAATTCAGAATCA (SEQ ID NO: 844) hD211_B2 TTGGGCAG TTACAGGAACTCCAGGATGGCA (SEQ ID NO: 845) hD7143_P3F5 GGAGCT CACCAGAGTAACAGTCTGAGTA (SEQ ID NO: 846) hD29-D6 TTTG CCTTTTTGGTATCTTACAGG (SEQ ID NO: 847) hD29-P3C8 TTTC CAATGGGAAAAAGTTAACAA (SEQ ID NO: 848) hD29-P3C9 TTTC TCCTTGTTTCTCAGGTAAAG (SEQ ID NO: 849) hD29-P3E9 TTTC CTTTCAGGTTTCCAGAGCTT (SEQ ID NO: 850) hD29_B8 TTTT AGCTCCTACTCAGACTGTTA (SEQ ID NO: 851) hD29_C8 TTTA GCTCCTACTCAGACTGTTAC (SEQ ID NO: 852) hD29 A9 TTTC CTTTTATTCTAGTTGAAAGA (SEQ ID NO: 853) hD36 P1D1 AAAGGTT AGCAGACAAATCTCCAGTGGAT (SEQ ID NO: 854) hD36_A2 CTAGGTC CCAATAGTGGTCAGTCCAGGAG (SEQ ID NO: 855) hD36 B2 TCAGGCT AGTGGTCAGTCCAGGAGCTAGG (SEQ ID NO: 856) hD36_H2 ACAGGTT GATGGCAGTTTCCTTAGTAACC (SEQ ID NO: 857) mhD119-28L-P4Hlb TTG GGAACATGCTAAATACAAA (SEQ ID NO: 858) hD119-28L-P4C5b TTA CAGGCAACAATGCAGGATT (SEQ ID NO: 859) hD119-28L-C9b TTA CCGCCTTCCACTCAGAGCT (SEQ ID NO: 860)- 79 - #593201Attomey Docket No. 00010.032.1801 Human guide ID PAM Spacer Target SequencehD119-28L-P4G1b TTA CCTACCCTTGTCGGTCCTT (SEQ ID NO: 861) mhD119-28L-P4A2b TTA CCTTAAGATACCATTTGTA (SEQ ID NO: 862) hD119-28L-P4B4b TTG TGTCACCAGAGTAACAGTC (SEQ ID NO: 863) hD119-28L-A9b TTG CCGCTGCCCAATGCCATCC (SEQ ID NO: 864) hD119-28L-g05b TCA GGTTTCCAGAGCTTTACCT (SEQ ID NO: 865) mhD119-28L-B9b TTA CAGGAACTCCAGGATGGCA (SEQ ID NO: 866) hD119-28L-P4C4b TTG AGGATATCAACGAGATGAT (SEQ ID NO: 867) hD119-28L-g09b TCA ACGAGATGATCATCAAGCA (SEQ ID NO: 868) hDABE103_Ex45_g4 ATGGCAT GGTATCTTACAGGAACTCCAGG (SEQ ID NO:869) hDABE106__Ex45__g5 TGGCATT GTATCTTACAGGAACTCCAGGA (SEQ ID NO: 870) hDABE180_Ex45_gl GGC GGTATCTTACAGGAACTCCAGGAT (SEQ ID NO:871)hDABE180 Ex45 g2 GGC TACAGGAACTCCAGGATGGCATTG (SEQ ID NO:872)hDABE180_Ex51_gl TAC TATTTTAGCTCCTACTCAGACTGT (SEQ ID NO:873)hDABE158_Ex45-g12 AGG TTTGGTATCTTACAGGAACTCC (SEQ ID NO: 874)hDABE 158_Ex45- TGG GTATCTTACAGGAACTCCAGGA (SEQ ID NO: 875) gl3hD119-28L-P4B5b TTG TTCTTACAGGCAACAATGC (SEQ ID NO: 876) hD119-28L-P4G2b TTG TTCTTCTAGCCTGGAGAAA (SEQ ID NO: 877) hD119-28L-P4D2b TTG GTATCTTACAGGAACTCCA (SEQ ID NO: 878) hD119-28L-P4G3b TTA AAGCAAAAAGTTCCCTACC (SEQ ID NO: 879) hD119-28L-P4F2b TTA GATCTGTCGCCCTACCTCT (SEQ ID NO: 880) hD119-28L-H8b TTA CCTGCAGGCGATTTGACAG (SEQ ID NO: 881) hD119-28L-g04b TCA AGCAGACAAATCTCCAGTG (SEQ ID NO: 882) mDl 19-28L-A9b TTG CCGCTGCCCAATGCCATCC (SEQ ID NO: 883) mDl 19-28L-P4B4b TTG TGTCACCAGAGTAACAGTC (SEQ ID NO: 884) mDl 19-28L-g05b TCA GGTTTCCAGAGCTTTACCT (SEQ ID NO: 885)Table 4. Sizes of MG Type II editor system components for AAV vectors.Editor Guide RNAEditor promoter Guide Poly A size promoter, ITRs, Total size Editor (bp) (EFla) (bp) RNA (bp) (bp) MCS (bp) (bp)ABE- 106 4803 280 110 49 646 5888- 80 - #593201Attorney Docket No. 00010.032.1801 (SEQ IDNO: 590)ABE- 103(SEQ IDNO: 589) 4782 280 110 49 646 5867 MG71-43(SEQ IDNO: 587) 4532 280 105 49 646 5612 MG29-1(SEQ IDNO: 586) 3939 280 70 49 646 4984 ABE-180(SEQ IDNO: 591) 3538 280 183 49 646 4696 MG3-6(SEQ IDNO: 582),MG3-6 / 3-4(SEQ IDNO: 583),MG3-6 / 3-8(SEQ IDNO: 584) 3501 280 110 49 646 4586 MG21-1(SEQ IDNO: 585) 3396 280 95 49 646 4466 ABE-158(SEQ IDNO: 592) 2971 280 143 49 646 4089 MG119-28(SEQ IDNO: 588) 1560 280 172 232 646 2890 General MethodsMyocyte transfection method and differentiation to myotubes

[0260] One day prior to transfection, frozen myocytes were revived in DMEM-high glucose with 10% KOSR, 1% GlutaMAX, 1% ITS-G, 1% N2-supplement, 10μM ROCK inhibitor and 1μg / μl doxycycline. Cells were counted, and 230,000 viable cells were added to each well in a TC-treated 24 well plate coated with a basement membrane matrix. Additional pre-equilibrated media was added to each well to bring the total volume to 500 LLL.

[0261] On the day of transfection, 0.6 ul of Lipofectamine messenger max solution per reaction was added to OptiMEM to make up 30 uL volume per reaction, vortexed, and incubated for at least 5 minutes at room temperature. In separate tubes, 600 ng of the mRNA and indicated pmole - 81 - #593201Attomey Docket No. 00010.032.1801 of guide RNA for each system were combined with OptiMEM media to make a total 30 uL reaction volume. After a brief vortex, the appropriate volume of messenger max solution was added to each RNA solution, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 minutes at room temperature and added directly to the myocytes. The cells were allowed to differentiate to multinucleated myotubes by culturing the cells in DMEM-high glucose with 10% KOSR, 1% GlutaMAX, 1% ITS-G, 1% N2-supplement for 10 days post-transfectionDNA, RNA and protein extraction to evaluate editing and expression restoration in disease model cells post-editing

[0262] For gDNA and RNA extraction, the media was aspirated from each well of myocytes and cells were lysed in 450 pL of RLT buffer from a RNA purification kit. 100 pL was transferred to 96-well 1 mL deep well plate for DNA purification. The remaining 350 uL of cell lysis was transferred to gDNA eliminator columns and processed using a kit. The region of the DMD gene targeted by the editing system was PCR amplified from gDNA samples for 29 cycles with Q5 high fidelity DNA polymerase using gene-specific primers with adapters complementary to the barcoded primers for next generation sequencing (NGS) (SEQ ID NOs: 557-574 and 679-784).The product of this first PCR reaction was PCR amplified for 10 cycles using the barcoded primers for NGS. The resulting product was subjected to NGS and the results were processed using a custom script to generate the percentage of sequencing reads that contain insertions or deletions (INDELS) at the targeted site in the DMD gene. The INDEL profiles obtained from the NGS sequencing were used to determine the percentage of INDELS that result in a splice acceptor disruption which potentially may result in exon skipping or in a coding region frameshift predicted to result in productive reframing of the exon. RNA was reversely transcribed using a reaction master mix. mRNA restoration was determined using a custom primer probe set with homology to dystrophin as well as a second primer probe set with homology to GAPDH, which was used as a reference gene. Using a digital droplet generator, thermocycler, and droplet reader, individual dropletized PCR reactions were analyzed for the number of DMD amplicons normalized to the number of GAPDH amplicons. DMD mRNA restoration was determined by comparing the ratio of DMD / GAPDH in treated samples versus the ratio of DMD / GAPDH in control samples. The sequences for ddPCR primer probe sets are listed below:DMD: forward primer- ACTCTACCAGGAGCCCAGAG (SEQ ID NO: 660);reverse primer- TCCGTGGCCTCTTGAAGTTC (SEQ ID NO: 661); probe-CTGCACTCCGCTGACTGGCA (SEQ ID NO: 662)GAPDH: forward primer- GCACCACCAACTGCTTAG (SEQ ID NO: 663);reverse primer- CCATCCACAGTCTTCTGGG (SEQ ID NO: 664); probe-CATGACCACAGTCCATGCCATC (SEQ ID NO: 665)- 82 - #593201Attorney Docket No. 00010.032.1801

[0263] For protein extraction, the media was aspirated from each well of myocytes and cells were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktail for protein extraction. Of the cell lysate, 5 pg was loaded onto a 3-8% Tris-Acetate gel and transferred to the nitrocellulose membrane. Blots were blocked with 5% milk, then probed with antibodies against the dystrophin (1:100) and vinculin (1:4000), detected using secondary antibody HRP-anti-mouse IgG (1:10000) & HRP-anti-rabbit IgG (1:5000) respectively. Blots were developed using a chemiluminescent substrate for chemiluminescence detection. The dystrophin signal from samples was normalized against vinculin and the relative protein expression was determined by using WT input as reference.

