US 12,227,742 B2
Nucleic acid modifiers
Amit Choudhary, Boston, MA (US); Donghyun Lim, Cambridge, MA (US); Kurt Cox, Cambridge, MA (US); and Soumyashree Ashok Gangopadhyay, Boston, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); and The Brigham and Women's Hospital, Inc., Boston, MA (US)
Appl. No. 16/758,840
Filed by THE BROAD INSTITUTE, INC., Cambridge, MA (US); and THE BRIGHAM AND WOMEN'S HOSPITAL, INC., Boston, MA (US)
PCT Filed Oct. 23, 2018, PCT No. PCT/US2018/057182
§ 371(c)(1), (2) Date Apr. 23, 2020,
PCT Pub. No. WO2019/135816, PCT Pub. Date Jul. 11, 2019.
Claims priority of provisional application 62/575,948, filed on Oct. 23, 2017.
Claims priority of provisional application 62/765,347, filed on Aug. 20, 2018.
Prior Publication US 2021/0214724 A1, Jul. 15, 2021
Int. Cl. C12N 15/11 (2006.01); A61K 9/51 (2006.01); A61K 31/18 (2006.01); A61K 31/336 (2006.01); A61K 31/655 (2006.01); A61K 31/7088 (2006.01); A61K 38/46 (2006.01); A61K 47/60 (2017.01); C12N 9/22 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/11 (2013.01) [A61K 9/5153 (2013.01); A61K 31/18 (2013.01); A61K 31/336 (2013.01); A61K 31/655 (2013.01); A61K 31/7088 (2013.01); A61K 38/465 (2013.01); A61K 47/60 (2017.08); C12N 9/22 (2013.01); C12N 15/902 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 26 Claims
 
1. A composition comprising:
a truncated CRISPR-Cas protein comprising a nucleic acid binding domain, wherein the nucleic acid binding domain comprises amino acids of
(i) a RuvC domain, a bridge helix (BH) domain, a REC1 domain, and a PAM-interacting (PI) domain of Streptococcus pyogenes Cas9 (SpCas9),
(ii) the RuvC, BH, REC, Wedge (WED), phosphate lock loop (PLL), and PI domains of Staphylococcus aureus Cas9 (SaCas9), or
(iii) a WED domain, a REC1 domain, a REC2 domain, a PI domain, a BH domain, and a RuvC domain of Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1);
a guide RNA capable of complexing with the truncated CRISPR-Cas protein;
a single-stranded oligo donor (ssODN) wherein the ssODN is connected to the truncated CRISPR-Cas protein by an adaptor oligonucleotide that is at least 10 nucleotides in length and capable of hybridizing to a portion of the ssODN;
one or more small molecule NHEJ inhibitors connected to the truncated CRISPR-Cas protein and selected from the group consisting of SCR7-G, SCR7, L189, PK 76, KU-0060648, KU inhibitor, and analogs thereof, an inhibitor of DNA ligase IV, KU70, or KU80;
one or more small molecule HDR activators connect to the truncated CRISPR-Cas protein and selected from the group consisting of RS1 or analogs thereof or stimulator of RAD51 activity, or a combination thereof.