Simple CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae.

CRISPR-Cas9 has emerged as a powerful method for editing the genome in a wide variety of species, since it can generate a specific DNA break when targeted by the Cas9-bound guide RNA. In yeast, Cas9-targeted DNA breaks are used to promote homologous recombination with a mutagenic template DNA, in order to rapidly generate genome edits (e.g., DNA substitutions, insertions, or deletions) encoded in the template DNA. Since repeated Cas9-induced DNA breaks select against unedited cells, Cas9 can be used to generate marker-free genome edits. Here, we describe a simple protocol for constructing Cas9-expressing plasmids containing a user-designed guide RNA, as well as protocols for using these plasmids for efficient genome editing in yeast.