iCASRED, a scarless DNA editing tool in E. coli for high-efficiency engineering of natural product biosynthetic gene clusters

• DOI: 10.1016/j.synbio.2025.03.008
• Publication Date: 2025-03-21T03:09:33Z
• Authors: Guosong Zheng; Jiafeng Xu; Hewei Liu; Huimin Hua; Andrei A. Zimin; Wenfang Wang; Yinhua Lu
• Keywords: Natural product

Efficient gene cluster editing tools are one of the key techniques for discovering novel compounds encoded by silent natural product (NP) biosynthetic clusters (BGCs) in microbial genomes. Currently, vivo BGC developed E. coli is most widely used, but they often introduces DNA scars into clusters, which may affect function target NP BGCs. Herein, a genome-integrated Cas9/λRed system-based scarless tool (iCASRED) was established BL23, constructed on basis BL21/DE3 with recA deletion and simultaneous integration an inducible sgRNA targeting plasmid (an all-in-one BGC-targeting sgRNAs repair templates). iCASRED achieved single targets three tested (44.2, 72.0, 76.2 kb) cloned either single-copy BAC or high-copy pCAP01 efficiencies 28.8 % ± 3.9 %-100 0 %. Furthermore, this could enable convenient, high-efficiency iterative editing. Finally, we 24.4 3.8 efficiency double-target replacing Cas9 nCas9 (Cas9D10A). Collectively, provides simple, cost-effective approach engineering facilitate discovery NPs strain improvements high-yield compounds.