[0264] For immunofluorescence, samples were fixed with 4% paraformaldehyde, permeabilized using 0.3% Triton X-100, and blocked with 10% goat serum in the permeabilization buffer. Primary antibody incubation was performed overnight at 4°C using a multiplexed mix of rabbit anti-dystrophin (1:250) and mouse anti-Myosin 4 (1:200) antibodies. Secondary antibodies, Alexa Fluor 488-conjugated donkey anti-rabbit and Alexa Fluor 647-conjugated goat anti-mouse (each at 1: 1000), were applied at 37°C in the dark for 1 hour. Nuclear staining was conducted with DAPI (1: 1000). followed by water wash and imaging via fluorescence microscopy.Example 2 - Guide screen for human DMD gene using mRNA transfection of human AEx44 or AEx52 myocytes

[0265] Guide RNAs hD368-Gl (SEQ ID NO: 9 or SEQ ID NO: 597), hD368_P1A5 (SEQ ID NO: 10 or SEQ ID NO: 599), hD21-1_P1B9 (SEQ ID NO: 11 or SEQ ID NO: 607) were transfected into human myocytes with a deletion of exon 44 of the DMD gene (“AEx44 myocytes”), and guide RNAs hD368-G2 (SEQ ID NO: 13 or SEQ ID NO: 598) and hD364-B7 (SEQ ID NO: 14 or SEQ ID NO: 595) were transfected into human AEx52 myocytes.

[0266] Myocyte Transfection Protocol

[0267] DMD deletion myocytes were transfected with MG3-6 / 3-4 or MG 3-6Z3-8 or MG21-1 nuclease mRNA plus guide RNA at 1:5 mRNA:gRNA molar ratio (1200 ng nuclease mRNA plus 175 ng guide RNA for MG3-6 / 3-4 and MG3-6 / 3-8 systems and 193 ng guide RNA for MG21-1 system) as follows.

[0268] One day prior to transfection, frozen myocytes were revived in DMEM-high glucose with 10% KOSR, 1% GlutaMAX, 1% ITS-G, 1% N2-supplement, 10 pM ROCK inhibitor and Ipg / pL doxycycline. Cells were counted, and 210,000 viable cells were added to each well in a TC-treated 24 well plate coated with Geltrex™. Additional pre-equilibrated media was added to each well to bring the total volume to 500 pL.- 83 - #593201Attomey Docket No. 00010.032.1801

[0269] On the day of transfection, 28.2 pL of OptiMEM media and 1.8 ul of Lipofectamine Messenger Max Solution per reaction were combined in a master mix solution, vortexed, and incubated for at least 5 mins at room temperature. In separate tubes. 1200 ng of the mRNA and 175 ng guide RNA for MG3-6 / 3-4 and MG3-6 / 3-8 systems or 193 ng guide RNA for MG21-1 system were combined with OptiMEM media to make a total 30 uL reaction volume. After a brief vortex, the appropriate volume of MessengerMax solution was added to each RNA solution, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 mins at room temperature and added directly to the myocytes.

[0270] Four days post transfection, the media was aspirated from each well of myocytes and replaced with extraction buffer. Cells were scraped and transferred to a 96-well plate and genomic DNA was purified by automated magnetic bead purification.

[0271] The region of the DMD gene targeted by the editing system was PCR amplified with a polymerase, such as Q5 high fidelity DNA polymerase, using gene-specific primers with adapters complementary to the barcoded primers for next generation sequencing (NGS) (SEQ ID NOs: 557-574). The product of this first PCR reaction was PCR amplified for 10 cycles using the barcoded primers for NGS. The resulting product was subjected to NGS and the results were processed using a custom script to generate the percentage of sequencing reads that contain insertions or deletions (INDELS) at the targeted site in the DMD gene.

[0272] The results indicate that all guides edited the DMD gene in SkM cells in the 30% to 70% range (Table 5, column 6). The INDEL profiles obtained from the NGS sequencing were used to determine the percentage of INDELS that result in a splice acceptor disruption which potentially may result in exon skipping or in a coding region frameshift predicted to result in productive reframing of the exon. Productive editing % data (Table 5, column 7 and 9) demonstrate that the MG3-6 / 3-4, MG3-6 / 3-8, MG 3-6, and MG21-1 nuclease systems with an appropriate single guide RNA have utility in skipping and / or refraining of selected exons of the DMD gene. The productive editing plotted in FIG. 1 is predicted to result in restoration of dystrophin transcript levels and protein in relevant disease model cells as indicated in Table 5, column 5. Individual guide RNAs along with their respective nuclease will be packaged in AAV vectors with editor and guide RNA promoters and other vector backbone elements using in-house optimized conditions for testing as AAV in disease model cells. The sizes for these components are indicated in Table 4.- 84 - #593201Attorney Docket No. 00010.032.1801 Table 5. Productive editing of guide RNAs at human DMD gene delivered by mRNA transfection.Human guide Target 5' Productive Applicable In WT human In AEx44 or AEx52 ID exon or edits deletion skeletal muscle cells myocytes3' modelEnd Indel% Productive Indel% Productive editing% editing% for the for the deletion deletion model model indicated indicated in bold in bold hD368-Gl 45 5 Exon 44 46 11 29 6.9 (SEQ ID NO: reframing597) and exonskippinghD368_PlA5 43 3 Exon 44 45.5 12.4 29 11.2 (SEQ ID NO: reframing599)hD21-l_PlB9 43 3 Exon 44 69 19.6 41 15.4 (SEQ ID NO: reframing607)hD21-l_PlF12 56 5 Exon 51-55 42.7 26.3 Not Not tested (SEQ ID NO: reframing tested612)11D368-G2 51 5 Exon 45-50, 48- 33 10 43 25 (SEQ ID NO: reframing 50, 49-50598)11D364-B7 51 5 Exon 45-50, 48- 35 4 43 30 (SEQ ID NO: reframing 50, 49-50,595) 46-50hD21-1_P1D9 45 3 Exon 46-47, 46- 46.8 10 Not Not tested (SEQ ID NO: reframing 50 tested609)hD21-l_PlC9 44 3 Exon 45, 45-50, 31.7 13.8 Not Not tested (SEQ ID NO: reframing 45-52 tested608)hD36_P1F1 50 3 Exon 51, 51-55 48.4 24.1 Not Not tested (SEQ ID NO: reframing tested623)hD368_P1B8 52 5 Exon 51 44.7 21.2 Not Not tested (SEQ ID NO: reframing tested602) and exonskippinghD21-1_P1F11 48 5 Exon 46-47 65.8 21.5 Not Not tested (SEQ ID NO: reframing tested611)hD368-E2 50 5 Exon 51, 51-55 61 4 Not Not tested (SEQ ID NO: skipping tested596)hD21-1_P1D11 48 5 Exon 46-47 62.2 14.3 Not Not tested (SEQ ID NO: reframing tested610)hD368_P1F5 45 3 Exon 46-47, 46- 54.5 5.8 Not Not tested (SEQ ID NO: reframing 50 tested601)hD368_P1E5 44 3 Exon 45, 45-50, 42.3 10.5 Not Not tested (SEQ ID NO: reframing 45-52 tested600)- 85 - #593201Attomey Docket No. 00010.032.1801Example 3 - Optimized MG119-28 sgRNA increases editing efficiency at therapeutic targets in K562 cells

[0273] Guide designs based on different scaffold / spacer length combinations

[0274] To assess whether a sgRNA spacer length different from 20 nt (e.g.. 18, 19, 21, 22, 23, 24 and 25 nt in length) would improve editing efficiency of the MG119-28 system, a guide RNA with a 22 nt spacer at hDMD was selected. The scaffold of the sgRNA was a truncated version (119 nt, SEQ ID NO: 193) ofthe original WT sgRNA (134 nt; SEQ ID NO: 192). For comparison, the WT sgRNA scaffold was also tested in combination with the eight different spacer lengths. Guides with either scaffold and each spacer length are described in SEQ ID NOs: 85-100.

[0275] Nuclease mRNA production

[0276] The sequence for the WT MG119-28 nuclease mRNA was codon optimized for human expression, then synthesized and cloned into a high copy ampicillin plasmid. Synthesized constructs encoding T7 promoter, UTRs, nuclease ORF, and NLS sequences were digested from the backbone with HindII and BamHI. and ligated into a pUC19 plasmid backbone with T4 DNA ligase and IX reaction buffer. The complete nuclease mRNA plasmid consists of an origin of replication, Kanamycin resistance cassette, the synthesized construct, and an encoded polyA tail. Nuclease mRNA was synthesized via in vitro transcription using the linearized nuclease mRNA plasmid. This plasmid was linearized by incubation at 37°C for 3 hours with Spel-HF enzyme. The linearization reaction consisted of a 200 pL reaction containing 50 ug pDNA, 50 units Spel-HF, and IX reaction buffer. Linearized plasmid was purified, precipitated in EtOH, and resuspended in nuclease free water at an adjusted concentration of 1000 ng / pL. The IVT reaction to generate nuclease mRNA was performed at 37 °C for 3 hours under the following conditions: 1.5 ug linearized plasmid; 5 mM ATP. CTP. GTP. and Nl-methyl pseudo-UTP; HiScribe T7 RNA Polymerase mix; 4 mM CleanCap AG; IX HiScribe transcription buffer; and 5 mM DTT. After 3 hours, IVT was stopped, and plasmid DNA was digested with the addition of 250 U / mL DNasel and incubated for 30 mins at 37 °C. Purification of nuclease mRNA was performed. Transcript concentration was determined by UV and further analyzed by capillary gel electrophoresis.

[0277] Cell culture, transfections, next generation sequencing, and indel analysis in mammalian cells.

[0278] For nucleofection with WT MG119-28 mRNA and guides targeting one human DMD site (DMD_H1; SEQ ID NOs: 85-100), 500 ng mRNA and 200 pmol of sgRNA were mixed together and incubated on ice until cells were prepared.- 86 - #593201Attorney Docket No. 00010.032.1801

[0279] Experiments were performed in K562 cells grown and passaged in Iscove's Modified Dulbecco's Medium supplemented with 10% (v / v) fetal bovine serum at 37 °C with 5% CO2. Approximately 1.2 x 105cells were transfected with mRNA plus sgRNA using a nucleofector. Transfected cells were grown for 3 days, harvested, and gDNA was extracted. Targeted regions for indels were amplified using Q5 High-Fidelity DNA polymerase with primers and extracted DNA as the templates. PCR products were purified. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced and analyzed with a proprietary algorithm to measure indel frequency.

