Multiplex Engineering of Industrial Yeast Genomes Using CRISPRm

Methods Enzymol. 2014;546:473-89. doi: 10.1016/B978-0-12-801185-0.00023-4.


Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Keywords: CRISPRm; Cas9; Chromosomal integration; DNA libraries; Directed evolution; Homology-directed repair; Industrial yeast; Multiplex genome editing; Saccharomyces cerevisiae; Synthetic biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Directed Molecular Evolution / methods*
  • Endonucleases / genetics
  • Gene Library
  • Genetic Engineering / methods*
  • Genome, Fungal
  • Industrial Microbiology / methods*
  • Molecular Sequence Data
  • Plasmids / genetics
  • RNA, Guide / genetics
  • Saccharomyces cerevisiae / genetics*


  • CRISPR-Associated Proteins
  • RNA, Guide
  • Endonucleases