CRISPR-Cas systems have been successfully applied to genome engineering in Saccharomyces cerevisiae. The study demonstrates that the type II bacterial CRISPR-Cas system can be used to target specific genomic loci in yeast, enabling precise genome modifications. The Cas9 gene, when expressed constitutively, and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. The study shows that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. Co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. The study also shows that CRISPR-Cas can stimulate recombination and select against wild-type sequences by mutating the protospacer-associated motif (PAM) sequence. The researchers calculated the frequency of gRNA target sites in yeast by analyzing all 12 bp 'seed' sequences crucial for gRNA genomic specificity proximal to a PAM sequence. The study also demonstrates that CRISPR-Cas can be used to target multiple genomic loci simultaneously, which could be useful for engineering whole metabolic pathways and large gene networks. The study highlights the potential of CRISPR-Cas as a powerful tool for site-specific mutagenesis and allelic replacement in yeast. The results suggest that CRISPR-Cas can be used for targeted knockouts, although further improvements are needed to achieve higher knockout rates. The study also shows that the use of a transient gRNA cassette can stimulate homologous recombination, allowing for the simultaneous targeting of multiple loci. The findings indicate that CRISPR-Cas is a promising tool for genome engineering in yeast, offering a simple and efficient method for site-specific modifications.CRISPR-Cas systems have been successfully applied to genome engineering in Saccharomyces cerevisiae. The study demonstrates that the type II bacterial CRISPR-Cas system can be used to target specific genomic loci in yeast, enabling precise genome modifications. The Cas9 gene, when expressed constitutively, and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. The study shows that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. Co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. The study also shows that CRISPR-Cas can stimulate recombination and select against wild-type sequences by mutating the protospacer-associated motif (PAM) sequence. The researchers calculated the frequency of gRNA target sites in yeast by analyzing all 12 bp 'seed' sequences crucial for gRNA genomic specificity proximal to a PAM sequence. The study also demonstrates that CRISPR-Cas can be used to target multiple genomic loci simultaneously, which could be useful for engineering whole metabolic pathways and large gene networks. The study highlights the potential of CRISPR-Cas as a powerful tool for site-specific mutagenesis and allelic replacement in yeast. The results suggest that CRISPR-Cas can be used for targeted knockouts, although further improvements are needed to achieve higher knockout rates. The study also shows that the use of a transient gRNA cassette can stimulate homologous recombination, allowing for the simultaneous targeting of multiple loci. The findings indicate that CRISPR-Cas is a promising tool for genome engineering in yeast, offering a simple and efficient method for site-specific modifications.