[0280] The percentage of amplicons from NGS amplicon sequencing that contain insertions or deletions from conditions including MG119-28 sgRNAs with two different scaffold lengths combined with eight different spacer lengths targeting one human DMD site is shown in FIG.2.The 19 nt spacer seemed to have an advantage over the other spacer lengths, in combination with the truncated scaffold (119 nt).Example 4 - Increased affinity for target DNA by rational design of the MG119-28 nuclease based on the Cryo-EM structure

[0281] Design of single point, double-, triple- and hexamutants and testing at therapeutic targets in K562 cells

[0282] Using available structures and phylogenetic sequence analysis, 52 different single-point mutation variants of MG119-28 were designed in an effort to improve the activity of MG119-28 (SEQ ID NOs: 104-155). This approach focused on increasing the positive charge of residues within hydrogen-bonding distance to the DNA substrate and / or the sgRNA. For the structure-guided designed variants, special attention was paid first to negatively-charged residues in the vicinity of the phosphodiester backbones of the sgRNA and / or the DNA substrate. Additional uncharged residues near the sgRNA and / or DNA binding sites were also selected for mutagenesis to primarily positively-charged residues. Most of these residues were located in the Wedge (WED) domain as seen in Table 6. Sequence alignments of MG119-28 with Casl2f homologs / orthologs also informed which residues could tolerate mutagenesis. Because MG119-28 exists as an asymmetric homodimer, special care was taken to ensure that the intended mutation would not disrupt (1) residues critical for the dimerization of the two protomers or (2) key protein-nucleic-acid interactions necessary for binding. As described below, these single-point mutant variants were tested in K562 cells targeting one human DMD site (SEQ ID NO: 89). The scaffold of the- 87 - #593201Attorney Docket No. 00010.032.1801 sgRNA was 119 nt long (short) and the spacer was 22 nt long. Table 7 summarizes the top six mutations and the domain they are in.Table 6. Domain distribution of single-point mutations.Domain # of residuesWED 14Rec 4RuvC 8Zn-finger 0Table 7. Domain location of the 6 single point mutations.MG119-28 WT residue Mutation Name Domain K93 R MG119-284 (SEQ ID NO: 110) Rec E202 K MG119-291 (SEQ ID NO: 117) Wed M204 K MG119-293 (SEQ ID NO: 119) Wed E236 K MG119-298 (SEQ ID NO: 124) Wed E255 K MG119-307 (SEQ ID NO: 133) WedN329 E MG119-319 (SEQ ID NO: 145) RuvC

[0283] The next step was to test combinations of two and three of the six point mutations for a total of 34 double- and triple-mutants of MG119-28 nuclease (SEQ ID NOs: 156-190). As introducing multiple mutations concurrently could negatively impact the protein's overall fold and activity and variants with fewer combinations of the top mutations were prioritized. A hexamutant (SEQ ID NO: 191) that combined all top six mutations was tested; this hexamutant (R-K-K-K-K-E) had dramatically reduced activity as seen in FIG.4. As described below, these combinatorial mutant variants were tested in K562 cells targeting one human DMD site (SEQ ID NO: 858 hD119-28L-P4Hlb; sgRNA sequence denoted by SEQ ID NO: 626). The scaffold of the sgRNA was 134 nt long and the spacer was 19 nt long.

[0284] Nuclease mRNA production

[0285] Sequences for the WT MG119-28 (SEQ ID NO: 194) and single point mutants (SPMs) mRNA were codon optimized for human expression. A plasmid encoding the WT MG119-28 nuclease was purified by midiprep. Sections of the MG119-28 DNA sequence were amplified with primers containing the mutated codons, purified by routine methods, and assembled in high throughput by a two-part Gibson assembly. In brief, using an acoustic liquid handler, equimolar amounts of the amplified DNA sequences containing the mutated codons were mixed with a Master Mix and water in a 5 pL total reaction volume in a 96 well PCR plate. The plate was incubated at 50 °C for 20 mins. The entire reaction mixture was then added to a 96 well plate - 88 - #593201Attomey Docket No. 00010.032.1801 containing 10-beta competent E. coli cells. The plate was incubated on ice for 20 mins, heat shocked at 42 °C for 30 seconds, and again placed on ice for 5 mins. The cells were then added to a 96 well deep well plate containing 200 pL of SOC recovery media in each well and incubated at 37°C. Subsequently, 100 pL of transformed cells were plated on kanamycin containing LB agar petri dishes and incubated overnight at 37°C.

[0286] Individual colonies were picked, grown, and plasmid DNA extracted by miniprep. The complete nuclease mRNA plasmid consists of an origin of replication, kanamycin resistance cassette, the synthesized construct, and an encoded polyA tail. MG119-28 SPM DNA sequences were confirmed by whole plasmid sequencing, amplified, purified with magnetic beads, and checked for purity prior to mRNA synthesis.

[0287] Nuclease mRNA was synthesized via in vitro transcription using 1,500 ng of amplified template from the plasmids described above. The IVT reaction to generate nuclease mRNA was performed at 37°C for 3 hours under the following conditions: 1,500 ng of amplified DNA template; 5 mM ATP, CTP, GTP, and Nl-methyl pseudo-UTP; RNA Polymerase Mix; 4 mM CleanCap AG: and IX transcription buffer. After 3 hours, IVT was stopped, and amplified template DNA was digested with the addition of 250 U / mL DNasel and incubated for 30 mins at 37°C. Purification of nuclease mRNA was performed. Transcript concentration was determined by UV and further analyzed by capillary gel electrophoresis.

[0288] Sequences for the WT MG119-28 and combinatorial mutants mRNA were codon optimized for human expression, and synthesized as gBlocks. Templates for mRNA production were obtained by PCR amplification, and the PCR products were cleaned and checked for purity by a DNA gel.

[0289] The IVT reaction to generate nuclease mRNA was performed at 50 °C for 1 hour under the following conditions: 2000 ng of amplified DNA template: 5 mM ATP, CTP, GTP, and Nl-methyl pseudo-UTP: 18750 U / mL Hi-T7 RNA Polymerase; 2.5 mM DTT: 4 mM CleanCap AG; 2.5 U / mL Inorganic E. coli pyrophosphatase; 1000 U / mL murine RNase Inhibitor; and IX transcription buffer. After 1 hour, IVT was stopped, and plasmid DNA was digested with the addition of 250 U / mL DNasel and incubated for 10 mins at 37°C. Purification of nuclease mRNA was performed. Transcript concentration was determined by UV and further analyzed by capillary’ gel electrophoresis.

[0290] Cell culture, transfections, next generation sequencing, and indel analysis in mammalian cells

[0291] For nucleofection with WT MG119-28 (SEQ ID NO: 194) and SPMs mRNA and sgRNA sequences targeting one human DMD site (SEQ ID NO: 89), 500 ng mRNA and 200 pmol of - 89 - #593201Attomey Docket No. 00010.032.1801 sgRNA were mixed together and incubated on ice until cells were prepared. The scaffold of the sgRNA was 119 nt long (short) and the spacer was 22 nt long.

[0292] For nucleofection with WT (SEQ ID NO: 194) and MG119-28 combinatorial mutant mRNA and the sgRNA sequence targeting one human DMD site (SEQ ID NO: 858), 500 ng mRNA and 200 pmol of sgRNA were mixed together and incubated on ice until cells were prepared. The scaffold of the sgRNA was 134 nt long and the spacer was 19 nt long.

[0293] Experiments were performed in K562 cells grown and passaged in Iscove's Modified Dulbecco's Medium supplemented with 10% (v / v) fetal bovine serum at 37 °C with 5% CO2. Approximately 1.2 x 105cells were transfected with mRNA plus sgRNA. Transfected cells were grown for 3 days, harvested, and gDNA was extracted. Targeted regions for indels were amplified using Q5 High-Fidelity DNA polymerase with primers and extracted DNA as the templates. PCR products were purified. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on and analyzed with a proprietary algorithm to measure indel frequency.

[0294] The percentage of amplicons from NGS sequencing that contained insertions or deletions resulting from each MG119-28 SPM and the DMD H1 guide (SEQ ID NO: 89) is shown in FIG.3. Two of the SPMs showed at least a fifteen-fold increase in editing efficiency compared to WT.

[0295] The percentage of amplicons from NGS sequencing that contained insertions or deletions resulting from each MG119-28 combinatorial mutant and the DMD H1 guide (SEQ ID NO: 626) is shown in FIG. 4. At least two-thirds of the combinatorial mutants with a sgRNA comprising the 134 nt scaffold and 19 nt spacer showed increased levels of editing efficiency compared to WT. Strikingly, one-third of combinatorial mutants resulted in > 80% InDeis.Example 5 - Identification of MG119-28 protein variant and guide RNA for therapeutic target in skeletal muscle cells

[0296] The SPMs and combinatorial mutants with the DMD guide w ere screened in K562 cells. To confirm the optimized MG119-28 system would lead to the same levels of editing efficiency at a disease-relevant cell line, the top six combinatorial mutants and the top SPM at DMD in K562 cells were tested in skeletal muscle (SkM) cells with the sgRNA comprising the 134 nt scaffold and 19 nt spacer combination. Four productive (exon skipping and / or exon reframing) guide RNAs (hDl 19-28L-P4Hlb (SEQ ID NO: 94 or SEQ ID NO: 626). hDl 19-28L-P4A2b (SEQ ID NO: 101 or SEQ ID NO: 630), hDl 19-28L-P4E2b (SEQ ID NO: 102 or SEQ ID NO: 634) and- 90 - #593201Attorney Docket No. 00010.032.1801 hD119-28L-P4C4b (SEQ ID NO: 103 or SEQ ID NO: 635) were identified based on their indel profile analysis from previous guide screen experiment were tested.

[0297] Human skeletal muscle cells (SkM) cell culture, transfections, and data analysis

[0298] SkM cells were cultured in Mesenchymal Stem Cell Basal Medium supplemented with Primary Skeletal Cell Muscle Growth Kit for 1-2 passages prior to lipofection.

[0299] SkM cells were transfected with MG119-28 mRNA (WT or variant) plus guide RNAs at 1:5 mRNA:gRNA molar ratio at four different mRNA masses as follows. Cells were trypsinized and counted, and the equivalent volume to 12,500 viable cells were added to each well in a 96-wp plate. Additional pre-equilibrated media was added to each well to bring the total volume to 100 pL.

[0300] On the day of transfection, 1.5 pL of Lipofectamine Messenger Max Solution per ug of mRNA was combined with OptiMEM media to make a total 50 pL master mix solution, vortexed, and incubated for at least 5 mins at room temperature. In separate tubes, different masses of each MG119-28 mRNA variant (400 ng, 200 ng, 100 ng and 50 ng) were mixed with 1:5 molar ratio of each guide RNA and combined with OptiMEM media to make a total 50 pL reaction volume. After a brief vortex, the appropriate volume of MessengerMax solution was added to the RNA solution mix, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 mins at room temperature and added directly to the SkM cells.

