US 12,264,341 B2
CRISPR-Cas enzymes with enhanced on-target activity
Benjamin Kleinstiver, Medford, MA (US); and Russell T. Walton, Waban, MA (US)
Assigned to The General Hospital Corporation, Boston, MA (US)
Filed by The General Hospital Corporation, Boston, MA (US)
Filed on Jan. 25, 2021, as Appl. No. 17/157,805.
Claims priority of provisional application 62/965,671, filed on Jan. 24, 2020.
Prior Publication US 2021/0261932 A1, Aug. 26, 2021
Int. Cl. C12N 9/22 (2006.01); C12N 9/10 (2006.01); C12N 9/78 (2006.01); C12N 15/90 (2006.01)
CPC C12N 9/22 (2013.01) [C12N 9/1007 (2013.01); C12N 9/78 (2013.01); C12N 15/907 (2013.01); C07K 2319/71 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 35 Claims
 
1. An isolated Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, comprising one of the following sets of mutations: A61R/N1317R, G1104K/N1317R, A61R/G1104K, S55R/G1104K, S55R/A61R, S55R/N1317R, G1104K/T1314R, A61R/T1314R, T1314R/N1317R, S55R/T1314R, A61R/G1104R, G1104R/N1317R, S55R/G1104R, G1104R/T1314R, G366R/G1104K, S55R/G366R, A61R/G366R, G366R/N1317R, G366R/T1314R, G366R/G1104R, G1104K/A1285R, A61R/A1285R, A1285R/N1317R, S55R/A1285R, A1285R/T1314R, G1104R/A1285R, G366R/A1285R, G1104K/A1285K, A61R/A1285K, A1285K/N1317R, S55R/A1285K, A1285K/T1314R, G1104R/A1285K, G366R/A1285K, G1104K/D1332R, A61R/D1332R, D1332R/N1317R, S55R/D1332R, G1104R/D1332R, G366R/D1332R, A1285K/D1332R, A1285R/D1332R, A61R/G1104K/N1317R, A61R/L1111R, A61R/A1322R, A61R/L1111R/A1322R, G1104K/L1111R, G1104K/A1322R, G1104K/L1111R/A1322R, N1317R/L1111R, N1317R/A1322R, N1317R/L1111R/A1322R, A61R/N1317R/L1111R, A61R/N1317R/A1322R, A61R/N1317R/L1111R/A1322R, G1104K/N1317R/L1111R, G1104K/N1317R/A1322R, G1104K/N1317R/L1111R/A1322R, A61R/G1104K/L1111R, A61R/G1104K/A1322R, A61R/G1104K/L1111R/A1322R, S55R/L1111R, S55R/A1322R, S55R/L1111R/A1322R, G366R/L1111R, G366R/A1322R, G366R/L1111R/A1322R, A1285K/L1111R, A1285K/A1322R, or A1285K/L1111R/A1322R.