RESUMEN
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 precloning in Escherichia coli, purification, validation, and cotransformation 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 precloning 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 through a quick protocol.