Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 100029, Beijing, China.
Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96, Gothenburg, Sweden.
Nat Commun. 2019 Mar 5;10(1):1053. doi: 10.1038/s41467-019-09005-3.
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.
随着 DNA 合成和测序技术的快速发展，菌株工程开始成为合成生物学的限速步骤。在这里，我们报告了一种用于 CRISPR-Cas9（GTR-CRISPR）的 gRNA-tRNA 阵列，用于酵母的高通量工程改造。使用已报道的有效 gRNA，该系统可实现 87%的效率同时敲除 8 个基因。我们进一步报道了一种加速的 Lightning GTR-CRISPR，它通过直接将 Golden Gate 反应混合物转化为酵母，避免了在大肠杆菌中的克隆步骤。使用已报道的 gRNA，该方法可在 3 天内以 60%的效率敲除 6 个基因，而使用未经优化的 gRNA 的效率为 23%。此外，我们将 Lightning GTR-CRISPR 应用于简化酵母脂质网络，仅使用两轮删除之前鉴定的八个基因，就可在 10 天内使游离脂肪酸产量增加 30 倍。GTR-CRISPR 应该是合成生物学和自动化工具箱中非常有价值的补充。
