Zheng Guosong, Xu Jiafeng, Liu Hewei, Hua Huimin, Zimin Andrei A, Wang Wenfang, Lu Yinhua
College of Life Sciences, Shanghai Normal University, Shanghai, 200234, China.
G.K. Scriabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, 142290, Russia.
Synth Syst Biotechnol. 2025 Mar 20;10(3):751-763. doi: 10.1016/j.synbio.2025.03.008. eCollection 2025 Sep.
Efficient gene cluster editing tools are one of the key techniques for discovering novel compounds encoded by silent natural product (NP) biosynthetic gene clusters (BGCs) in microbial genomes. Currently, BGC editing tools developed in . is the most widely used, but they often introduces DNA scars into gene clusters, which may affect the function of target NP BGCs. Herein, a genome-integrated Cas9/λRed system-based scarless gene cluster editing tool (iCASRED) was established in BL23, which was constructed on the basis of BL21/DE3 with deletion and simultaneous integration of an inducible sgRNA targeting the editing plasmid (an all-in-one plasmid with the BGC-targeting sgRNAs and repair templates). iCASRED achieved scarless editing of single targets in three tested gene clusters (44.2, 72.0, and 76.2 kb) cloned in either a single-copy BAC plasmid or a high-copy plasmid pCAP01 with the efficiencies of 28.8 % ± 3.9 %-100 % ± 0 %. Furthermore, this tool could enable convenient, high-efficiency iterative editing. Finally, we achieved 24.4 % ± 3.8 % efficiency for simultaneous double-target editing by replacing Cas9 by nCas9 (Cas9). Collectively, iCASRED provides a simple, convenient, and cost-effective approach for engineering gene clusters, which may facilitate the discovery of novel NPs and strain improvements for high-yield of target compounds.
高效的基因簇编辑工具是发现微生物基因组中沉默天然产物（NP）生物合成基因簇（BGC）所编码的新型化合物的关键技术之一。目前，在……中开发的BGC编辑工具使用最为广泛，但它们常常会在基因簇中引入DNA疤痕，这可能会影响目标NP BGC的功能。在此，我们在BL23中建立了一种基于基因组整合Cas9/λRed系统的无疤痕基因簇编辑工具（iCASRED），它是在BL21/DE3的基础上构建的，缺失了……并同时整合了一个靶向编辑质粒（一种携带BGC靶向sgRNA和修复模板的一体化质粒）的可诱导sgRNA。iCASRED在克隆于单拷贝BAC质粒或高拷贝质粒pCAP01中的三个测试基因簇（44.2、72.0和76.2 kb）中实现了单个靶点的无疤痕编辑，效率为28.8%±3.9% - 100%±0%。此外，该工具能够实现便捷、高效的迭代编辑。最后，通过用nCas9（Cas9）替代Cas9，我们实现了同时双靶点编辑的效率为24.4%±3.8%。总体而言，iCASRED为基因簇工程提供了一种简单、便捷且经济高效的方法，这可能有助于新型NP的发现以及目标化合物高产菌株的改良。
