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. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Constructing the guide RNA expression vector Basic Protocol 2: Preparing double-stranded oligonucleotide repair template Alternate Protocol 1: Preparing a single-stranded oligonucleotide repair template Basic Protocol 3: Induce genome editing by co-transformation of yeast Basic Protocol 4: Screening for edited cells Basic Protocol 5: Removing sgRNA/CAS9 expression vector Alternate Protocol 2: Removing pML107-derived sgRNA/CAS9 expression vector.