Catalytically inactive Cas9 attenuates DNA end resection: A potential application for region-restricted random mutagenesis.

ABSTRACT: Gene duplication followed by sequence diversification is a key driver of innovation in genome evolution. To mimic this process in genome engineering, a method for region-restricted mutagenesis is needed to selectively mutate one copy of a duplicated gene. Notably, regions flanking a double-strand break (DSB) become hypersensitive to mutagens due to end resection, which converts them into single-stranded DNA (ssDNA). Blocking end resection could, therefore, confine hypermutation to a limited region. To achieve this, we investigated a catalytically inactive variant of Streptococcus pyogenes Cas9 (dSpCas9) and demonstrated its ability to attenuate end resection in the budding yeast Saccharomyces cerevisiae using ssDNA-specific quantitative PCR, live-cell imaging, and Sout

SUBMITTER: Towa S 

PROVIDER: S-EPMC12164011 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature