A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1

Technologies development for seamless gene editing and marker recycling has allowed frequent genomic engineering in Saccharomyces cerevisiae for desired laboratory strains and cell factory. Alternative new approaches are still required for complicated scenarios. In this study, we report that inducible overexpression of cell wall protein 1 (Cwp1) by galactose addition confers yeast cells a robust growth inhibition. Direct repeats flanking the Gal‐CWP1:selectable marker cassette allow for its homology recombination excision and counter selection upon galactose addition, therefore enable seamless gene editing and marker recycling. We used this strategy and efficiently generated scarless Ade8 deletion mutants. Our results highlight the utility of lethal effect of Cwp1 overexpression a new counter selection strategy and a simple and efficient method for seamless gene editing and marker recycling in S. cerevisiae and potentially other fungi.