	US 11,685,935 B2
	Compact scaffold of Cas9 in the type II CRISPR system
Philippe Duchateau, Draveil (FR); and Claudia Bertonati, Paris (FR)
Assigned to CELLECTIS, Paris (FR)
Appl. No. 14/892,707
Filed by CELLECTIS, Paris (FR)
PCT Filed May 28, 2014, PCT No. PCT/EP2014/061181 § 371(c)(1), (2) Date Nov. 20, 2015, PCT Pub. No. WO2014/191521, PCT Pub. Date Dec. 4, 2014.
Claims priority of application No. PA201370297 (DK), filed on May 29, 2013; and application No. PA201370771 (DK), filed on Dec. 13, 2013.
Prior Publication US 2016/0102324 A1, Apr. 14, 2016
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01)

CPC C12N 15/907 (2013.01) [C12N 9/22 (2013.01); C12N 15/902 (2013.01); C12N 15/1082 (2013.01); C12N 15/63 (2013.01); C12Y 301/00 (2013.01); C12Y 301/26004 (2013.01)]

28 Claims

1. A method of genome targeting in a mammalian cell comprising:

(a) selecting a target nucleic acid sequence comprising a PAM motif,

(b) providing a guide RNA comprising a sequence complementary to the target nucleic acid sequence;

(c) providing two nucleic acids encoding two separate Split Cas9, one Split Cas9 providing a RuvC domain and another Split Cas9 providing an HNH domain,

wherein said RuvC domain comprises at least one RuvC motif sequence D-I-G-T-N-S-V-G-W-A (amino acids 10-19 of SEQ ID NO:52) and has at least 90% identity with the amino acid sequence of SEQ ID NO:52,

wherein said HNH domain comprises at least one HNH motif sequence Y-D-V-D-H-I-V-P-Q-S-F-L-K-D-D-S (amino acids 589-604 of SEQ ID NO:53) and has at least 90% identity with the amino acid sequence of SEQ ID NO:53 and

wherein one of the Split Cas9 is less than 1000 amino acids long; and

(d) introducing into the cell said guide RNA and said two nucleic acids encoding two separate Split Cas9; such that said two separate Split Cas9 process the target nucleic acid sequence in the cell.