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

Journal article, 2023

Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9technology, with its ability to target a specific DNA locus usingguide RNAs (gRNAs), is particularly suited for targeted mutagenesis.The targeted diversification of nucleotides in Saccharomycescerevisiae using a CRISPR-guided error-prone DNA polymerase calledyEvolvR was recently reported. Here, we investigate the effectof multiplexed expression of gRNAs flanking a short stretch of DNAon reversion and mutation frequencies using yEvolvR. Phenotypic assaysdemonstrate that higher reversion frequencies are observed when expressingmultiple gRNAs simultaneously. Next generation sequencing revealsa synergistic effect of multiple gRNAs on mutation frequencies, whichis more pronounced in a mutant with a partially defective DNA mismatchrepair system. Additionally, we characterize a galactose-inducibleyEvolvR, which enables temporal control of mutagenesis. This studydemonstrates that multiplex expression of gRNAs and induction of mutagenesisgreatly improves the capabilities of yEvolvR for generation of geneticlibraries in vivo.