EasyGuide plasmids support in vivo assembly of gRNAs for CRISPR/Cas9 applications in Saccharomyces cerevisiae

Most CRISPR/Cas9 applications in yeast rely on a plasmid-based expression of Cas9 and its guide RNA (gRNA) containing a 20-nucleotides (nts) spacer tailored to each genomic target. The lengthy assembly of this customized gRNA requires at least 3–5 days for its pre-cloning in Escherichia coli , purification, validation, and co-transformation with Cas9 into a yeast strain. Here, we constructed a series of 12 EasyGuide plasmids to simplify CRISPR/Cas9 applications in Saccharomyces cerevisiae . The new vectors provide templates for generating PCR fragments that can assemble up to six functional gRNAs directly into yeasts via homologous recombination between the 20-nts spacers. By dispensing pre-cloning in E. coli , yeast in vivo gRNA assembly significantly reduces the CRISPR/Cas9 experimental workload. A highly efficient yeast genome editing procedure, involving PCR amplification of gRNAs and donors, followed by their transformation into a Cas9-expressing strain, can be easily accomplished in a single day.