US 12,123,014 B2
Class II, type V CRISPR systems
Brian Thomas, Emeryville, CA (US); Christopher Brown, Emeryville, CA (US); Audra Devoto, Emeryville, CA (US); Cristina Butterfield, Emeryville, CA (US); Lisa Alexander, Emeryville, CA (US); Daniela S. A. Goltsman, Emeryville, CA (US); Justine Albers, Emeryville, CA (US); Alan Brooks, Emeryville, CA (US); Greg Cost, Emeryville, CA (US); Morayma Temoche-Diaz, Emeryville, CA (US); Cindy Castelle, Emeryville, CA (US); and Rebecca Lamothe, Emeryville, CA (US)
Assigned to Metagenomi, Inc., Emeryville, CA (US)
Filed by METAGENOMI, INC., Emeryville, CA (US)
Filed on Apr. 18, 2023, as Appl. No. 18/302,387.
Application 18/302,387 is a continuation of application No. 17/706,466, filed on Mar. 28, 2022, granted, now 11,713,471.
Application 17/706,466 is a continuation of application No. PCT/US2021/021259, filed on Mar. 6, 2021.
Claims priority of provisional application 62/986,477, filed on Mar. 6, 2020.
Claims priority of provisional application 63/022,276, filed on May 8, 2020.
Claims priority of provisional application 63/045,815, filed on Jun. 29, 2020.
Claims priority of provisional application 63/068,316, filed on Aug. 20, 2020.
Claims priority of provisional application 63/069,699, filed on Aug. 24, 2020.
Claims priority of provisional application 63/116,157, filed on Nov. 19, 2020.
Prior Publication US 2024/0093239 A1, Mar. 21, 2024
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/90 (2013.01) [C12N 9/22 (2013.01); C12N 15/1068 (2013.01); C12N 15/11 (2013.01); C12N 15/111 (2013.01); C12N 15/902 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 28 Claims
 
1. A method of modifying a target polynucleotide, the method comprising contacting the target polynucleotide with an engineered nuclease system comprising:
(a) a class 2, type V Cas endonuclease configured to be selective for a protospacer adjacent motif (PAM) sequence comprising 5′-YYn-3′ (SEQ ID NO:3871), wherein the endonuclease comprises a WED II domain and a PAM-interacting domain having at least 80% sequence identity to a WED II domain and a PAM-interacting domain, respectively, of the amino acid sequence of SEQ ID NO: 215; and
(b) an engineered guide ribonucleic acid, wherein the engineered guide ribonucleic acid is configured to form a complex with the endonuclease and the engineered guide ribonucleic acid comprises a spacer sequence configured to hybridize to a region of the target polynucleotide;
thereby modifying the target polynucleotide.