Abstract
Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection–counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection–counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research.
Key points
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By combining recombineering, ccdB counterselection and exonuclease-mediated in vitro annealing, RedEx achieves seamless mutagenesis of large DNA molecules, including plasmids, fosmids and BACs.
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Compared with CRISPR-based approaches, this method allows the efficient editing of highly repetitive, multimodular gene clusters and represents a powerful tool for modifying biosynthesis pathways and generating new natural products.
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Data availability
The data supporting the findings of this study are available in the supporting primary research paper12.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (32122049); Natural Science Foundation of Shandong Province (ZR2022JQ11 and ZR2019ZD22); National Key Research and Development Program of China (2018YFA0900400, 2021YFC2101000 and 2019YFA0904000); the Fund for Distinguished Young Scholars of SDU; the Fundamental Research Funds of Shandong University (2023QNTD001); Qingdao Key Technology Research and Industrialization Demonstration Project (22-3-4-xxgg-1-nsh); and the 111 Project (B16030). The authors acknowledge J. Qu, J. Zhu and Z. Li of the Core Facilities for Life and Environmental Sciences, State Key laboratory of Microbial Technology of Shandong University for their technical assistance.
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J.L., A.F.S., J.F., Y.Z. and H.W. designed and supervised the project. J.L., C.S., Y.L., R.H., R.G., Q.C., C.J., X.L. and K.H. performed the experiments. J.L. and H.W. wrote the manuscript. All authors reviewed and approved the manuscript.
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Key references using this protocol
Wang, H. et al. Nucleic Acids Res. 42, e37 (2014): https://doi.org/10.1093/nar/gkt1339
Song, C. et al. Nucleic Acids Res. 48, e130 (2020): https://doi.org/10.1093/nar/gkaa956
Zhang, Y. et al. Nat. Genet. 20, 123–128 (1998): https://doi.org/10.1038/2417
Muyrers, J. P. P. et al. EMBO Rep. 1, 239–243 (2000): https://doi.org/10.1093/embo-reports/kvd049
Bird, A. W. et al. Nat. Methods 9, 103–109 (2012): https://doi.org/10.1038/nmeth.1803
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Luan, J., Song, C., Liu, Y. et al. Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method. Nat Protoc 19, 3360–3388 (2024). https://doi.org/10.1038/s41596-024-01016-9
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DOI: https://doi.org/10.1038/s41596-024-01016-9
