The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea.
ACS Synth Biol. 2023 Aug 18;12(8):2353-2366. doi: 10.1021/acssynbio.3c00188. Epub 2023 Jul 4.
CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in . We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of chassis and beyond.
CRISPR 工具，特别是 Cas9n-sgRNA 引导的胞嘧啶脱氨酶碱基编辑器，如 CRISPR-BEST，极大地简化了链霉菌的遗传操作。CRISPR 碱基编辑技术的一个主要优势是有可能在基因组不稳定的物种中进行多重实验。在这里，我们展示了使用 CRISPR-mcBEST 在. 中放大的基于 Csy4 的多路基因组编辑。我们通过在单个实验中同时靶向 9、18 个，最后是所有 28 个预测的特殊代谢物生物合成基因簇，来评估该系统。我们提供了在不同规模下基于 Csy4 的多路基因组编辑性能的重要见解。使用多组学分析，我们研究了这种广泛编辑实验的系统范围影响，并揭示了 CRISPR-mcBEST 的巨大潜力和重要瓶颈。所提出的分析为多路碱基编辑作为一种新型高通量工程 底盘和超越的范例的发展提供了关键数据和见解。
