IP Library › Granted Patent US 12,435,323
Granted Patent B2
US 12,435,323 · App. 18/586,929 · Granted Oct 7, 2025

Enzymes with RUVC domains

Inventors: Lisa Alexander (Albany, CA); Daniela S. A. Goltsman (Oakland, CA); Sarah Laperriere (Berkeley, CA); Morayma Temoche-Diaz (Oakland, CA)
Assignee: Metagenomi, Inc.
C12N9/22C12N15/102C12N15/11C12N2310/20
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Quick Facts
Patent No.
US 12,435,323
App. No.
18/586,929
Granted
Oct 7, 2025
Kind
B2
Abstract

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

Claims (42)

1. An engineered nuclease system comprising:

a) an endonuclease or a ribonucleic acid encoding said endonuclease, wherein said endonuclease comprises a sequence having at least 80% sequence identity to SEQ ID NO: 1433; and

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

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

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

wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 11145.

2. The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 90% sequence identity to non-degenerate nucleotides of SEQ ID NO: 11145.

3. The engineered nuclease system of claim 2 , wherein said engineered guide ribonucleic acid structure comprises the non-degenerate nucleotides of SEQ ID NO: 11145.

4. The engineered nuclease system of claim 1 , wherein said tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 11201.

5. The engineered nuclease system of claim 4 , wherein said tracr ribonucleic acid sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 11201.

6. The engineered nuclease system of claim 5 , wherein said tracr ribonucleic acid sequence comprises the sequence of SEQ ID NO: 11201.

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

a) at least two ribonucleic acid polynucleotides; or

b) one ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence.

8. The engineered nuclease system of claim 2 , wherein said tracr ribonucleic acid sequence comprises the sequence of SEQ ID NO: 11201.

9. The engineered nuclease system of claim 6 , wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1433.

10. The engineered nuclease system of claim 9 , wherein said endonuclease comprises the sequence of SEQ ID NO: 1433.

11. The engineered nuclease system of claim 6 , wherein said endonuclease comprises a RuvC_III domain, wherein said RuvC_III domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 3253.

12. The engineered nuclease system of claim 11 , wherein said RuvC_III domain comprises the sequence of SEQ ID NO: 3253.

13. The engineered nuclease system of claim 12 , wherein said endonuclease comprises an HNH domain, wherein said HNH domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 5068.

14. The engineered nuclease system of claim 13 , wherein said HNH domain comprises the sequence of SEQ ID NO: 5068.

15. The engineered nuclease system of claim 6 , wherein said endonuclease is a class 2, type II Cas endonuclease.

16. A method of editing a locus in a cell, said method comprising contacting to said cell:

a) an endonuclease or a ribonucleic acid encoding said endonuclease, wherein said endonuclease comprises a sequence having at least 80% sequence identity to SEQ ID NO: 1433; and

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

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

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

wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 80% sequence identity to non-degenerate nucleotides of SEQ ID NO: 11145.

17. The method of claim 16 , wherein said engineered guide ribonucleic acid structure comprises a sequence having at least 90% sequence identity to non-degenerate nucleotides of SEQ ID NO: 11145.

18. The method of claim 17 , wherein said engineered guide ribonucleic acid structure comprises the non-degenerate nucleotides of SEQ ID NO: 11145.

19. The method of claim 16 , wherein said tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO: 11201.

20. The method of claim 19 , wherein said tracr ribonucleic acid sequence comprises a sequence having at least 90% sequence identity to SEQ ID NO: 11201.

21. The method of claim 20 , wherein said tracr ribonucleic acid sequence comprises the sequence of SEQ ID NO: 11201.

22. The method of claim 17 , wherein said tracr ribonucleic acid sequence comprises the sequence of SEQ ID NO: 11201.

23. The method of claim 21 , wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1433.

24. The method of claim 23 , wherein said endonuclease comprises the sequence of SEQ ID NO: 1433.

25. The method of claim 21 , wherein said endonuclease comprises a RuvC_III domain, wherein said RuvC_III domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 3253.

26. The method of claim 25 , wherein said RuvC_III domain comprises the sequence of SEQ ID NO: 3253.

27. The method of claim 26 , wherein said endonuclease comprises an HNH domain, wherein said HNH domain comprises a sequence having at least 80% sequence identity to SEQ ID NO: 5068.

