US 12,168,765 B2
S. pyogenes CAS9 mutant genes and polypeptides encoded by same
Christopher Anthony Vakulskas, North Liberty, IA (US); Michael Allen Collingwood, North Liberty, IA (US); Garrett Richard Rettig, Coralville, IA (US); and Mark Aaron Behlke, Coralville, IA (US)
Assigned to Integrated DNA Technologies, Inc., Coralville, IA (US)
Filed by INTEGRATED DNA TECHNOLOGIES, INC., Coralville, IA (US)
Filed on Jul. 21, 2022, as Appl. No. 17/870,190.
Application 16/931,889 is a division of application No. 15/729,491, filed on Oct. 10, 2017, granted, now 10,717,978, issued on Jul. 17, 2020.
Application 17/870,190 is a continuation of application No. 16/931,889, filed on Jul. 17, 2020, granted, now 11,427,818.
Claims priority of provisional application 62/405,601, filed on Oct. 7, 2016.
Prior Publication US 2023/0133277 A1, May 4, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01)
CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 15/902 (2013.01); C12N 15/113 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 7 Claims
 
1. A nucleic acid vector encoding an isolated mutant Cas9 protein, wherein the isolated mutant Cas9 protein is active in a CRISPR/Cas endonuclease system, wherein the CRISPR/Cas endonuclease system displays a lower ratio of off-target editing activity to on-target editing activity for at least one target relative to the corresponding ratio of off-target editing activity to on-target editing activity of a wild-type CRISPR/Cas endonuclease system having the wild-type Cas9 protein of SEQ ID NO: 5,
wherein the isolated mutant Cas9 protein comprises a substitution mutation selected from the group consisting of
(a) a single substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO: 5 selected from the following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, S793, N803, N854, S872 and R925; or
(b) a double substitution mutation introduced into the WT-Cas9 protein of SEQ ID NO: 5 selected from two of following positions: R494, N522, N588, N612, T657, S663, R691, N692, S730, T740, R765, T770, N776, R778, R783, S793, N803, S845, N854, S872 and R925.