[0301] Three days post-transfection, the media was aspirated and genomic DNA was harvested. The resulting gDNA was used as a template for NGS PCR. Targeted sequences were amplified with NGS primers. Amplicons around 250 bp long were checked in a DNA gel, sequenced, and analyzed to measure gene editing outcomes.

[0302] Analysis of NGS results indicate that the 7 variants (MG119-28 variant with K93R-M204K-E236K " MG I 19-350’7(denoted by SEQ ID NO: 176); MG119-28 variant with M204K-E236K-N329E (denoted by SEQ ID NO: 172); MG119-28 variant with K93R-E202K-E236K (denoted by SEQ ID NO: 183); MG119-28 vanant with K93R-E236K-E255K (denoted by SEQ ID NO: 179); MG119-28 variant with M204K-E236K (denoted by SEQ ID NO: 158); MG119-28 variant with E236K-E255K (denoted by SEQ ID NO: 166); MG119-28 variant with E236K (denoted by SEQ ID NO: 124) tested in the experiment performed better than the WT MG 119-28 across four different guide RNAs targeting DMD locus (FIGs. 5A-5D).Example 6 - Structure-guided engineering of small ABEs improves editing in human cells

[0303] Base editors (BEs) are gene editors that directly modify a nucleobase to install point mutations into cellular nucleic acid molecules, without generating double-stranded DNA - 91 - #593201Attomey Docket No. 00010.032.1801 (dsDNA) breaks. Adenine base editors (ABEs) comprise a nickase (nuclease with one of two catalytic sites disabled) fused to an adenosine deaminase (ADA) enzyme. Prior genomic mining revealed small archaeal-associated. arginine-rich nuclease systems (SMART) that are distinguished by their small size (450 aa - 1050 aa), in contrast to the substantially larger size of Cas9, whose 1,368 aa preclude facile AAV packaging. Here, SMART nuclease variants were fused to either a monomeric or dimeric ADA (MG68-4) and tested in human cells to identify active variants. These base editors are referred to as SMART ABEs. Two high performing nuclease candidates (MG34-29 and MG102-71, 748 aa and 943 aa in length, respectively) were further engineered, both the nickase and the ADA components.

[0304] Using available structures and phylogenetic sequence analysis, 48 different single-point mutation variants were designed from MG34-29 (SEQ ID NO: 789) (Table 8).Table 8: Designed single point mutations in MG34-29.Name Mutation Name Mutation Name Mutation Name Mutation MG34-108 MG34-120 MG34-132 MG34-144(SEQ ID NO: T33R (SEQ ID NO: Q227R (SEQ ID NO: A524R (SEQ ID NO: N604R 790) 802) 814) 826)MG34-109 MG34-121 MG34-133 MG34-145(SEQ ID NO: E49R (SEQ ID NO: Q228R (SEQ ID NO: D527R (SEQ ID NO: E624Q 791) 803) 815) 827)MG34-110 MG34-122 MG34-134 MG34-146(SEQ ID NO: Q58R (SEQ ID NO: N232R (SEQ ID NO: E542N (SEQ ID NO: E624R 792) 804) 816) 828)MG34-111 MG34-123 MG34-135 MG34-147(SEQ ID NO: N72R (SEQ ID NO: G247R (SEQ ID NO: D556Q (SEQ ID NO: M627R 793) 805) 817) 829)MG34-112 MG34-124 MG34-136 MG34-148(SEQ ID NO: S89R (SEQ ID NO: E267R (SEQ ID NO: N564R (SEQ ID NO: P638R 794) 806) 818) 830)MG34-113 MG34-125 MG34-137 MG34-149(SEQ ID NO: E103R (SEQ ID NO: T277R (SEQ ID NO: E575Q (SEQ ID NO: H654Q 795) 807) 819) 831)MG34-114 MG34-126 MG34-138 MG34-150(SEQ ID NO: Q132R (SEQ ID NO: S281R (SEQ ID NO: E577R (SEQ ID NO: N655K 796) 808) 820) 832)MG34-115 MG34-127 MG34-139 MG34-151(SEQ ID NO: E139R (SEQ ID NO: N307R (SEQ ID NO: A583R (SEQ ID NO: E672R 797) 809) 821) 833)MG34-116 MG34-128 MG34-140 MG34-152(SEQ ID NO: N153R (SEQ ID NO: L348R (SEQ ID NO: E588R (SEQ ID NO: L673R 798) 810) 822) 834)MG34-117 MG34-129 MG34-141 MG34-153(SEQ ID NO: S164R (SEQ ID NO: D357R (SEQ ID NO: D598K (SEQ ID NO: G700R 799) 811) 823) 835)- 92 - #593201Attorney Docket No. 00010.032.1801 Name Mutation Name Mutation Name Mutation Name Mutation MG34-118 MG34-130 MG34-142 MG34-154(SEQ ID NO: N195R (SEQ ID NO: N401R (SEQ ID NO: I602T (SEQ ID NO: T733R 800) 812) 824) 836)MG34-119 MG34-131 MG34-143 MG34-155(SEQ ID NO: T217R (SEQ ID NO: Q435R (SEQ ID NO: I602E (SEQ ID NO: S737T 801) 813) 825) 837)

[0305] Domain-walking of the monomeric MG68-4 variant along the MG34-29 (and variants) and MG102-71 (and variants) nickase chassis was performed to identify the optimal insertion point for MG68-4. Further rational engineering of the nickase identified SMART ABEs with robust A-G conversion activity. SMART ABEs are active at a therapeutically relevant splice acceptor site in the dystrophin gene and have relevance for restoring gene expression in the treatment of Duchenne muscular dystrophy.

[0306] Methods for HEK293T cell fluorescence screen

[0307] A mammalian fluorescence-based screen was developed for evaluation of base editing activity in cells (FIG.6A). A pEditor plasmid encodes ABE-T2A-GFP in which an ABE and GFP reporter were co-expressed and separated by a T2A ribosomal skipping peptide and a pReporter plasmid encodes BFP-T2A-target-mCherry containing a target spacer sequence (FIG. 6B). In cells, an ABE expressed from a pEditor edits an in-frame stop codon to a sense codon on a pReporter, and mCherry expression acted as an indicator of ABE activity. HEK293T cells were transfected with a transfection reagent and 100 pmol synthetic gRNA. This study used flow cytometry to measure the fraction of dually-transfected cells, which was the sub-population positive for both GFP and BFP, that also expressed mCherry. This fraction of mCherry -positive cells in the dually-transfected sub-population was increased for ABE variants with improved activity. Synthetic gRNAs containing phosphorothioate and 2'-O-methyl chemical modifications were synthesized by IDT.

[0308] Domain walking of improved ADA MG68-4 in SMART nickase MG 34-29 and MG 102-71

[0309] Methods for designing constructs, set of variants

[0310] To generate pEditor plasmids, a monomeric ADA MG68-4_Var91 (V83S, D109N, T112R, R154P) or MG68-4_Var95 (V83S, D109N, T112R, R153P, R154P) was inserted at unstructured loops of the MG34-29 (SEQ ID NOs: 220-272) and MG102-71 (SEQ ID NOs. 273-303) nickases that were proximal to the substrate binding site. Unstructured loops were modeled in AlphaFold2. Specific inlay sites in MG102-71 were in the loop separating RuvC-II from HNH (D360), HNH domain (G389, K410, D426, L457), and RuvC-III (D563, N599, D621, E626).- 93 - #593201Attomey Docket No. 00010.032.1801 Specific inlay sites in MG34-29 were in the loop separating RuvC-I from REC lobe (D38), REC lobe (P102, E103, E104, K105. El 12, DI 14, N203), loop separating REC lobe from RuvC-II (E279), RuvC-III (S475. E491, K496, L516. E530); additional tested fusion sites were the N- and C -termini.

[0311] Arginine scan ofMG102-71 ABE

[0312] Methods for designing constructs, set of variants

[0313] Arginine substitutions were performed on MG102-71 variants that contained either improved monomeric ADA MG68-4_Var91 (V83S, D109N, T112R, R154P) or MG68-4_Var95 (V83S, D109N, T112R, R153P, R154P) at the N599 inlay site (SEQ ID NOs. 195-217). Sites for mutagenesis were identified based on (1) the lack of positively charged residues in MG102-71; (2) the presence in Recognition Domain, WED domain, or Pam-interacting domain; (3) high frequency ofR / Kin other MG102 homologs; and (4) a high solvent accessibility in the AlphaFold-predicted structure of MG102-71. A total of 23 positions were identified in MG102-71 to mutate to arginine and were introduced into either MG68-4_Var91 or MG68-4_Var95 ADA constructs with N599 inlay site (Table 9).Table 9. MG102-71 Arginine mutations and associated ADA variant tested.Mutation ADA Nucleotide Amino Mutation ADA Nucleotide Amino variant SEQ ID Acid variant SEQ ID Acid SEQ NO: SEQ ID NO: ID NO:NO:S12R var91 195 376 S185R var95 210 391 T132R var91 196 377 Q202R var95 209 390 Q158R var91 197 378 W367R var95 214 395 E277R var91 198 379 Q657R var95 215 396 T278R var91 199 380 T681R var95 216 397 N372R var91 200 381 S697R var95 217 398 L598R var91 201 382 S748R var95 208 389 E672R var91 202 383 G846R var95 211 392 L696R var91 203 384 L828R var95 212 393 N701R var91 204 385 P797R var95 213 394 T378R var91 205 386N745R var91 206 387P820R var91 207 388

[0314] Experimental Results

[0315] HEK293T cell fluorescence screen results for MG34-29 and MG102-71 domain walking

[0316] Based on the fraction of mCherry-positive cells in the dually-transfected sub-population, and using guide RNAs of SEQ ID NOs: 171-181, for target spacers SEQ ID NOs: 352-362, the - 94 - #593201Attorney Docket No. 00010.032.1801 study identified improved MG102-71 ABE variants. A total of 18 new MG102-71 ABE variants were compared to ABE-192 (denoted by SEQ ID NO: 476, which is identical to SEQ ID NO: 538). where 13 variants performed better at DMD exon 45 (FIG. 7A) and 2 variants were on par for activity at DMD exon 51 (FIG. 7B). Based on the combined activity (FIG. 8A), the study identified N599 as the best inlay site.

