US 12,435,320 B2
CRISPR having or associated with destabilization domains
Feng Zhang, Cambridge, MA (US); Bernd Zetsche, Cambridge, MA (US); and Amit Choudhary, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Oct. 27, 2020, as Appl. No. 17/081,387.
Application 17/081,387 is a continuation of application No. 15/633,126, filed on Jun. 26, 2017, abandoned.
Application 15/633,126 is a continuation in part of application No. PCT/US2015/067177, filed on Dec. 21, 2015.
Claims priority of provisional application 62/181,151, filed on Jun. 17, 2015.
Claims priority of provisional application 62/096,656, filed on Dec. 24, 2014.
Prior Publication US 2021/0139872 A1, May 13, 2021
Int. Cl. C12N 9/22 (2006.01); A01K 67/0275 (2024.01); A61K 48/00 (2006.01); C07K 14/72 (2006.01); C12N 9/06 (2006.01); C12N 15/10 (2006.01); C12N 15/90 (2006.01)
CPC C12N 9/22 (2013.01) [A01K 67/0275 (2013.01); A61K 48/005 (2013.01); C07K 14/721 (2013.01); C12N 9/003 (2013.01); C12N 15/102 (2013.01); C12N 15/907 (2013.01); C12Y 105/01003 (2013.01); A01K 2207/10 (2013.01); A01K 2217/05 (2013.01); A01K 2227/105 (2013.01); C07K 2319/00 (2013.01); C07K 2319/095 (2013.01)] 29 Claims
 
1. A composition comprising:
(a) a non-naturally occurring or engineered Cas9 attached to at least two destabilization domains (DD); wherein a first DD is attached to the N-terminus of the Cas9 and a second DD is attached to the C-terminus of the Cas9, the first and second DDs being the same or different; wherein the Cas9 is attached to the at least two DDs via fusion, tether, or non-covalent bond;
(b) a guide RNA that forms a CRISPR complex with the Cas9; and
(c) at least one stabilizing ligand that binds to at least one of the destabilization domains;
wherein binding of the at least one stabilizing ligand to at least one of the destabilization domains inhibits degradation of the Cas9 by proteasome, and wherein the guide RNA directs sequence-specific binding of the CRISPR complex to a target DNA.