Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

DiCarlo, James E.; Norville, Julie E.; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

doi:10.1093/nar/gkt135

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Journal Article · Sun Mar 03 23:00:00 EST 2013 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkt135· OSTI ID:1904692

DiCarlo, James E. ^[1]; Norville, Julie E. ^[2]; Mali, Prashant ^[2]; Rios, Xavier ^[2]; Aach, John ^[2]; Church, George M. ^[2]

Boston University, MA (United States); Harvard Medical School, Boston, MA (United States); Harvard Medical School, Boston, MA (United States)
Harvard Medical School, Boston, MA (United States)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease 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-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using 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. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

View Accepted Manuscript (DOE)

Research Organization:: Harvard University, Cambridge, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Science Foundation (NSF); National Institutes of Health (NIH)

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1904692

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 7 Vol. 41; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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