[0317] Similarly, based on the fraction of mCherry -positive cells in the dually-transfected subpopulation. the study identified improved MG34-29 ABE variants. A total of 32 new MG34-29 ABE variants were compared to one of the previous-generation ABE variants, ABE-87: 13 variants performed better for DMD exon 45 (FIG. 7D). Based on the combined activity (FIG.8B), the study identified El 12 and L516 as the best inlay sites.

[0318] Combining the variants from SMART BE screening and adenine deaminase variants resulted in systems with up to 40% editing based on stop-to-sense codon conversion analysis performed using a mammalian fluorescence reporter encoding a DMD therapeutic target.

[0319] HEK293T cell fluorescence screen results for MG 102-71 arginine scanning

[0320] Similarly, based on the fraction of mCherry -positive cells in the dually-transfected subpopulation. the study identified improved MG102-71 ABE arginine point mutants (SEQ ID NOs.195-217). A total of 23 new MG102-71 ABE arginine point mutants were compared to one of the previous-generation ABE variants, ABE-192; 20 arginine point mutants performed better than ABE-192 for DMD exon 45 (FIG. 7C). Inclusion of the arginine point mutations provided a further 1.4-fold activity boost, enabling the SMART BE to achieve up to 73% apparent editing based on stop-to-sense codon conversion analysis.Example 7 - Small ABEs are active at DMD in SkM cells

[0321] This example demonstrates the robust editing across multiple ABEs using guide RNAs designed to position the conserved splice acceptor adenines of exons 45 and 51 within the active deamination window of ABEs and tested in human skeletal muscle cells.

[0322] Human skeletal muscle cells (SkM) cell culture, transfections, and data analysis

[0323] SkM cells were cultured in Mesenchymal Stem Cell Basal Medium supplemented with Primary Skeletal Cell Muscle Growth Kit for 1-2 passages prior to lipofection. SkM cells were transfected with ABE mRNA plus guide RNA hD_ABE_Ex45-g!2 (SEQ ID NO: 375 / SEQ ID NO: 642) at 1:5 mRNA:gRNA molar ratio at four different mRNA masses as follows. Cells were trypsinized and counted, and the equivalent volume to 12,500 viable cells were added to each well in a 96-wp plate. Additional pre-equilibrated media was added to each well to bring the total volume to 100 pL. On the day of transfection, 1.5 uL of Lipofectamine Solution per ug of mRNA was combined with OptiMEM media to make a total 50 uL master mix solution, vortexed, and - 95 - #593201Attomey Docket No. 00010.032.1801 incubated for at least 5 minutes at room temperature. In separate tubes, different masses of ABE-163 mRNA (400 ng. 200 ng, 100 ng and 50 ng) were mixed with 1:5 molar ratio of hD ABE Ex45-gl2 guide RNA and combined with OptiMEM media to make a total 50 pL reaction volume. After a brief vortex, the appropriate volume of MessengerMax solution was RNA solution mix, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 minutes at room temperature and added directly to the SkM cells. Three days post-transfection, the media was aspirated and genomic DNA was harvested. Resulting gDNA was used as a template for NGS PCR. Targeted sequences were amplified with NGS primers. Amplicons around 250 bp long were sequenced, and then analyzed to measure gene editing outcomes.

[0324] Experimental Results

[0325] Rational engineering of the nickase chassis for SMART ABEs identified several improved variants with base editing activity at the DMD target locus. A dose titration experiment of SMART ABE variant mRNA indicated successful editing, with an A to G substitution rate of over 10% with the ABE-163 using a DMD exon 45 targeting guide hD_ABE_Ex45-gl2 (FIG. 9). Further experiments with 400 ng of ABE mRNA for selected rationally engineered variants tested at DMD in SkM cells showed a marked increase in base editing activity with MG34 family ABEs (ABE-211 (denoted by SEQ ID NO: 491), ABE-218 (denoted by SEQ ID NO: 498), ABE-267 (denoted by SEQ ID NO: 539), ABE-268 (denoted by SEQ ID No: 540), ABE-269 (denoted by SEQ ID No: 541), ABE-270 (denoted by SEQ ID No: 542). ABE-271 (denoted by SEQ ID No: 543). ABE-272 (denoted by SEQ ID No: 544), and ABE-273 (denoted by SEQ ID No: 545)) with 30% total base editing conversion and up to 7% on-target A to G conversion at DMD exon 45 (FIG. 10A-FIG. 10B). For the MG102 family ABEs (ABE-245 (denoted by SEQ ID NO: 525), ABE-254 (denoted by SEQ ID NO: 534), ABE-274 (denoted by SEQ ID NO: 391), and ABE-275 (denoted by SEQ ID NO: 397)), the study observed 36% total base editing with 21% on-target A to G conversion at exon 45 at the DMD gene (FIG. 10C-FIG. 10D).

[0326] At exon 45, the ABE variant, ABE-275 (denoted by SEQ ID NO: 397) exhibited the highest activity achieving splice-site A to G editing of 20.3% and total A to G editing of 34.6%. In contrast, the miniABE variant ABE-271 (denoted by SEQ ID No: 543) showed comparable total A to G editing (31.6%) but reduced splice-site specific editing (7%), highlighting differences in editing specificity between editor architecture. Editing efficiencies were also guide-dependent, with miniABE gRNA gl2 (SEQ ID NO: 375 / SEQ ID NO: 642) exhibiting approximately threefold higher activity than gRNA gl3 (FIGs. 10B-10D).- 96 - #593201Attorney Docket No. 00010.032.1801

[0327] Consistent with exon 45 results, ABE-275 again demonstrated the highest activity, achieving splice-site A to G editing of 19.2% and total A to G editing of 28.5% (FIG. 10D).Example 8 - Dystrophin protein restoration with MG119-28 nucleases in human AEx52 myocytes

[0328] Guide RNAs hD119-28-P4C4 (SEQ ID NO: 785), hDl 19-28-P4B4 (SEQ ID NO: 786), hDl 19-28-g09b (SEQ ID NO: 787) were transfected into human myocytes with a deletion of exon 52 of the DMD gene (c'AEx52 myocytes”).

[0329] Myocyte Transfection Protocol

[0330] DMD deletion myocytes were transfected with MG119-28 nuclease mRNA (SEQ ID NO: 785) plus guide RNA at 1:5 mRNA:gRNA molar ratio (600 ng nuclease mRNA plus 5.08 pmole guide RNA) as follows.

[0331] One day prior to transfection, frozen myocytes were revived in DMEM-high glucose with 10% KOSR, 1% GlutaMAX, 1% ITS-G, 1 % N2-supplement, 10 pM ROCK inhibitor and 1 pg / pl doxycycline. Cells were counted, and 230,000 viable cells were added to each well in a TC-treated 24 well plate coated with GeltrexTM. Additional pre-equilibrated media was added to each well to bring the total volume to 500 pL.

[0332] On the day of transfection, 27.3 pL of OptiMEM media and 0.6 ul of Lipofectamine MessengerMax Solution per reaction were combined in a master mix solution, vortexed, and incubated for at least 5 minutes at room temperature. In separate tubes, 600 ng of the mRNA and 5.08 pmole of guide RNA were combined with OptiMEM media to make a total 30 uL reaction volume. After a brief vortex, the appropriate volume of MessengerMax solution was added to each RNA solution, mixed by flicking the tube, and briefly spun down at a low speed. The complete editing reagent solutions were incubated for 10 minutes at room temperature and added directly to the myocytes.

[0333] Eleven days post transfection, the media was aspirated from each well of myocytes and cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail for protein extraction. Of the cell lysate, 5 pg was loaded onto a 3-8% Tris-Acetate gel and transferred to the nitrocellulose membrane using an iBlot-2. Blots were blocked with 5% milk, then probed with antibodies against the dystrophin (1:100) and vinculin (1:4000), detected using secondary antibody HRP-anti-mouse IgG (1:10000) & HRP-anti-rabbit IgG (1:5000) respectively. Blots were developed using SuperSignal West Dura Chemiluminescent Substrate for chemiluminescence detection using ChemiDoc MP (FIG. 11A). The dystrophin signal from- 97 - #593201Attorney Docket No. 00010.032.1801 samples was normalized against vinculin and the relative protein expression was determined by using WT input as reference (FIGs. 11B and 11C).

[0334] The Western blot results indicate that wild-type myocytes express dystrophin protein while the age-matched mock treated AEx52 myocytes which underwent the same differentiation and transfection procedure do not express dystrophin protein. Upon editing with MG119-28 candidates, hD119-28-P4C4 (SEQ ID NO: 786) shows 50% protein restoration as compared to WT expression in AEx52 myocytes. hD119-28-P4B4 (SEQ ID NO: 787) and hD119-28-g09b (SEQ ID NO: 788) showed 11% and 4% protein restoration, respectively (FIGs. 11A-11C). Example 9 - Dystrophin expression restoration in human AEx44 myocytes with MG29-1, MG3-6 / 3-8, MG21-1, MG3-6x ABE candidates

[0335] Guide RNAs hD29-P3C8 (SEQ ID NO: 616), hD368-Gl (SEQ ID NO: 597), hD368_P! A5 (SEQ ID NO: 599), hD211 P1B9 (SEQ ID NO: 607), hDABE103_Ex45_g4 (SEQ ID NO: 637), hD ABE 106_Ex45_g5 (SEQ ID NO: 638) were transfected into human myocytes with a deletion of exon 44 of the DMD gene (“AEx44 myocytes”).

[0336] Myocyte Transfection Protocol

[0337] DMD deletion myocytes were transfected with MG29-1 nuclease mRNA (SEQ ID NO: 586) / MG3-6 / 3-8 nuclease mRNA (SEQ ID NO: 584) / MG21-1 nuclease mRNA (SEQ ID NO: 585) / ABE-103 (SEQ ID NO: 589) / ABE-106 (SEQ ID NO: 590) plus guide RNA at 1:5 mRNA:gRNA molar ratio (600 ng nuclease mRNA each system plus 2.2 pmole guideRNA for MG29-1. 2.5 pmole guide RNA for MG3-6 / 3-8, 2.6 pmole guide RNA for MG21-1, 1.9 pmole for ABE 103 and ABE106 candidates). Human AEx44 myocytes were plated and transfected with the above listed nuclease mRNA and respective guideRNAs following the procedure described in general methods section titled “Myocyte transfection method and differentiation to myotubes”. Eleven days post-transfection, genomic DNA (gDNA), RNA and protein were extracted from biological replicates (one well each from a 24-well plate format) of differentiated AEx44 myotubes as described in general methods section titled “DNA, RNA and protein extraction to evaluate editing and expression restoration in disease model cells post-editing”.

