High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

ABSTRACT: The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H muta

SUBMITTER: Yuen CTL 

PROVIDER: S-EPMC8860571 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature