Compact scaffold of Cas9 in the type II CRISPR system
Inventors
Duchateau, Philippe • Bertonati, Claudia
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11685935-B2
Publication Date
2023-06-27
Expiration Date
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.
Core Innovation
The invention relates to genome targeting in a mammalian cell using a guide RNA and two nucleic acids encoding two separate Split Cas9 components. A target nucleic acid sequence comprising a PAM motif is selected, and a guide RNA comprising a sequence complementary to the target nucleic acid sequence is provided, such that the two separate Split Cas9 process the target nucleic acid sequence in the cell.
The Split Cas9 are functionally assigned by domain content, with one Split Cas9 providing a RuvC domain and the other Split Cas9 providing an HNH domain. The RuvC domain comprises at least one RuvC motif sequence D-I-G-T-N-S-V-G-W-A and has at least 90% identity with the amino acid sequence of SEQ ID NO:52, and the 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 and has at least 90% identity with the amino acid sequence of SEQ ID NO:53.
A size constraint is imposed, wherein one of the Split Cas9 is less than 1000 amino acids long, with dependent refinements to less than 800 amino acids long or less than 500 amino acids long. The description also includes dependent refinements directed to generating a double-strand break in the target nucleic acid and repairing the double-strand break to induce a genetic modification.
Claims Coverage
The independent claims cover genome targeting in a mammalian cell using a PAM-containing target, a complementary guide RNA, and two nucleic acids encoding separate Split Cas9 components providing RuvC and HNH domains. Across the independent claims, the main inventive features are the split RuvC/HNH architecture with motif and sequence-identity requirements and the split protein size thresholds (<1000, <800, <500 amino acids).
PAM-comprising target selection and complementary guide RNA
Selecting a target nucleic acid sequence comprising a PAM motif and providing a guide RNA comprising a sequence complementary to the target nucleic acid sequence.
Two nucleic acids encoding separate Split Cas9 for RuvC and HNH
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 the two separate Split Cas9 process the target nucleic acid sequence in the cell.
RuvC motif and sequence identity requirement
The RuvC domain comprises at least one RuvC motif sequence D-I-G-T-N-S-V-G-W-A and has at least 90% identity with the amino acid sequence of SEQ ID NO:52.
HNH motif and sequence identity requirement
The 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 and has at least 90% identity with the amino acid sequence of SEQ ID NO:53.
Split Cas9 length constraint
One of the Split Cas9 is less than 1000 amino acids long, or less than 800 amino acids long, or less than 500 amino acids long.
Across the independent claims, the invention is directed to genome targeting in a mammalian cell by selecting a PAM-containing target and providing a complementary guide RNA together with two separate Split Cas9 nucleic acids that are assigned to RuvC and HNH domains. The RuvC and HNH domains are defined by motif sequences with at least 90% identity to SEQ ID NO:52 and SEQ ID NO:53, respectively, and the claims further constrain one Split Cas9 to be below specified length thresholds.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?