title: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.
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.
authors: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
authors: James E
authors: JE
authors: DiCarlo
authors: Julie E
authors: JE
authors: Norville
authors: Prashant
authors: P
authors: Mali
authors: Xavier
authors: X
authors: Rios
authors: John
authors: J
authors: Aach
authors: George M
authors: GM
authors: Church
mesh_terms: D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005818:Genetic Engineering; D056426:Genetic Loci; D016681:Genome, Fungal; D059765:Homologous Recombination; D055029:Inverted Repeat Sequences; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058727:RNA, Small Untranslated
chemical_list: D004706:Endodeoxyribonucleases; D058727:RNA, Small Untranslated