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...

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Bibliographic Details
Published in:Nature communications 2019-03, Vol.10 (1), p.1053-10, Article 1053
Main Authors: Zhang, Yueping, Wang, Juan, Wang, Zibai, Zhang, Yiming, Shi, Shuobo, Nielsen, Jens, Liu, Zihe
Format: Article
Language:English
Subjects:
45/41
631/553/552
631/61/318
Acid production
Automation
Baking yeast
Biology
Cloning
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
Disruption
DNA
DNA biosynthesis
DNA sequencing
E coli
Gene Editing - methods
Genes
Genome editing
Genomes
gRNA
Humanities and Social Sciences
Lightning
Lipids
multidisciplinary
Multiplexing
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Transfer - genetics
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Science
Science (multidisciplinary)
Sequence Deletion
Synthetic biology
Synthetic Biology - methods
Time Factors
tRNA
Yeast
Yeasts
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Summary: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. Strain engineering is increasingly the bottleneck step in synthetic biology workflows. Here the authors present GTR-CRISPR for rapid, multiplexed engineering of yeast metabolic pathways.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-019-09005-3