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.