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

ACS Synth Biol. 2023 Aug 18;12(8):2278-2289. doi: 10.1021/acssynbio.2c00690. Epub 2023 Jul 24.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Gene Editing
  • Mutagenesis / genetics
  • Mutagenesis, Site-Directed
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism

Substances

  • adenine deaminase