US 12,168,789 B2
Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
Feng Zhang, Cambridge, MA (US); Winston Yan, Cambridge, MA (US); Osamu Nureki, Tokyo (JP); Kaijie Zheng, Cambridge, MA (US); Le Cong, Cambridge, MA (US); Hiroshi Nishimasu, Tokyo (JP); Fei Ran, Cambridge, MA (US); and Yinqing Li, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US); and UNIVERSITY OF TOKYO, Tokyo (JP)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); President and Fellows of Harvard College, Cambridge, MA (US); and University of Tokyo, Tokyo (JP)
Filed on Jan. 26, 2023, as Appl. No. 18/101,867.
Application 18/101,867 is a continuation of application No. 15/838,720, filed on Dec. 12, 2017, granted, now 11,578,312.
Application 15/838,720 is a continuation in part of application No. PCT/US2016/038252, filed on Jun. 17, 2016.
Claims priority of provisional application 62/207,318, filed on Aug. 19, 2015.
Claims priority of provisional application 62/181,659, filed on Jun. 18, 2015.
Prior Publication US 2023/0287373 A1, Sep. 14, 2023
Int. Cl. C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01); G16B 15/00 (2019.01); G16B 15/20 (2019.01); G16B 15/30 (2019.01)
CPC C12N 9/22 (2013.01) [C12N 15/102 (2013.01); C12N 15/11 (2013.01); G16B 15/00 (2019.02); G16B 15/20 (2019.02); G16B 15/30 (2019.02); C07K 2299/00 (2013.01); C07K 2319/09 (2013.01); C07K 2319/095 (2013.01); C07K 2319/20 (2013.01); C07K 2319/70 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 25 Claims
 
1. A non-naturally occurring and engineered SaCas9 protein comprising at least one modification or mutation compared to a wild-type SaCas9 protein, wherein the modification or mutation comprises mutation of at least one amino acid of the SaCas9 protein that interacts with sugar-phosphate backbone of an sgRNA or sugar-phosphate backbone of a target DNA strand, wherein the at least one amino acid comprises Thr238, Tyr239, Lys248, Tyr256, Arg314, Asn394, Gln414, Tyr211, Trp229, Tyr230, Gly235, Arg245, Gly391, Thr392, Asn419, Leu446, Tyr651, or Arg654.