US 12,258,595 B2
Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
Silvana Konermann, Cambridge, MA (US); Alexandro Trevino, Cambridge, MA (US); Mark Brigham, Cambridge, MA (US); Fei Ran, Cambridge, MA (US); Patrick Hsu, Cambridge, MA (US); Chie-Yu Lin, Cambridge, MA (US); Osamu Nureki, Tokyo (JP); Hiroshi Nishimasu, Tokyo (JP); Ryuichiro Ishitani, Tokyo (JP); and Feng Zhang, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTIT JTE OF TECHNOLOGY, Cambridge, MA (US); UNIVERSITY OF TOKYO, Tokyo (JP); 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); University of Tokyo, Tokyo (JP); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Dec. 27, 2022, as Appl. No. 18/089,023.
Application 16/592,744 is a division of application No. 15/179,912, filed on Jun. 10, 2016, granted, now 10,550,372, issued on Feb. 4, 2020.
Application 18/089,023 is a continuation of application No. 16/592,744, filed on Oct. 3, 2019, granted, now 11,597,919.
Application 15/179,912 is a continuation in part of application No. PCT/US2014/070175, filed on Dec. 12, 2014.
Claims priority of provisional application 61/939,256, filed on Feb. 12, 2014.
Claims priority of provisional application 61/980,012, filed on Apr. 15, 2014.
Claims priority of provisional application 61/915,251, filed on Dec. 12, 2013.
Claims priority of provisional application 61/939,242, filed on Feb. 12, 2014.
Claims priority of provisional application 62/055,484, filed on Sep. 25, 2014.
Claims priority of provisional application 61/930,214, filed on Jan. 22, 2014.
Claims priority of provisional application 61/915,267, filed on Dec. 12, 2013.
Claims priority of provisional application 62/087,537, filed on Dec. 4, 2014.
Prior Publication US 2024/0035007 A1, Feb. 1, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); A61K 38/46 (2006.01); A61K 48/00 (2006.01); A61P 43/00 (2006.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01); C12Q 1/6876 (2018.01)
CPC C12N 9/22 (2013.01) [A61K 38/465 (2013.01); A61K 48/005 (2013.01); C12N 15/113 (2013.01); C12N 15/902 (2013.01); C12N 15/907 (2013.01); C12Q 1/6876 (2013.01); C12Y 301/00 (2013.01); C12N 2310/20 (2017.05)] 30 Claims
 
1. A method for modifying a eukaryotic cell, comprising introducing a CRISPR-Cas system into the eukaryotic cell, wherein the CRISPR-Cas system comprises:
an engineered Cas9 protein comprising at least one mutation such that the Cas9 protein substantially lacks DNA cleavage activity, or a polynucleotide encoding the Cas9 protein;
a chimeric RNA comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in the eukaryotic cell, and a modified loop comprising an insertion of an aptamer sequence into a tetraloop and/or a stem-loop 2 of the chimeric RNA, or a polynucleotide encoding the chimeric RNA; and
an adaptor protein fused to at least one heterologous functional domain, or a polynucleotide encoding the adaptor protein;
wherein the Cas9 protein forms a CRISPR complex with the chimeric RNA, wherein the adaptor protein binds to the aptamer sequence, and wherein the guide sequence direct sequence-specific binding of the CRISPR complex to the target sequence.