US 12,123,032 B2
CRISPR enzyme mutations reducing off-target effects
Feng Zhang, Cambridge, MA (US); Linyi Gao, Cambridge, MA (US); Bernd Zetsche, Cambridge, MA (US); and Ian Slaymaker, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); and MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); and Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Nov. 26, 2019, as Appl. No. 16/697,018.
Application 16/697,018 is a continuation of application No. 16/158,295, filed on Oct. 11, 2018, granted, now 10,494,621.
Application 16/158,295 is a continuation of application No. 15/844,528, filed on Dec. 16, 2017, granted, now 10,876,100.
Application 15/844,528 is a continuation in part of application No. PCT/US2016/038034, filed on Jun. 17, 2016.
Claims priority of provisional application 62/269,876, filed on Dec. 18, 2015.
Claims priority of provisional application 62/255,256, filed on Nov. 13, 2015.
Claims priority of provisional application 62/237,360, filed on Oct. 5, 2015.
Claims priority of provisional application 62/207,312, filed on Aug. 19, 2015.
Claims priority of provisional application 62/181,453, filed on Jun. 18, 2015.
Prior Publication US 2020/0087641 A1, Mar. 19, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/90 (2006.01)
CPC C12N 9/22 (2013.01) [C12N 15/111 (2013.01); C12N 15/902 (2013.01); C12Y 301/00 (2013.01); C07K 2319/09 (2013.01); C12N 2310/20 (2017.05)] 13 Claims
 
1. An engineered S. pyogenes Cas9 protein comprising an HNH domain, a RuvC domain, and at least one modified amino acid residue in a groove between the HNH and RuvC domains, wherein the modified amino acid residue is an Alanine, and the unmodified amino acid residue is a K, R, or Q at a corresponding position of wild-type S. pyogenes Cas9 which has been substituted with said Alanine.