[0338] Results

[0339] Total and productive editing efficiencies for each guideRNA candidate are summarized in Table 10. A strong correlation was observed between productive editing and restoration of mRNA and protein expression levels (FIG. 12A). Immunofluorescence analysis of selected samples further validated the extent of protein restoration observed in Western blot analysis (FIG. 12B-12C)- 98 - #593201Attomey Docket No. 00010.032.1801 Example 10 - Dystrophin expression restoration in human AEx44 myocytes with MG21-1 and MG119-28 candidates

[0340] Guide RNAs hD211 P1B9 (SEQ ID NO: 607). hD119-28L-P4G1b (SEQ ID NO: 629), hD119-28L-A9b (SEQ ID NO: 632), mhD119-28L-B9b (SEQ ID NO: 634) were transfected into human myocytes with a deletion of exon 44 of the DMD gene (“AEx44 myocytes”).

[0341] Myocyte Transfection Protocol

[0342] DMD deletion myocytes were transfected with MG21-1 nuclease mRNA (SEQ ID NO: 585) or MG119-28 nuclease mRNA (SEQ ID NO: 588) plus guide RNA at 1:5 mRNA:gRNA molar ratio (600 ng nuclease mRNA each system plus 2.6 pmole guide RNA for MG21-1, 5.1 pmole guideRNA for MG119-28 candidates). Human AEx44 myocytes were plated and transfected with the above listed nuclease mRNA and respective guideRNAs following the procedure described in general methods section titled " Myocyte transfection method and differentiation to myotubes”. Eleven days post-transfection, genomic DNA (gDNA), RNA and protein were extracted from biological replicates (one well each from a 24-well plate format) of differentiated AEx44 my otubes as described in general methods section titled “DNA, RNA and protein extraction to evaluate editing and expression restoration in disease model cells postediting”.

[0343] Results

[0344] Total and productive editing efficiencies for each guideRNA candidate are summarized in Table 10. All tested guideRNAs successfully restored dystrophin expression at both the transcript and protein levels, though to varying degrees (FIG. 13A). In most cases, protein restoration as detected by western blot closely correlated to mRNA restoration as measured by ddPCR except hD119-28L-P4Glb which showed lower protein restoration compared to its transcript-level recovery (FIG. 13B). Notably, guideRNA hDl 19-28L-A9b (SEQ ID NO: 632) achieved nearcomplete restoration of dystrophin protein expression comparable to wild-type levels in AEx44 myocytes (FIG. 13A-13B).Example 11 - Dystrophin expression restoration in human AEx44 myocytes with SMART ABEs and MG119-28 and MG21-1 candidates

[0345] Guide RNAs hDABE180_Ex45_gl (SEQ ID NO: 639), hDABE180_Ex45_g2 (SEQ ID NO: 640), hD211_P1B9 (SEQ ID NO: 607), hDl 19-28L-A9b (SEQ ID NO: 632) were transfected into human myocytes with a deletion of exon 44 of the DMD gene (“AEx44 myocytes”).

[0346] Myocyte Transfection Protocol

[0347] DMD deletion myocytes were transfected with MG21-1 nuclease mRNA (SEQ ID NO: 585) / MG119-28 nuclease mRNA (SEQ ID NO: 588) / ABE-180 (SEQ ID NO: 591) plus guide - 99 - #593201Attomey Docket No. 00010.032.1801 RNA at 1:5 mRNA:gRNA molar ratio (600 ng nuclease mRNA each system plus 2.6 pmole guide RNA for MG21-1. 13.2 pmole guideRNA for ABE180, 15.9 pmole guideRNA for MG119-28 candidates). Human AEx44 myocytes were plated and transfected with the above listed nuclease mRNA and respective guideRNAs following the procedure described in general methods section titled ‘'Myocyte transfection method and differentiation to myotubes”. Eleven days posttransfection, genomic DNA (gDNA), RNA and protein were extracted from biological replicates (one well each from a 24-well plate format) of differentiated AEx44 myotubes as described in general methods section titled “DNA, RNA and protein extraction to evaluate editing and expression restoration in disease model cells post-editing.”

[0348] Results

[0349] Total and productive editing efficiencies for each guideRNA candidate are summarized in Table 10. All tested guideRNAs successfully restored dystrophin expression at the transcript level to varying extents (FIG. 14A). In most cases, protein restoration as detected by western blot closely correlated to mRNA restoration as measured by ddPCR. An exception was guideRNA hD211_P1B9 which exhibited low protein restoration relative to its transcript-level recovery (Figure 4B). Specifically, guideRNA hD119-28L-A9b (SEQ ID NO: 632) reproducibly achieved high levels of protein restoration, reaching approximately 67% of wild-type expression in AEx44 myocytes (FIGs. 14A-14B).Example 12 - Dystrophin expression restoration in human AEx52 myocytes with MG3-6 / 3-4, MG3-6 / 3-8, MG29-1 and MG119-28 candidates

[0350] Guide RNAs hD364_B9 (SEQ ID NO: 594), hD368_E3 (SEQ ID NO: 605), hD368_H3 (SEQ ID NO: 606), hD29_A9 (SEQ ID NO: 621), hD119-28L-P4C4b (SEQ ID NO: 635), hD119-28L-P4B4b (SEQ ID NO: 631), hD119-28L-g09b (SEQ ID NO: 636) were transfected into human myocytes with a deletion of exon 52 of the DMD gene (“AEx52 myocytes”).

[0351] Myocyte Transfection Protocol

[0352] DMD deletion myocytes were transfected with MG3-6 / 3-4 nuclease (SEQ ID NO: 583) / MG3-6 / 3-8 (SEQ ID NO: 584) / MG29-1 nuclease mRNA (SEQ ID NO: 586) / MG119-28 nuclease mRNA (SEQ ID NO: 588) plus guide RNA at 1:5 mRNA:gRNA molar ratio (600 ng nuclease mRNA each system plus 2.5 pmole guideRNA for MG3-6 / 3-8. MG3-6 / 3-4 candidates, 2.2 pmole guideRNA for MG29-1 candidates, 5.1 pmole guideRNA for MG119-28 candidates). Human AEx52 myocytes were plated and transfected with the above listed nuclease mRNA and respective guideRNAs following the procedure described in general methods section titled “Myocyte transfection method and differentiation to myotubes”. Eleven days post-transfection, genomic DNA (gDNA), RNA and protein were extracted from biological replicates (one well - 100 - #593201Attorney Docket No. 00010.032.1801 each from a 24-well plate format) of differentiated AEx52 myotubes as described in general methods section titled “DNA. RNA and protein extraction to evaluate editing and expression restoration in disease model cells post-editing.”

[0353] The productive editing profile of hD119-28L-P4C4b at the transcript level was assessed by amplifying an 11 kb region encompassing the full DMD coding sequence using long-range PCR, followed by Oxford Nanopore Technology (ONT) long-read sequencing. Amplification was performed using primers 5'-CTTTCCCCCTACAGGACTCAG-3' (Primer 1, targeting Exon 1 of the Dp427m isoform, SEQ ID NO: 666) and 5'-CCAAATCATCTGCCATGTGG-3' (Primer 2, targeting the 3' UTR, SEQ ID NO: 667) to generate the full-length DMD cDNA. Following quality trimming, sequencing reads were aligned to the reference genome and annotated. The annotated reads were subsequently analyzed to assess splicing patterns and productive exon refraining, including canonical splice junctions, novel splice junctions, in-frame exon skipping events, and small in-frame insertions or deletions contributing to productive transcript restoration.

[0354] Results

[0355] Total and productive editing efficiencies for each guideRNA candidate are summarized in Table 10. GuideRNAs 11D364 B9. hD29_A9, hD119-28L-P4C4b, hD119-28L-P4B4b successfully restored dystrophin expression at both transcript and protein levels to varying extents (FIG. 15A). Specifically, guideRNA hD119-28L-P4C4b achieved high levels of protein restoration, reaching >50% of wild-type expression in AEx52 myocytes (FIGs. 15A-15B) Immunofluorescence analysis of selected samples further validated the extent of protein restoration observed in western blot analysis (FIG. 15C). Long-read sequencing analysis of the DMD transcript following editing with hD119-28L-P4C4b revealed that approximately 42% of the total transcripts exhibited exon skipping, while around 24% resulted in productive exon reframing (FIG. 15D).Example 13 - MG119-28 guide screen for human guideRNA candidates in human skeletal muscle cells and editing efficiency of lead candidates in mouse myoblasts

[0356] A guide RNA screening was conducted in human skeletal muscle cells using the MG119-28 guideRNA library' targeting the human DMD gene. Top-performing guideRNAs with high editing efficiency against human DMD were identified from this screen. Based on these results, mouse surrogate guideRNAs were selected to target the mouse Dmd gene, leveraging sequence homology. Specifically, human guideRNAs with fewer than two mismatches between the human and mouse DMD sequences and with exact PAM sequence matches were prioritized. Five guideRNAs met these criteria and were designated as mouse surrogate guideRNAs for targeting mDMD: mD119-28L-P4B4b (SEQ ID NO: 651). mhD119-28L-P4A2b (SEQ ID NO: 630),- 101 - #593201Attomey Docket No. 00010.032.1801 mDl 19-28L-A9b (SEQ ID NO: 650), mhDl 19-28L-B9b (SEQ ID NO: 634), and mD119-28L-gO5b (SEQ ID NO: 652). These guideRNAs were subsequently evaluated for editing efficiency at the mDMD locus in C2C12 mouse myoblasts, across four different dosing conditions.

[0357] SkM and C2C12 Transfection method

[0358] MG119-28 guide screen for human guideRNA candidates was performed in hSkM cells in 96-well plate format as described in Example 1. Briefly, 200 ng of mRNA and 83 ng of guide RNA were used per transfection. A 5.0 pL RNA reaction was prepared and combined with 4.7 pL OptiMEM and 0.3 pL Messenger Max per well. Transfection complexes were incubated for 10 minutes at room temperature before being added to the cells. Cells were harvested three days posttransfection. Media was aspirated, and 50 pL of QuickExtract was added to each well. Plates were incubated at 65 °C for 6 minutes at 300 rpm, followed by 98 °C for 3 minutes. Target regions of the DMD gene were PCR-amplified using a DNA Polymerase with gene-specific primers and adapters for next-generation sequencing (NGS). A second PCR using barcoded primers was performed for 10 cycles, and amplicons were analyzed. Data were processed using a custom script to quantify insertion / deletion (INDEL) frequencies at the target sites.

