Shi Shuobo, Liang Youyun, Ang Ee Lui, Zhao Huimin
Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Methods Mol Biol. 2019;1927:73-91. doi: 10.1007/978-1-4939-9142-6_6.
Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).
尽管酿酒酵母基因工程取得了进展，但大型生化途径的多拷贝基因组整合仍然是一项挑战。在此，我们开发了一种基于成簇规律间隔短回文重复序列（CRISPR）和CRISPR相关系统（Cas）切割δ位点的双CRISPR（δ整合CRISPR-Cas）平台，以实现将大型生化途径以前所未有的高效率、多拷贝、无标记整合到酿酒酵母基因组中。文中提供了关于整个工作流程的详细方案，包括pDi-CRISPR质粒和供体DNA构建、双CRISPR介导的整合以及通过流式细胞术和定量聚合酶链反应（qPCR）分析整合效率和拷贝数。
