Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

ABSTRACT: There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitativ

SUBMITTER: Bak SY 

PROVIDER: S-EPMC8565309 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature