A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae
Journal article, 2019

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.

Author

Yueping Zhang

Beijing University of Chemical Technology

Juan Wang

Beijing University of Chemical Technology

Zibai Wang

Beijing University of Chemical Technology

Yiming Zhang

Beijing University of Chemical Technology

Shuobo Shi

Beijing University of Chemical Technology

Jens Christian Froslev Nielsen

Technical University of Denmark (DTU)

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Beijing University of Chemical Technology

Zihe Liu

Beijing University of Chemical Technology

Nature Communications

2041-1723 (ISSN)

Vol. 10 1 1053

Subject Categories

Biochemistry and Molecular Biology

Organic Chemistry

Other Industrial Biotechnology

DOI

10.1038/s41467-019-09005-3

PubMed

30837474

More information

Latest update

7/21/2019