[0359] Mouse guideRNA surrogates were transfected into C2Cf2 cells using a 96-well plate format. C2C12 cells were seeded at a density of 5,000 cells per well. mRNA was transfected at doses of 400 ng, 200 ng, 100 ng, and 50 ng per well, along with guideRNAs at a 1:5 molar ratio relative to the mRNA. Transfection complexes were prepared in a total volume of 5 pL, using 1.5 pL of Lipofectamine MessengerMAX per pg of RNA cargo, diluted in Opti-MEM medium. Complexes were incubated for 10 minutes at room temperature before being added to the cells. Cells were harvested three days post-transfection. Media was removed, and 50 pL of DNA Extraction Solution was added to each well. Plates were incubated at 65 °C for 6 minutes with shaking at 300 rpm, followed by incubation at 98 °C for 3 minutes. Target regions of the DMD gene were PCR-amplified using a DNA Polymerase and gene-specific primers containing adapter sequences for next-generation sequencing (NGS). A secondary PCR with barcoded primers was performed for 10 cycles. Final amplicons were sequenced. INDEL frequencies at the target sites were quantified using a custom analysis pipeline.

[0360] Results

[0361] Of the human guideRNAs tested for MG119-28, 17 guideRNAs (SEQ ID NOs: 626-635, 643-649) demonstrated over 10% editing efficiency at the hDMD locus (FIG. 16A). From this subset, mouse surrogate guideRNAs were selected based on two criteria: (1) they originated from human guideRNAs showing >10% editing, and (2) they contained fewer than two mismatches between the human and mouse DMD sequences, along with an exact PAM sequence match. These - 102 - #593201Attorney Docket No. 00010.032.1801 mouse guideRNAs exhibited dose-dependent editing activity in C2C12 cells, with mD119-28L-P4B4b (SEQ ID NO: 651), mDl 19-28L-A9b (SEQ ID NO: 650), and mhl 19-28L-B9b (SEQ ID NO: 634) showing the highest levels of editing (FIG. 16B).Example 14 - In-vivo editing at DMD locus by MG119-28 mouse guideRNA candidates delivered via AAV

[0362] All-in-one AA V cargo design

[0363] AAV cargo constructs were designed to express MG119-28 and a guide RNA from a single AAV. The payload is flanked by two, 141 bp inverted terminal repeats (ITRs). A U6 polymerase III promoter is placed upstream of the guide RNA sequence in the reverse orientation at the 5’ end of the cargo. This is followed by a CMV promoter driving the MG119-28 open reading frame. MG119-28 is N-terminally tagged with a V5 epitope. A synthetic poly-adenylation sequence (SpA) follows MG119-28 (FIG. 17A).

[0364] AA V production

[0365] AAVs were produced using a transient triple co-transfection method involving a plasmid encoding adenoviral helper proteins, a plasmid encoding AAV rep protein and capsid proteins, and a plasmid encoding guide and MG119-28 flanked between two AAV2 ITRs (SEQ ID NOs: 653-659). One day prior to transfection, Viral Production Cells 2.0 were seeded at 1.5e6 cells / mL in HEK293 medium supplemented with GlutaMAX supplement at 1:100 dilution. On the day of transfection, the three plasmids were mixed at a 1:1:1 mass ratio with a total plasmid mass of 2 pg per mL of cell culture volume. The plasmid mixture was diluted in 100 pL of IX PBS per mL of cell culture volume. To make the final transfection mixture, 1 pL of RevIT AAV enhancer and 2 pL of TransIT Transfection Reagent per mL of cell culture volume were mixed into the diluted plasmid mixture, and incubated at room temperature for 30 minutes. The entire transfection mixture was added into the cells and incubated at 37°C for 72 hours prior to harvest. The cells were harvested by centrifuging at 1500 g for 15 minutes at 4°C and resuspended in a TMS buffer containing 50 mM of Tris-HCl, 2 mM MgC12, and 150 mM NaCl. The cell pellets in the TMS buffer were subjected to physical lysis through 3 rapid freeze and thaw cycles, resulting in a crude viral lysate.

[0366] AA V Purification

[0367] The crude viral lysate was treated with 100 U / rnL of DENARASE® for 1 hour to digest exogenous nucleic acids. The treated viral lysate was clarified by centrifugation at 12000 g for 60 minutes at 4°C in two 30-minute steps, with the supernatant retained after each centrifugation step. The clarified viral lysate was further purified via cesium chloride density gradient ultracentrifugation. To prepare the gradient, 5 mL of cesium chloride in IX DPBS at a density of - 103 - #593201Attomey Docket No. 00010.032.1801 1.3 g / cm3 was added to the bottom of a 17 mL open-top ultracentrifuge tube, 5 mL of cesium chloride in IX DPBS at a density of 1.5 g / cm3 was then slowly underlaid below the cesium chloride at 1.3 g / cm3 density, and 7 mL of the clarified viral lysate is slowly overlaid on top of the cesium chloride at 1.3 g / cm3 density. The ultracentrifuge tubes were loaded on an Ultracentrifuge and centrifuged at 32000 rpm for 6 hours at room temperature. To retrieve AAV particles from the gradients, an 18G needle in a 5 mL syringe is punctured into the side of the ultracentrifuge tube at the interface of the two different cesium chloride densities, and 2.5 mL of the sedimented AAV is pulled. The AAV underwent 3 rounds of dialysis in a solution of 0.001% Pluronic F-68 in IX DPBS with calcium and magnesium at pH 7.10. The AAV suspension was finally sterilized with a 0.45 pm syringe filter.

[0368] Systemic delivery of myotropic AAVs carrying MG 119-28 mouse DMD albumin / Rosa26 guideRNAs

[0369] Six- to eight-week-old C57BL / 6J mice (N = 5 per group) were systemically administered myotropic AAVs encoding mDMD or mRosa26 guideRNAs, or AAV8 vectors encoding mAlbumin or mRosa26 guideRNAs, along with the MG119-28 nuclease. Injections were performed via the tail vein at a dose of 1 x io12vector genomes (VG) per mouse in a volume of 0.2 mL. Fourteen days post-injection, animals were euthanized, and tissues including quadriceps, gastrocnemius, heart, diaphragm, and liver were harvested from mice dosed with mDMD and mRosa26 guideRNA AAVs. Liver tissues were collected from the mAlbumin AAV 8-dosed group. All tissues were stored in DNA / RNA Shield stabilization solution until further processing.

[0370] For the mDMD guideRNA-injected groups, a portion of heart tissue was additionally processed to generate single-nuclei suspensions using the Single Nuclei Isolation Kit, protocol optimized for adult brain tissue. Isolated nuclei w ere fixed in ice-cold 70% ethanol for 10 minutes to preserve sample integrity. Nuclei were subsequently sorted based on GFP expression using a cell sorter. Schematics illustrating the in vivo delivery of MG119-28 candidates are shown in FIG.17B and FIG. 18A. Both unenriched and EGFP-enriched nuclei samples were subjected to nextgeneration sequencing (NGS). INDEL events at the target sites were identified and quantified using a custom in-house analysis pipeline.

[0371] Results

[0372] Maximum genome editing (-10%) was observed in heart tissue across all tested DMD candidate guides. Among the four evaluated DMD candidate guides, P4A2b and B9b exhibited the highest editing efficiency followed by P4B4b (FIG. 17D). Importantly, both P4A2b and B9b spacer sequences were fully conserved between mouse and human, containing no mismatches. Lower editing efficiencies were detected in diaphragm, quadriceps, and gastrocnemius tissues - 104 - #593201Attorney Docket No. 00010.032.1801 across all evaluated DMD candidates (FIG. 17C). As anticipated, no editing was detectable in liver lobes with DMD-targeting guides, consistent with the absence of DMD gene expression in liver tissue. At the Rosa26 locus, editing efficiency (-13% in heart) was slightly higher compared to the DMD locus in heart, diaphragm, gastrocnemius and quadriceps muscles (FIGs. 17C-17D).Approximately 13% editing efficiency at Rosa26 was observed in liver tissues following myotropic vector delivery, aligning with expectations due to the ubiquitous nature of Rosa26 expression. At the Albumin locus, the top-performing mouse albumin-targeted guide consistently achieved approximately 18% editing efficiency across all three liver lobes tested. Additionally, higher editing efficiencies were recorded when targeting the mRosa26 locus delivered via AAV8 vectors specifically in liver tissues (FIG. 18B). For mhll9-28L-P4A2b and mhll9-28L-B9b guideRNA which have no mismatch between mouse and human sequences, around l / 3rd of the total editing outcomes were productive edits in the EGFP enriched heart tissue samples highlighting their applicability in restoring dystrophin expression in transgenic DMD disease mouse models with a more efficient AAV serotype for effective delivery in muscle tissues as well as heart.Table 10. Productive DMD editing by guideRNA candidates in disease cell models delivered by mRNA transfection.Target % PRODUCTIVE Example Sample exon INDELs editing%hD29-P3C8 3’ of Ex43 78 16.7hD368-Gl 5’ of Ex45 45 5.4 hD368_PlA5 3’ of Ex43 52 9.1 hD211_PlB9 3’ of Ex43 79 17.526.4% (A: G base hDABEI03_Ex45_g4 5’ of Ex45 - editing) Example 9 in 50% (A: G base AEx44 myocytes hDABEI06_E\45_g5 5’ of Ex45 - editing)hD211_PlB9 3’ of Ex43 76.4 17.38hD119-28L-P4G1b 3’ of Ex43 87.5 11.12hD119-28L-A9b 5’ of Ex45 81.1 16.32 Example 10 inAEx44 myocytes mhD119-28L-B9b 5’ of Ex45 71.8 6.73- 105 - #593201Attomey Docket No. 00010.032.1801 hDABEI80_Ex45_g1 5’ of Ex45 48.8 23.6 hDABEI80_Ex45_g2 5’ of Ex45 29.4 2.1 hD211_PlB9 3’ of Ex43 23.8 2.3Example 11 inAEx44 myocytes hDl 19-28L-A9b 5’ of Ex45 17.4 6.7hD364_B9 5’ of Ex53 19 5.2 hD368_E3 5’ of Ex53 7.4 1 hD368_H3 5’ of Ex53 1.4 0.6hD29_A9 5’ of Ex53 62 7.4hD119-28L-P4C4b 3’ of Ex51 69.8 4hD119-28L-P4B4b 3’ of Ex51 87.9 5.1Example 12 inAEx52 myocytes hD119-28L-g09b 3’ ofEx51 16.6 8

[0373] While embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all embodiments of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein, in some embodiment, are be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.- 106 - #593201

Claims

Attorney Docket No. 00010.032.1801CLAIMS WHAT IS CLAIMED IS:

1. An engineered nuclease system, comprising:a) (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304- 364, and 589-590 or comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 397, 457, 479. 398, 376-396, 399-456, 458-478, 480-484, and 485- 545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176. 124, 158, 166. 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

2. An engineered nuclease system, comprising:a) (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304- 364, and 589-590 or comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485- 545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity’ to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642. and 786-788.

