Targeted Diversification in the S. cerevisiae Genome with CRISPR-Guided DNA Polymerase I

New technologies to target nucleotide diversification in vivo are promising enabling strategies to perform directed evolution for engineering applications and forward genetics for addressing biological questions. Recently, we reported EvolvR—a system that employs CRISPR-guided Cas9 nickases fused to nick-translating, error-prone DNA polymerases to diversify targeted genomic loci—in E. coli. As CRISPR-Cas9 has shown activity across diverse cell types, EvolvR has the potential to be ported into other organisms, including eukaryotes, if nick-translating polymerases can be active across species. Here, we implement and characterize EvolvR's function in Saccharomyces cerevisiae, representing a key first step to enable EvolvR-mediated mutagenesis in eukaryotes. This advance will be useful for mutagenesis of user-defined loci in the yeast chromosomes for both engineering and basic research applications, and it furthermore provides a platform to develop the EvolvR technology for performance in higher eukaryotes.