Genome Engineering in Saccharomyces cerevisiae using CRISPR/Cas systems

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James DiCarlo, Julie Norville, Prashant Mali, Xavier Rios, George Church

Boston University/Harvard University, United States

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) systems in bacteria and archaea employ RNA-guided DNA endonuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here we report the use of Type II bacterial CRISPR/Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA guided endonuclease activity at targeted endogenous loci in yeast. Using a constitutive Cas9 expression and a transient gRNA cassette we show that targeted double-strand breaks can increase homologous recombination rates of single and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.