3. The engineered nuclease system of claim 1 or 2, wherein the base editor is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457. 479, 398, 376-396, 399-456,- 107 - #593201Attorney Docket No. 00010.032.1801458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

4. The engineered nuclease system of claim 1 or 2, wherein the base editor is encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275. 277-297. 299-303. 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

5. The engineered nuclease system of claim 1 or 2, wherein the base editor is encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or the endonuclease is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

6. The engineered nuclease system of any one of claims 1-5, wherein the endonuclease comprises an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 194, 176. 124, 158. 166, 172, 179, 183, 104-123, 125-157. 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891.

7. The engineered nuclease system of any one of claims 1-5, wherein the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891.

8. The engineered nuclease system of any one of claims 1-5, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891.

9. The engineered nuclease system of any one of claims 1-5. wherein the endonuclease comprises an amino acid sequence having at least 100% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891.

10. The engineered nuclease system of any one of claims 1-9. wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.- 108 - #593201Attorney Docket No. 00010.032.180111. The engineered nuclease system of any one of claims 1-9, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

12. The engineered nuclease system of any one of claims 1-9. wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

13. The engineered nuclease system of any one of claims 1-9, wherein the engineered guide polynucleotide comprises a sequence having 90% sequence identity' to any one of SEQ ID NOs: 632, 9-23, 85-103. 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

14. The engineered nuclease system of any one of claims 1-9, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

15. An engineered nuclease system, comprising:a) an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 194; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

16. The engineered nuclease system of claim 15, wherein the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 194.

17. The engineered nuclease system of claim 15, wherein the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E255K, and N329E as compared to SEQ ID NO: 194.

18. The engineered nuclease system of claim 15, wherein the endonuclease comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 156-191.

19. The engineered nuclease system of claim 15, wherein the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 156-191.

20. The engineered nuclease system of claim 15, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 156-191.

21. The engineered nuclease system of claim 15, wherein the endonuclease comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 156-191.

22. The engineered nuclease system of claim 15, wherein the endonuclease comprises one or more of the following sets of amino acid modifications selected from the group consisting of:- 109 - #593201Attomey Docket No. 00010.032.1801i) E236K and E255K;ii) M204K and E236K;iii) K93R, E236K, and E255K;iv) K93R, E202K, and E236K;v) M204K. E236K, and N329E; andvi) K93R, M204K, and E236K as compared to SEQ ID NO: 194.

23. An engineered nuclease system, comprising:a) an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 176, 124, 158. 166, 172, 179. 183, 104-123, 125-157, 159- 165, 167-171, 173-175, 177, 178, and 180-191; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 626-636, 643-652, and 786-788.

24. The engineered nuclease system of claim 23, wherein the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 176, 124, 158, 166. 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191.

25. The engineered nuclease system of claim 23, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191.

26. The engineered nuclease system of claim 23, wherein the endonuclease comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs:

176. 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, and 180-191.

27. The engineered nuclease system of any one of claims 15-26, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

28. The engineered nuclease system of any one of claims 15-27, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

29. The engineered nuclease system of any one of claims 15-27, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

30. The engineered nuclease system of any one of claims 1-29, wherein the engineered guide polynucleotide is a single guide nucleic acid.- 110 - #593201Attorney Docket No. 00010.032.180131. The engineered nuclease system of any one of claims 1-29, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

32. The engineered nuclease system of any one of claims 1-29, wherein the engineered guide polynucleotide is RNA.

33. The engineered nuclease system of any one of claims 1-32, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

34. The engineered nuclease system of any one of claims 1-32, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

35. The engineered nuclease system of any one of claims 1-32, wherein the endonuclease is fused to the engineered guide polynucleotide.

36. The engineered nuclease system of any one of claims 1-35, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193. 626-636. 643-652, and 786-788.

37. The engineered nuclease system of any one of claims 1-35, wherein the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 626-636, 643-652, and 786-788.

38. The engineered nuclease system of any one of claims 1-35, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 626-636, 643-652, and 786-788.

39. A method for modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with the engineered nuclease system of any one of claims 1-38.

40. A method for modifying a target nucleic acid sequence within a dystrophin gene of a mammalian cell comprising contacting the mammalian cell with the engineered nuclease system of any one of claims 1-38.

41. The method of claim 39 or 40, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence.

42. The method of any one of claims 39-41, wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

43. The method of any one of claims 39-42, wherein the modification is in vitro, in vivo, or ex vivo.

44. The method of any one of claims 39-43, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.- I l l - #593201Attomey Docket No. 00010.032.180145. The method of any one of claims 39-43, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

46. A method of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: a) (i) a base editor encoded by a sequence having at least 70% sequence identify to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 397, 457, 479, 398, 376-396, 399-456, 458-478, 480-484, and 485-545, or (li) an endonuclease encoded by a sequence having at least 70% sequence identify to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence.

47. A method of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: a) (i) a base editor encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 216, 276, 298, 195-215, 217, 218-275, 277-297, 299-303, 304-364, and 589-590 or comprising a sequence having at least 70% sequence identify to any one of SEQ ID NOs:

397.

457.

479. 398, 376-396, 399-456, 458-478. 480-484, and 485-545, or (ii) an endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587 or an endonuclease comprising an amino acid sequence having at least 70% sequence identify to any one ofSEQ IDNOs: 194, 176, 124, 158, 166, 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178, 180-191, 5-8, and 886-891; and b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

48. The method of claim 46 or 47, wherein the endonuclease is encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

49. The method of claim 46 or 47, wherein the endonuclease is encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs:

588. 785, 1-4, and 582-587.- 112 - #593201Attorney Docket No. 00010.032.180150. The method of claim 46 or 47, wherein the endonuclease is encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 588, 785, 1-4, and 582-587.

51. The method of any one of claims 46-50, wherein the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs:

194. 176, 124, 158, 166.

172. 179, 183. 104-123. 125-157. 159-165. 167-171. 173-175, 177, 178. 180-191. 5-8, and 886-891.

52. The method of any one of claims 46-50, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166. 172, 179, 183. 104-123. 125-157, 159-165, 167-171, 173-175, 177, 178. 180-191. 5-8, and 886-891.

53. The method of any one of claims 46-50, wherein the endonuclease comprises an amino acid sequence having at least 100% sequence identity to any one of SEQ ID NOs: 194, 176, 124, 158, 166. 172, 179, 183, 104-123, 125-157, 159-165, 167-171, 173-175, 177, 178. 180-191, 5-8, and 886-891.

54. The method of any one of claims 46-50, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578. and 838-885.

55. The method of any one of claims 46-50, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

56. The method of any one of claims 46-50, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

57. The method of any one of claims 46-56, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

58. The method of any one of claims 46-56, wherein the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581. 593-625. 626-631, 633-636. 643-652. 637-642. and 786-788.

59. The method of any one of claims 46-56, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.- 113 - #593201Attomey Docket No. 00010.032.180160. A method of modifying atarget nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: a) an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 194; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence within a dystrophin gene.

61. A method of modifying a target nucleic acid sequence within a dystrophin gene comprising contacting the target nucleic acid sequence with an engineered nuclease system comprising: a) an endonuclease comprises an amino acid sequence having at least 70% sequence identify to any one of SEQ ID NOs: 156-191; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to the target nucleic acid sequence, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

62. The method of claim 60 or 61, wherein the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 194.

63. The method of claim 60 or 61, wherein the endonuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 156-191.

64. The method of claim 60 or 61, wherein the endonuclease comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 156-191.

65. The method of claim 60 or 61, wherein the endonuclease comprises an amino acid sequence having at least 100% sequence identity to any one of SEQ ID NOs: 156-191.

66. The method of claim 60, wherein the endonuclease comprises one or more of the following sets of amino acid modifications selected from the group consisting of:i) E236K and E255K;li) M204K and E236K;iii) K93R, E236K, and E255K;iv) K93R, E202K, and E236K;v) M204K, E236K, and N329E; andvi) K93R, M204K, and E236K as compared to SEQ ID NO: 194.

67. The method of one of claims 60-66, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.- 114 - #593201Attorney Docket No. 00010.032.180168. The method of any one of claims 60-66, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

69. The method of any one of claims 60-66, wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 24-38, 575, 578, and 838-885.

70. The method of any one of claims 60-69, wherein the engineered guide polynucleotide is a single guide nucleic acid.

71. The method of any one of claims 60-69, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

72. The method of any one of claims 60-69, wherein the engineered guide polynucleotide is RNA.

73. The method of any one of claims 60-72, wherein the engineered endonuclease binds non-covalently to the engineered guide polynucleotide.

74. The method of any one of claims 60-73, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

75. The method of any one of claims 60-73, wherein the endonuclease is fused to the engineered guide polynucleotide.

76. The method of any one of claims 60-75, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

77. The method of any one of claims 60-75, wherein the engineered guide polynucleotide comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

78. The method of any one of claims 60-75. wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 632, 9-23, 85-103, 192, 193, 365-368, 375, 369-374, 581, 593-625, 626-631, 633-636, 643-652, 637-642, and 786-788.

79. A cell comprising the engineered nuclease system of any one of claims 1-38.

80. The cell of claim 79, wherein the cell is a eukaryotic cell, a mammalian cell, a muscle cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell.- 115 - #593201Attomey Docket No. 00010.032.180181. The cell of claim 80, wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

82. The cell of claim 80, wherein the cell is an engineered cell and / or a stable cell.

83. A viral vector comprising the engineered nuclease system of any one of claims 1-38.

84. The viral vector of claim 83, wherein the viral vector is an adeno-associated viral (AAV) vector.

85. The viral vector of claim 84, wherein the AAV is AAV1, AAV2, AAV3. AAV4, AAV5, AAV6. AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12. AAV 13, AAV 14, AAV 15, AAV 16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7. AAV-HSC8, AAV-HSC9. AAV-HSC10. AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof.- 116 - #593201

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