US 12,215,365 B2
Cas variants for gene editing
David R. Liu, Cambridge, MA (US); and Alexis Christine Komor, Cambridge, MA (US)
Assigned to President and Fellows of Harvard College, Cambridge, MA (US)
Filed by President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Aug. 20, 2021, as Appl. No. 17/408,306.
Application 17/408,306 is a continuation of application No. 16/374,634, filed on Apr. 3, 2019, granted, now 11,124,782.
Application 16/374,634 is a continuation of application No. 15/103,608, granted, now 10,465,176, previously published as PCT/US2014/070038, filed on Dec. 12, 2014.
Application 15/103,608 is a continuation of application No. 14/325,815, filed on Jul. 8, 2014, granted, now 11,053,481.
Application 15/103,608 is a continuation of application No. 14/326,109, filed on Jul. 8, 2014, granted, now 9,840,699.
Application 15/103,608 is a continuation of application No. 14/326,140, filed on Jul. 8, 2014, abandoned.
Application 15/103,608 is a continuation of application No. 14/326,269, filed on Jul. 8, 2014, granted, now 9,068,179.
Application 14/325,815 is a continuation of application No. 14/326,290, filed on Jul. 8, 2014, abandoned.
Application 14/325,815 is a continuation of application No. 14/326,318, filed on Jul. 8, 2014, abandoned.
Application 14/325,815 is a continuation of application No. 14/326,303, filed on Jul. 8, 2014, abandoned.
Application 15/103,608 is a continuation of application No. 14/325,815, filed on Jul. 8, 2014, granted, now 11,053,481.
Application 14/325,815 is a continuation of application No. 14/326,109, filed on Jul. 8, 2014, granted, now 9,840,699.
Application 14/325,815 is a continuation of application No. 14/326,140, filed on Jul. 8, 2014, abandoned.
Application 14/325,815 is a continuation of application No. 14/326,290, filed on Jul. 8, 2014, abandoned.
Application 14/325,815 is a continuation of application No. 14/326,318, filed on Jul. 8, 2014, abandoned.
Application 14/326,109 is a continuation of application No. 14/326,303, filed on Jul. 8, 2014, abandoned.
Application 15/103,608 is a continuation of application No. 14/326,269, filed on Jul. 8, 2014, granted, now 9,068,179.
Claims priority of provisional application 61/980,333, filed on Apr. 16, 2014.
Claims priority of provisional application 61/915,386, filed on Dec. 12, 2013.
Prior Publication US 2022/0119785 A1, Apr. 21, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); A61K 38/46 (2006.01); A61K 38/50 (2006.01); A61K 47/61 (2017.01); C12N 9/64 (2006.01); C12N 9/78 (2006.01); C12N 15/01 (2006.01); C12N 15/10 (2006.01); C12P 19/34 (2006.01); C12Q 1/6883 (2018.01)
CPC C12N 9/22 (2013.01) [A61K 38/465 (2013.01); A61K 38/50 (2013.01); A61K 47/61 (2017.08); C12N 9/6472 (2013.01); C12N 9/78 (2013.01); C12N 15/01 (2013.01); C12N 15/102 (2013.01); C12P 19/34 (2013.01); C12Q 1/6883 (2013.01); C12Y 301/00 (2013.01); C12Y 304/22062 (2013.01); C12Y 305/04 (2013.01); C12Y 305/04001 (2013.01); C12Y 305/04004 (2013.01); C12Y 305/04005 (2013.01); C07K 2319/00 (2013.01); C07K 2319/80 (2013.01); C12Q 2600/156 (2013.01); C12Y 301/22 (2013.01)] 37 Claims
 
1. A multi-molecular complex comprising:
(a) a cytidine deaminase;
(b) a Cas9 variant protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37, wherein the Cas9 variant comprises one or more nuclease inactivating mutations within a RuvC1 subdomain and/or a HNH subdomain; and
(c) a single guide RNA (sgRNA) comprising a region complementary to a strand of the target nucleic acid molecule;
wherein the cytidine deaminase, the target nucleic acid molecule, the Cas9 variant, and the sgRNA associate in a multi-molecular complex; and
wherein the multi-molecular complex deaminates a cytidine in the target nucleic acid molecule.