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