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

## TL;DR

This paper shows how a modified Cas9 protein can block DNA repair processes to limit mutations to specific regions in yeast.

## Contribution

The study introduces a novel use of catalytically inactive Cas9 to control DNA end resection and restrict mutagenesis.

## Key findings

- Positioning dCas9 near a DSB inhibits end resection in yeast.
- dCas9 limits the region susceptible to DSB-coupled bisulfite mutagenesis.
- End resection machinery is stalled at the dCas9-binding site.

## 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 Southern blot analysis. By leveraging the bisulfite sensitivity of ssDNA, we further validated the concept of DSB-coupled, dSpCas9-mediated region-restricted mutagenesis. We anticipate that dSpCas9-mediated modulation of end resection at induced DSB sites will have valuable applications in both genome engineering and mechanistic studies.

•Positioning dCas9 near a double-strand break (DSB) inhibits end resection•The end resection machinery is stalled at the dCas9-binding site•dCas9 can limit the region susceptible to DSB-coupled bisulfite mutagenesis

Positioning dCas9 near a double-strand break (DSB) inhibits end resection

The end resection machinery is stalled at the dCas9-binding site

dCas9 can limit the region susceptible to DSB-coupled bisulfite mutagenesis

Techniques in genetics; Molecular genetics; Biological sciences research methodologies; Model organism