On July 15, 2024, Nature Protocols (5-Year Impact Factor=17.4) published a research paper entitled ‘Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method’ online from Hailong Wang’s laboratory at State Key Laboratory of Microbial Technology of Shandong University. Prof. Hailong Wang is the corresponding author, and associate researcher Ji Luan is the first author.

        Multimodular polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and their hybrids produce a variety of valuable compounds useful for pharmaceuticals and agriculture, such as the antibiotics erythromycin and daptomycin, the immunosuppressants rapamycin and cyclosporine, the insecticides avermectin and spinosad, and the anticancer compounds epothilone and bleomycin. Multimodular PKSs and NRPSs both consist of single or multiple modules, each comprising a set of domains. Rational engineering of them by genetic modification is expected to develop novel drug analogs. However, the presence of identical domains in multiple modules results in a vast number of large DNA direct repeats within PKS and NRPS gene clusters, which hinders their engineering.

        To tackle these problems, Hailong Wang Lab developed the RedEx method published in Nucleic Acids Research (48 (22), e130) in 2020. By combining Redαβ mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing, we introduced seamless DNA insertion and deletion in the multimodular PKS gene cluster (79-kb) of the biopesticide spinosad and the recombined PKS gene clusters produced butenyl-spinosyn and spinetoram precursor with better insecticidal activity.

Figure 1 Overview of the RedEx strategy for seamless site-directed mutagenesis in a BAC.

         In the paper latest published in Nature Protocols, we take BAC vector as an example to describe in detail the seamless site-directed mutagenesis using RedEx. This protocol can be performed within 3 weeks. Overall, the process includes three parts (Figure 1): (i) insertion of the RedEx cassette containing the desired mutation into the target site by recombineering, (ii) removal of the selection–counterselection markers in the BAC by restriction digestion, and (iii) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. RedEx seamless site-directed mutagenesis method can be used for point mutations, insertions, deletions, and swaps of highly repetitive and multimodular gene clusters (Figure 2). RedEx achieves seamless mutagenesis of large DNA molecules, including plasmids, fosmids, and BACs. This protocol will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis and to accelerate their research.

Figure 2 Design templates for point mutations, insertions, deletions, and swaps using RedEx.

        This work was supported by Prof. Youming Zhang, Prof. Jun Fu from Shandong University and Prof. A. Francis Stewart from Technische Universität Dresden and Shandong University, and supported by the National Natural Science Foundation of China, Natural Science Foundation of Shandong Province, National Key Research and Development Project of China, and Shandong University Outstanding Young Scholars, etc.

          Paper links:  https://www.nature.com/articles/s41596-024-01016-9