Targeted In Vivo Mutagenesis in Yeast Using CRISPR/Cas9 and Hyperactive Cytidine and Adenine Deaminases

Journal article, 2023

Directed evolution is a preferred strategy to improvethe functionof proteins such as enzymes that act as bottlenecks in metabolic pathways.Common directed evolution approaches rely on error-prone PCR-basedlibraries where the number of possible variants is usually limitedby cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help toovercome limitations in library generation. In the current study,we aimed to develop a high-efficiency time-controllable targeted mutagenesistoolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused thedCas9 protein with hyperactive variants of adenine and cytidine deaminasesaiming to create an inducible CRISPR-based mutagenesis tool targetinga specific DNA sequence in vivo with extended editingwindows and high mutagenesis efficiency. We also investigated theeffect of guide RNA multiplexing on the mutagenesis efficiency bothphenotypically and on the DNA level.