Publications
ACS synthetic biologyJul 2023 DOI:
10.1021/acssynbio.2c00690

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

Skrekas, Christos; Limeta, Angelo; Siewers, Verena; David, Florian
Product Used
Genes
Abstract
Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based Variant Libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence in vivo with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.
Product Used
Genes

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