Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

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Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

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Title: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
Author: DiCarlo, James; Norville, Julie; Mali, Prashant; Rios Villanueva, Xavier; Aach, John Dennis; Church, George McDonald

Note: Order does not necessarily reflect citation order of authors.

Citation: DiCarlo, James E., Julie E. Norville, Prashant Mali, Xavier Rios, John Aach, and George M. Church. 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Research 41(7): 4336-4343.
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Abstract: 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.
Published Version: doi:10.1093/nar/gkt135
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627607/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:11181141
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