Abstract
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, in vivo BGC editing tools developed in E. coli 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 in vivo scarless gene cluster editing tool (iCASRED) was established in E. coli BL23, which was constructed on the basis of BL21/DE3 with recA 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(D10A)). 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.