Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

TLDR

CRISPR-Cas systems use RNA‑guided nuclease activity to provide adaptive immunity against foreign nucleic acids in bacteria and archaea. The study reports applying a type II bacterial CRISPR‑Cas system to Saccharomyces cerevisiae for genome engineering. The Cas9–gRNA system in yeast produced robust, specific endonuclease activity, boosting homologous recombination by 5‑fold for single‑strand and 130‑fold for double‑strand donors, achieving nearly 100 % donor integration and establishing a versatile platform for site‑specific mutagenesis and allelic replacement.

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.