	US 12,351,838 B2
	Target-specific CRISPR mutant
Joon Sun Lee, Seoul (KR); Min Hee Jung, Seoul (KR); Yu Ri Choi, Gyeonggi-do (KR); and Jeong Joon Lee, Gyeonggi-do (KR)
Assigned to TOOLGEN INCORPORATED, Seoul (KR)
Appl. No. 17/606,502
Filed by TOOLGEN INCORPORATED, Seoul (KR)
PCT Filed Apr. 26, 2019, PCT No. PCT/KR2019/005110 § 371(c)(1), (2) Date Oct. 26, 2021, PCT Pub. No. WO2020/218657, PCT Pub. Date Oct. 29, 2020.
Prior Publication US 2022/0204955 A1, Jun. 30, 2022
Int. Cl. C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/85 (2006.01); C12N 15/88 (2006.01)

CPC C12N 9/22 (2013.01) [C12N 15/11 (2013.01); C12N 15/85 (2013.01); C12N 15/88 (2013.01); C12N 2310/20 (2017.05); C12N 2800/107 (2013.01); C12N 2800/80 (2013.01)]

11 Claims

1. An artificially engineered SpCas9 (Streptococcus pyogenes Cas9) variant comprising:

an amino acid sequence having a mutated sequence of SEQ ID NO: 30 wherein SEQ ID NO: 30 has substitutions of F539S, M763I and K890N,

wherein the mutated sequence consists of mutation selected from the group consisting of:

i) mutation at site 112th position, 492th position, 671th position and 735th position in SEQ ID NO: 30, wherein the mutations are that,

lysine at 112th position (K112) is substituted with asparagine,

isoleucine at 492th position (1492) is substituted with phenylalanine,

arginine at 671th position (R671) is substituted with histidine, and

lysine at 735th position (K735) is substituted with threonine;

ii) mutation at site 350th position, 1007th position, and 1021th position in SEQ ID NO: 30, wherein the mutations are that,

isoleucine at 350th position (1350) is substituted with valine,

glutamic acid at 1007th position (E1007) is substituted with valine, and

methionine at 1021th position (M1021) is substituted with cysteine;

iii) mutation at site 1277th position in SEQ ID NO: 30, wherein the mutation is that,

serine at 1277th position (S1277) is substituted with glycine;

iv) mutation at site 1007th position, 1192th position in SEQ ID NO: 30, wherein the mutations are that,

glutamic acid at 1007th position (E1007) is substituted with glycine, and

lysine at 1192th position (K1192) is substituted with arginine;

v) mutation at site 889th position in SEQ ID NO: 30, wherein the mutation is that, alanine at 889th position (A889) is substituted with valine;

vi) mutation at site 1191th position in SEQ ID NO: 30, wherein the mutation is that, lysine at 1191th position (K1191) is substituted with glutamic acid; and

vii) mutation at site 1007th position in SEQ ID NO: 30, wherein the mutation is that,

glutamic acid at 1007th position (E1007) is substituted with one amino acid selected from alanine, asparagine, aspartic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.