Multiplex engineering of industrial yeast genomes using CRISPRm

Owen W Ryan; Jamie H D Cate

doi:10.1016/B978-0-12-801185-0.00023-4

Multiplex engineering of industrial yeast genomes using CRISPRm

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

Authors

Owen W Ryan¹, Jamie H D Cate²

Affiliations

¹ Energy Biosciences Institute, University of California, Berkeley, California, USA.
² Energy Biosciences Institute, University of California, Berkeley, California, USA; Department of Molecular and Cell Biology, University of California, Berkeley, California, USA; Department of Chemistry, University of California, Berkeley, California, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. Electronic address: [email protected].

PMID: 25398354
DOI: 10.1016/B978-0-12-801185-0.00023-4

Abstract

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, CRISPR-Cas Systems / genetics
Saccharomyces cerevisiae / genetics*

Substances

CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
Endonucleases