Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

Nucleic Acids Res. 2013 Apr;41(7):4336-43. doi: 10.1093/nar/gkt135. Epub 2013 Mar 4.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Genes, Bacterial
  • Genetic Engineering*
  • Genetic Loci
  • Genome, Fungal
  • Homologous Recombination*
  • Inverted Repeat Sequences
  • Mutagenesis
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • RNA, Guide / metabolism*
  • Saccharomyces cerevisiae / genetics*

Substances

  • RNA, Guide
  • Endodeoxyribonucleases