Journal article
A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae
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.
Language: | English |
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Publisher: | Nature Publishing Group UK |
Year: | 2019 |
Pages: | 1053 |
ISSN: | 20411723 |
Types: | Journal article |
DOI: | 10.1038/s41467-019-09005-3 |
ORCIDs: | 0000-0003-3204-6428 and 0000-0002-9955-6003 |