CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells

Owen W Ryan; Snigdha Poddar; Jamie H D Cate

doi:10.1101/pdb.prot086827

CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells

Cold Spring Harb Protoc. 2016 Jun 1;2016(6). doi: 10.1101/pdb.prot086827.

Authors

Owen W Ryan¹, Snigdha Poddar¹, Jamie H D Cate²

Affiliations

¹ Energy Biosciences Institute, University of California, Berkeley, California 94720;
² Energy Biosciences Institute, University of California, Berkeley, California 94720; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; Department of Chemistry, University of California, Berkeley, California 94720.

PMID: 27250940
DOI: 10.1101/pdb.prot086827

Abstract

This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.

MeSH terms

CRISPR-Cas Systems*
Gene Targeting / methods*
Genome, Fungal*
Recombination, Genetic
Saccharomyces cerevisiae / genetics*