IP Library › Granted Patent US 12,410,449
Granted Patent B2
US 12,410,449 · App. 18/669,712 · Granted Sep 9, 2025

Endonuclease systems

Inventors: Brian C. Thomas (Berkeley, CA); Christopher Brown (Albany, CA); Cristina Butterfield (Oakland, CA); Lisa Alexander (Albany, CA); Daniela S. A. Goltsman (Oakland, CA); Rebecca Lamothe (Albany, CA); Isabel Nocedal (Oakland, CA)
Assignee: Metagenomi, Inc.
C12N15/907C12N9/22C12N15/11C12N15/902C12N2310/20C12N2310/531C12N2800/80
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Quick Facts
Patent No.
US 12,410,449
App. No.
18/669,712
Granted
Sep 9, 2025
Kind
B2
Abstract

The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.

Claims (39)

1. An engineered nuclease system comprising:

(a) an endonuclease or a nucleic acid encoding said endonuclease, wherein said endonuclease comprises a RuvC domain and an HNH domain, wherein said endonuclease comprises a sequence having at least 85% sequence identity to SEQ ID NO: 1316; and

(b) an engineered guide ribonucleic acid structure or a nucleic acid encoding said engineered guide ribonucleic acid structure, wherein said engineered guide ribonucleic acid structure is configured to form a complex with said endonuclease, wherein said engineered guide ribonucleic acid structure comprises:

(i) a guide ribonucleic acid sequence configured to hybridize to a target nucleic acid sequence; and

(ii) a tracr ribonucleic acid sequence configured to bind to said endonuclease.

2. The engineered nuclease system of claim 1 , wherein said endonuclease is an archaeal endonuclease.

3. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises one or more of: an arginine-rich region comprising an RRxRR motif (SEQ ID NO: 1361), a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain.

4. The engineered nuclease system of claim 3 , wherein said arginine-rich region, said domain with PF14239 homology, said recognition (REC) domain, said bridge helix (BH) domain, said wedge (WED) domain, or said PAM interacting (PI) domain comprises a sequence having at least 85% sequence identity to an arginine-rich region comprising an RRxRR motif (SEQ ID NO: 1361), a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain, respectively, of SEQ ID NO: 1316.

5. The engineered nuclease system of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N-terminus or a C-terminus of said endonuclease.

6. The engineered nuclease system of claim 1 , wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1316.

7. The engineered nuclease system of claim 6 , wherein said endonuclease comprises a sequence of SEQ ID NO: 1316.

8. The engineered nuclease system of claim 1 , wherein said tracr ribonucleic acid sequence comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 200.

9. The engineered nuclease system of claim 8 , wherein said tracr ribonucleic acid sequence comprises a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 200.

10. The engineered nuclease system of claim 9 , wherein said tracr ribonucleic acid sequence comprises a polynucleotide sequence of SEQ ID NO: 200.

11. The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 613, 615, or 616.

12. The engineered nuclease system of claim 11 , wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 90% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 613, 615, or 616.

13. The engineered nuclease system of claim 12 , wherein said engineered guide ribonucleic acid structure comprises non-degenerate nucleotides any one of SEQ ID NOs: 613, 615, or 616.

14. The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises:

(a) at least two ribonucleic acid polynucleotides; or (b) a single ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence.

15. The engineered nuclease system of claim 1 , wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, a fungal, a plant, a mammalian, or a human genomic sequence.

16. The engineered nuclease system of claim 1 , further comprising a single-stranded or double-stranded deoxyribonucleic acid repair template.

17. The engineered nuclease system of claim 16 , wherein said single-stranded or double-stranded deoxyribonucleic acid repair template comprises a transgene donor.

18. The engineered nuclease system of claim 1 , wherein said sequence identity is determined by a BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.

19. A method of modifying a target nucleic acid locus, said method comprising contacting said target nucleic acid locus with:

(a) an endonuclease comprising a RuvC domain and an HNH domain, wherein said endonuclease comprises a sequence having at least 85% sequence identity to SEQ ID NO: 1316; and

(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease, wherein said engineered guide ribonucleic acid structure comprises:

(i) a guide ribonucleic acid sequence configured to hybridize to a portion of said target nucleic acid locus; and

(ii) a tracr ribonucleic acid sequence configured to bind to said endonuclease,

wherein said complex modifies said target nucleic acid locus.

20. The method of claim 19 , wherein said endonuclease further comprises one or more of: an arginine-rich region comprising an RRxRR motif (SEQ ID NO: 1361), a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain.

21. The method of claim 20 , wherein said arginine-rich region, said domain with PF14239 homology, said recognition (REC) domain, said bridge helix (BH) domain, said wedge (WED) domain, or said PAM interacting (PI) domain comprises a sequence having at least 85% sequence identity to an arginine-rich region comprising an RRxRR motif (SEQ ID NO: 1361), a domain with PF14239 homology, a recognition (REC) domain, a bridge helix (BH) domain, a wedge (WED) domain, or a PAM interacting (PI) domain, respectively, of SEQ ID NO: 1316.

22. The method of claim 19 , wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1316.

23. The method of claim 22 , wherein said endonuclease comprises a sequence of SEQ ID NO: 1316.

24. The method of claim 19 , wherein said tracr ribonucleic acid sequence comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 200.

25. The method of claim 24 , wherein said tracr ribonucleic acid sequence comprises a polynucleotide sequence of SEQ ID NO: 200.

26. The method of claim 19 , wherein said modifying comprises binding, nicking, cleaving, or marking said target nucleic acid locus.

27. The method of claim 19 , wherein said target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

28. The method of claim 19 , wherein said target nucleic acid locus is within a cell.

29. The method of claim 28 , wherein said cell is a eukaryotic cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 23, 2025
From: NOCEDAL, ISABEL
To: METAGENOMI, INC.
Reel/Frame 071808/0593 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 3, 2025
From: THOMAS, BRIAN C.; BROWN, CHRISTOPHER; DEVOTO, AUDRA; BUTTERFIELD, CRISTINA; ALEXANDER, LISA; GOLTSMAN, DANIELA S.A.; LAMOTHE, REBECCA
To: METAGENOMI, INC.
Reel/Frame 071304/0931 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 11, 2025
From: THOMAS, BRIAN C.; BROWN, CHRISTOPHER; DEVOTO, AUDRA; BUTTERFIELD, CRISTINA; ALEXANDER, LISA; GOLTSMAN, DANIELA S.A.; LAMOTHE, REBECCA
To: METAGENOMI, INC.
Reel/Frame 070473/0857 →
Continuity (5)
Continuation PCTUS2022080437 · Nov 23, 2022
Provisional Application 63356908 · Jun 29, 2022
Provisional Application 63289981 · Dec 15, 2021
Provisional Application 63282999 · Nov 24, 2021
Related Publication 20240294948A1 · Sep 5, 2024
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