A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae

Yueping Zhang; Juan Wang; Zibai Wang; Yiming Zhang; Shuobo Shi; Jens Nielsen; Zihe Liu

doi:10.1038/s41467-019-09005-3

A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae

Nat Commun. 2019 Mar 5;10(1):1053. doi: 10.1038/s41467-019-09005-3.

Authors

Yueping Zhang¹, Juan Wang¹, Zibai Wang¹, Yiming Zhang¹, Shuobo Shi¹, Jens Nielsen^{1

2

3}, Zihe Liu⁴

Affiliations

¹ Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 100029, Beijing, China.
² Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96, Gothenburg, Sweden.
³ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK2800, Lyngby, Denmark.
⁴ Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 100029, Beijing, China. zihe@mail.buct.edu.cn.

Abstract

With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics*
Gene Editing / methods*
RNA, Guide, CRISPR-Cas Systems / genetics*
RNA, Transfer / genetics*
Saccharomyces cerevisiae / genetics*
Sequence Deletion
Synthetic Biology / methods
Time Factors

Substances

RNA, Guide, CRISPR-Cas Systems
RNA, Transfer