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 and Zihe Liu ()
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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 Nielsen: Beijing University of Chemical Technology
Zihe Liu: Beijing University of Chemical Technology

Nature Communications, 2019, vol. 10, issue 1, 1-10

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-09005-3

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