Journal
NATURE COMMUNICATIONS
Volume 10, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41467-019-09005-3
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Funding
- Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology
- Beijing Municipal Natural Science Foundation [5182017]
- Fundamental Research Funds for the Central Universities [buctrc201801]
- State Key Laboratory of Microbial Technology Open Projects Fund [M2017-06]
- State Key Laboratory of Chemical Resource Engineering
- International Cooperation Program of Beijing Municipal Science & Technology Commission
- Novo Nordisk Foundation [NNF10CC1016517]
- Knut and Alice Wallenberg Foundation
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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.
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