28. The method of claim 27 , wherein said HNH domain comprises the sequence of SEQ ID NO: 5068.

29. The method of claim 21 , wherein said endonuclease is a class 2, type II Cas endonuclease.

30. The method of claim 24 , further comprising contacting said cell with a deoxyribonucleic acid repair template.

Assignments (4)
RELEASE OF SECURITY INTEREST Recorded Dec 12, 2025
From: CATALIO NEXUS FUND III, LP
To: AFFINI-T THERAPEUTICS, INC.
Reel/Frame 073201/0168 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 10, 2025
From: LAPERRIERE, SARAH; TEMOCHE-DIAZ, MORAYMA
To: METAGENOMI, INC.
Reel/Frame 072212/0813 →
SECURITY INTEREST Recorded Mar 4, 2025
From: AFFINI-T THERAPEUTICS, INC.
To: CATALIO NEXUS FUND III, LP
Reel/Frame 070403/0862 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 3, 2024
From: THOMAS, BRIAN C.; BROWN, CHRISTOPHER; KANTOR, ROSE; DEVOTO, AUDRA; BUTTERFIELD, CRISTINA; ALEXANDER, LISA; GOLTSMAN, DANIELA S.A.; LIU, JASON; LAMOTHE, REBECCA; ESPINOSA, DIEGO; STORLIE, MEGHAN; COST, GREG
To: METAGENOMI, INC.
Reel/Frame 066991/0419 →
Continuity (10)
Continuation PCTUS2022041755 · Aug 29, 2022
Provisional Application 63369858 · Jul 29, 2022
Provisional Application 63322944 · Mar 23, 2022
Provisional Application 63319681 · Mar 14, 2022
Provisional Application 63316895 · Mar 4, 2022
Provisional Application 63282909 · Nov 24, 2021
Provisional Application 63252956 · Oct 6, 2021
Provisional Application 63245629 · Sep 17, 2021
Provisional Application 63237791 · Aug 27, 2021
Related Publication 20240200047A1 · Jun 20, 2024
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Uniprotkb/trembl: A0A1F0KNW4 ⋅ A0A1F0KNW4_9MICC HNHc domain-containing protein. [cited by applicant]
Uniprotkb/trembl: A0A1S1DAD0 ⋅ A0A1S1DAD0_9MICC HNH Cas9-type domain-containing protein. [cited by applicant]
Uniprotkb/trembl: A1F0PN46 ⋅ A0A1F0PN46_9MICC HNH Cas9-type domain-containing protein. [cited by applicant]
U.S. Appl. No. 17/193,173 Final Office Action dated Apr. 17, 2023. [cited by applicant]
U.S. Appl. No. 17/193,173 Non-Final Office Action dated Oct. 7, 2022. [cited by applicant]
U.S. Appl. No. 17/857,923 Advisory Action dated Aug. 8, 2023. [cited by applicant]
U.S. Appl. No. 17/857,923 Final Office Action dated Jun. 13, 2023. [cited by applicant]
U.S. Appl. No. 17/857,923 Non-Final Office Action dated Feb. 28, 2023. [cited by applicant]
U.S. Appl. No. 16/917,837 Office Action dated Aug. 26, 2020. [cited by applicant]
U.S. Appl. No. 16/917,838 Office Action dated Jul. 28, 2020. [cited by applicant]
U.S. Appl. No. 17/431,135 Office Action dated Feb. 16, 2024. [cited by applicant]
Weinberg, Z. et al, Extraordinary Structured Noncoding RNAs Revealed by Bacterial Metagenome Analysis, Nature 2009, vol. 462, No. 7273, pp. 656-659. [cited by applicant]
Xiao, N. et la., protr/ProtrWeb: R package and web server for generating various numerical representation schemes of protein sequences, Bioinformatics, 2015, vol. 31, No. 11, pp. 1857-1859. [cited by applicant]
Yan et al., Functionally diverse type V CRISPR-Cas systems. Science 363: 88-91 (2019). [cited by applicant]
Yang et al.: New CRISPR-Cas systems discovered. Cell Res. 27(3):313-314 doi:10.1038/cr.2017.21 (2017). [cited by applicant]
Yang, Z., PAML 4: Phylogenetic Analysis by Maximum Likelihood, Molecular Biology and Evolution, 2007, vol. 24, No. 8, pp. 1586-1591. [cited by applicant]
Kortleve, D. et al. Orthotopic editing of T-cell receptors. Cell Therapy vol. 3: 949-950 (2019). [cited by applicant]
NCBI GenBank: OGP48943.1—MAG: hypothetical protein A2022_01700 [Deltaproteobacteria bacterium GWF2_42_12], pp. 1-2 (Oct. 20, 2016). [cited by applicant]
Co-pending U.S. Appl. No. 19/028,339, inventors Thomas; Brian C. et al., filed Jan. 17, 2025. [cited by applicant]
Makarova, Kira S. et al. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biology Direct 6:38, 1-27 (2011). [cited by applicant]