Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae

[Image: see text] Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase—called yEvolvR—was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.