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US 11,685,935 B2Granted
Compact scaffold of Cas9 in the type II CRISPR system
AssigneeCellectis
InventorsPhilippe Duchateau, Claudia Bertonati
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Abstract
The present invention is in the field of CRISPR-Cas system for genome targeting. The present invention relates to new engineered Cas9 scaffolds and uses thereof. More particularly, the present invention relates to methods for genome targeting, cell engineering and therapeutic application. The present invention also relates to vectors, compositions and kits in which the new Cas9 scaffolds of the present invention are used.
Claims28
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.
6 more elements
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 800 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.
6 more elements
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 500 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.
6 more elements
Dependencies are inferred from the claim text.