Structural basis for mismatch surveillance by CRISPR-Cas9.

ABSTRACT: CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage^1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood^5-7. Although Cas9 variants with greater discrimination against mismatches have been designed^8-10, these suffer from substantially reduced rates of on-target DNA cleavage^5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that conta

SUBMITTER: Bravo JPK 

PROVIDER: S-EPMC8907077 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature