IP Library › Granted Patent US 12,431,216
Granted Patent B2
US 12,431,216 · App. 16/326,132 · Granted Sep 30, 2025

Methods for identifying class 2 crispr-cas systems

Inventors: Sergey Shmakov (Moscow, RU); Kira S. Makarova (Bethesda, MD); Yuri I. Wolf (Bethesda, MD); Aaron Smargon (Cambridge, MA); Neena Pyzocha (Cambridge, MA); David Cox (Cambridge, MA); Winston Yan (Brookline, MA); David Scott (Cambridge, MA); Konstantin Severinov (Piscataway, NJ); Feng Zhang (Cambridge, MA); Eugene V. Koonin (Bethesda, MD)
Assignees: Broad Institute, Inc.; Massachusetts Institute of Technology; Rutgers, The State University of New Jersey; The United States of America, as represented by the Secretary, Department of Health and Human Services
G16B20/20C12N9/22C12N15/11G16B30/10G16B40/30C12N2310/20C12N2800/80
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Quick Facts
Patent No.
US 12,431,216
App. No.
16/326,132
Granted
Sep 30, 2025
Kind
B2
Abstract

Disclosed here is a method of identifying novel CRISPR effectors, comprising: identifying sequences in a genomic or metagenomic database encoding a CRISPR array; identifying one or more Open Reading Frames (ORFs) in said selected sequences within 10 kb of the CRISPR array; discarding all loci encoding proteins which are assigned to known CRISPR-Cas subtypes and, optionally, all loci encoding a protein of less than 700 amino acids; and identifying putative novel CRISPR effectors, and optionally classifying them based on structure analysis.

Claims (6)

1. An engineered, or non-naturally occurring composition comprising a Class 2, Type V-U Cas protein encoded by a sequence selected from SEQ ID NO: 14 and a guide RNA capable of forming a complex with the Class 2, Type V-U Cas protein and comprising a guide sequence heterologous to a naturally occurring guide sequence, wherein the guide sequence directs sequence-specific binding of the complex to a target sequence other than a naturally occurring protospacer of the Class 2, Type V-U Cas protein/guide RNA complex.

2. The composition of claim 1 , wherein the Class 2, Type V-U Cas protein is a nuclease adapted to modify a DNA strand.

3. The composition of claim 1 , wherein the Class 2, Type V-U Cas protein is a TnpB homolog.

4. The composition of claim 1 , wherein the Class 2, Type V-U Cas protein is fused to one or more nuclear localization sequences.

5. The composition of claim 1 , wherein the guide sequence further comprises a tracr sequence and a tracr mate sequence.

6. The composition of claim 1 , wherein the guide sequence is about 10-30 nucleotides long.

Assignments (8)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 10, 2021
From: KOONIN, EUGENE V
To: UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT OF HEALTH AND HUMAN SERVICES, THE; THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HEALTH AND HUMAN SERVICES
Reel/Frame 057440/0319 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 9, 2021
From: KOONIN, EUGENE V
To: UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT OF HEALTH AND HUMAN SERVICES, THE; THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HEALTH AND HUMAN SERVICES
Reel/Frame 057431/0634 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 23, 2021
From: WOLF, YURI I
To: UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT OF HEALTH AND HUMAN SERVICES, THE
Reel/Frame 056955/0374 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 20, 2021
From: MAKAROVA, KIRA S.
To: UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT OF HEALTH AND HUMAN SERVICES, THE
Reel/Frame 056910/0085 →
CONFIRMATORY LICENSE Recorded Mar 9, 2020
From: BROAD INSTITUTE, INC.
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 052122/0043 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 16, 2019
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 050730/0449 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 15, 2019
From: COX, DAVID BENJAMIN TURITZ
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 050720/0447 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 26, 2019
From: SEVERINOV, KONSTANTIN
To: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
Reel/Frame 050504/0312 →
Continuity (2)
Provisional Application 62376387 · Aug 17, 2016
Related Publication 20210166783A1 · Jun 3, 2021
References Cited (90)
US 8697359B1 · Zhang · 2014 [cited by applicant]
US 8771945B1 · Zhang · 2014 [cited by applicant]
US 8795965B2 · Zhang · 2014 [cited by applicant]
US 8865406B2 · Zhang et al. · 2014 [cited by applicant]
US 8871445B2 · Cong et al. · 2014 [cited by applicant]
US 8889356B2 · Zhang · 2014 [cited by applicant]
US 8889418B2 · Zhang et al. · 2014 [cited by applicant]
US 8895308B1 · Zhang et al. · 2014 [cited by applicant]
US 8906616B2 · Zhang et al. · 2014 [cited by applicant]
US 8932814B2 · Cong et al. · 2015 [cited by applicant]
US 8945839B2 · Zhang · 2015 [cited by applicant]
US 8993233B2 · Zhang et al. · 2015 [cited by applicant]
US 8999641B2 · Zhang et al. · 2015 [cited by applicant]
US 10815730B2 · Liu et al. · 2020 [cited by applicant]
US 11041173B2 · Zhang et al. · 2021 [cited by applicant]
US 20040171156A1 · Hartley et al. · 2004 [cited by applicant]
US 20110265198A1 · Gregory et al. · 2011 [cited by applicant]
US 20120017290A1 · Cui et al. · 2012 [cited by applicant]
US 20130236946A1 · Gouble · 2013 [cited by applicant]
US 20140170753A1 · Zhang · 2014 [cited by applicant]
US 20140179006A1 · Zhang · 2014 [cited by applicant]
US 20140179770A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186843A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186919A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186958A1 · Zhang et al. · 2014 [cited by applicant]
US 20140189896A1 · Zhang et al. · 2014 [cited by applicant]
US 20140227787A1 · Zhang · 2014 [cited by applicant]
US 20140234972A1 · Zhang · 2014 [cited by applicant]
US 20140242664A1 · Zhang et al. · 2014 [cited by applicant]
US 20140242699A1 · Zhang · 2014 [cited by applicant]
US 20140242700A1 · Zhang et al. · 2014 [cited by applicant]
US 20140248702A1 · Zhang et al. · 2014 [cited by applicant]
US 20140256046A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273231A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273232A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273234A1 · Zhang et al. · 2014 [cited by applicant]
US 20140287938A1 · Zhang et al. · 2014 [cited by applicant]
US 20140310830A1 · Zhang et al. · 2014 [cited by applicant]
US 20150247150A1 · Zhang et al. · 2015 [cited by applicant]
US 20150291965A1 · Zhang et al. · 2015 [cited by applicant]
US 20160251648A1 · Wang et al. · 2016 [cited by applicant]
US 20170198269A1 · Zhang et al. · 2017 [cited by applicant]
US 20170349894A1 · Dahlman et al. · 2017 [cited by applicant]
US 20170349914A1 · Cox et al. · 2017 [cited by applicant]
US 20180010134A1 · Sharp et al. · 2018 [cited by applicant]
US 20180044662A1 · Platt et al. · 2018 [cited by applicant]
US 20180057810A1 · Zhang et al. · 2018 [cited by applicant]
US 20190203212A1 · Zhang et al. · 2019 [cited by applicant]
US 20190330605A1 · Zhang et al. · 2019 [cited by applicant]
US 20200318123A1 · Zhang · 2020 [cited by applicant]
US 20200354751A1 · Zhang et al. · 2020 [cited by applicant]
EP 2784162A1 · 2014 [cited by applicant]
EP 2771468B1 · 2015 [cited by applicant]
EP 2764103B1 · 2015 [cited by applicant]
WO WO2012164565A1 · 2012 [cited by examiner]
WO 2014018423A2 · 2014 [cited by applicant]
WO 2014093595A1 · 2014 [cited by applicant]
WO 2014093622A2 · 2014 [cited by applicant]
WO 2014093635A1 · 2014 [cited by applicant]
WO 2014093655A2 · 2014 [cited by applicant]
WO 2014093661A2 · 2014 [cited by applicant]
WO 2014093694A1 · 2014 [cited by applicant]
WO 2014093701A1 · 2014 [cited by applicant]
WO 2014093709A1 · 2014 [cited by applicant]
WO 2014093712A1 · 2014 [cited by applicant]
WO 2014093718A1 · 2014 [cited by applicant]
WO 2014204723A1 · 2014 [cited by applicant]
WO 2014204724A1 · 2014 [cited by applicant]
WO 2014204725A1 · 2014 [cited by applicant]
WO 2014204726A1 · 2014 [cited by applicant]
WO 2014204727A1 · 2014 [cited by applicant]
WO 2014204728A1 · 2014 [cited by applicant]
WO 2014204729A1 · 2014 [cited by applicant]
WO 2015065964A1 · 2015 [cited by applicant]
WO 2018035250A1 · 2018 [cited by applicant]
Ngo et al. in The Protein Folding Problem and Tertiary Structure Prediction, 1994, Merz et al. (ed.), Birkhauser, Boston, MA, pp. 433 and 492-495. [cited by examiner]
Stella et al. (Nature Structural & Molecular Biology, vol. 24 No. 11, pp. 882-892, Nov. 2017). [cited by examiner]
Doggett et al., GenBank: AFQ19722.1. Jul. 26, 2016. [cited by examiner]
Database EMBL [OnLine], “ [cited by applicant]
Rahmatabadi et al., “Studying the features of 57 confirmed CRISPR loci in 29 strains of [cited by applicant]
Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, vol. 60, No. 3, Nov. 1, 2015, pp. 385-397. [cited by applicant]
The Broad Institute, Inc., “Communication pursuant to Article 94(3) EPC for EP 17764475.4”, May 14, 2021, 3 pages. [cited by applicant]
The Broad Institute, Inc., “Communication Pursuant to Rule 164(2)(b) and Article 94(3) EPC for EP 17764475.4”, Jun. 25, 2020, 8 pages. [cited by applicant]
Shmakov, et al., “Diversity and Evolution of Class 2 CRISPR-Cas Systems”, Nature Reviews, Microbiology, vol. 15, No. 3, Mar. 2017, pp. 169-182. [cited by applicant]
Shmakov, et al., “Supplementary Information S3 (figure). Multiple Alignment of Representatives from Five V-U Families”, Nature Reviews, Microbiology, vol. 15, No. 3, Jan. 2017, 17 pages. [cited by applicant]
The Broad Institute, Inc. “International Preliminary Report on Patentability received in PCT Application No. PCT/US2017/047193”, mailed on Feb. 28, 2019, 11 pages. [cited by applicant]
The Broad Institute, Inc. “International Search Report & Written Opinion received in PCT Application No. PCT/US2017/047193”, mailed on Nov. 18, 2018, 15 pages. [cited by applicant]
Chung, et al., “Polycistronic RNA polymerase II Expression Vectors for RNA Interference Based on BIC/miR-155”, Nucleic Acids Research, vol. 34, No. 7, e53, Apr. 13, 2006, 14 pages. [cited by applicant]
Shmakov, et al., “Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems”, Mol Cell. 60(3), 2015, pp. 385-397. [cited by applicant]
Gao, et al., “Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition”, Cell Res 2016, 26, 901-913. [cited by applicant]