AbstractSeamless 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.

摘要无缝定点诱变是研究蛋白质功能，调节酶催化活性和多轮修饰遗传元件的重要技术，因为它可以在靶位点插入，删除或取代核苷酸，DNA片段甚至整个基因，而不会引入任何不必要的变化。

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.

为了促进具有重复序列的大质粒和细菌人工染色体（BAC）中的无缝定点诱变，我们最近开发了RedEx策略。与以前的方法相比，我们的方法通过避免重复序列之间不需要的重组，以高精度恢复正确的重组体。

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.

RedEx在大型多模式聚酮合酶基因簇中无缝DNA插入和缺失的准确率很容易达到80%以上，由于编码几乎相同酶的模块中存在大量重复DNA序列，因此这些基因簇是最困难的靶标之一。在这里，我们通过详细描述BAC载体中的无缝定点诱变来介绍RedEx方法。

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.

总体而言，该过程包括三个部分：（1）通过重组工程将含有所需突变的RedEx盒与侧翼为独特限制性位点和20 bp重叠序列的选择-反选择标记一起插入靶位点，（2）通过限制性消化去除BAC中的选择-反选择标记，以及（3）通过核酸外切酶介导的体外DNA退火使线性BAC环化。

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.

该方案可以在3周内完成，并将使具有DNA克隆经验的研究人员能够掌握无缝定点诱变，以加速他们的研究。关键点。

By combining recombineering, ccdB counterselection and exonuclease-mediated in vitro annealing, RedEx achieves seamless mutagenesis of large DNA molecules, including plasmids, fosmids and BACs.

通过结合重组工程，ccdB反选择和核酸外切酶介导的体外退火，RedEx实现了大DNA分子（包括质粒，fosmid和BAC）的无缝诱变。

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.

与基于CRISPR的方法相比，该方法可以有效编辑高度重复的多模式基因簇，并且是修饰生物合成途径和产生新天然产物的有力工具。

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Fig. 1: Overview of the RedEx strategy for seamless site-directed mutagenesis in a BAC.Fig. 2: Analysis of DNA direct repeats in pBAC-Target using the Unipro UGENE software.Fig. 3: Analysis of restriction enzymes that did not cleave pBAC-Target (noncutters) by SnapGene software.Fig. 4: Design templates for point mutations, insertions, deletions and swaps using RedEx.Fig.

图1:BAC中无缝定点诱变的RedEx策略概述。图2：使用Unipro UGENE软件分析pBAC靶标中的DNA直接重复序列。图3：通过SnapGene软件分析未切割pBAC靶标（非切割）的限制酶。图4：使用RedEx设计点突变，插入，缺失和交换的模板。图。

5: Preparation of the RedEx cassette.Fig. 6: Restriction analysis of pBAC-ampccdB-Mut recombinants after LCHR.Fig. 7: Restriction analysis of pBAC-Mut recombinants..

5： RedEx盒的准备。图6:LCHR后pBAC-ampccdB-Mut重组体的限制性分析。图7:pBAC-Mut重组体的限制性分析。。

Data availability

数据可用性

The data supporting the findings of this study are available in the supporting primary research paper12.

支持本研究结果的数据可在支持性主要研究论文12中找到。

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Download referencesAcknowledgementsThis 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).

下载参考文献致谢这项工作得到了国家自然科学基金（32122049）的支持；山东省自然科学基金（ZR2022JQ11和ZR2019ZD22）；国家重点研究发展计划（2018YFA0900400、2021YFC2101000和2019YFA0904000）；SDU杰出青年学者基金；山东大学基础研究基金（2023QNTD001）；青岛市关键技术研究与产业化示范项目（22-3-4-xxgg-1-nsh）；和111项目（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.Author informationAuthors and AffiliationsState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, ChinaJi Luan, Chaoyi Song, Yan Liu, Ruoting He, Ruofei Guo, Qingwen Cui, Chanjuan Jiang, Xiaochen Li, Kexin Hao, Jun Fu, Youming Zhang & Hailong WangGenomics, Center for Molecular and Cellular Bioengineering, Biotechnology Center, Technische Universität Dresden, Dresden, GermanyA.

作者感谢山东大学微生物技术国家重点实验室生命与环境科学核心设施的J.Qu，J.Zhu和Z.Li的技术援助。作者信息作者和附属机构山东大学亥姆霍兹国际抗感染实验室微生物技术研究所微生物技术国家重点实验室-山东大学亥姆霍兹生物技术研究所，山东青岛，中国济銮，宋朝毅，刘燕，何若婷，郭若飞，崔庆文，蒋春娟，李晓晨，郝可欣，傅军，张有明，王海龙基因组学，分子与细胞生物工程中心，德累斯顿工业大学生物技术中心，德国德累斯顿。

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PubMed Google ScholarContributionsJ.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.Corresponding authorCorrespondence to.

PubMed谷歌学术贡献。五十、 ，A.F.S.，J.F.，Y.Z.和H.W.设计并监督了该项目。J、 L.，C.S.，Y.L.，R.H.，R.G.，Q.C.，C.J.，X.L.和K.H.进行了实验。J、 L.和H.W.写了手稿。所有作者都审查并批准了手稿。对应作者对应。

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Nature Protocols thanks Imre Berger, Hang Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Nature Protocols感谢Imre Berger，Hang Wu和其他匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Related linksKey references using this protocolWang, H. et al. Nucleic Acids Res. 42, e37 (2014): https://doi.org/10.1093/nar/gkt1339Song, C. et al.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。使用此协议的相关linksKey参考文献Wang，H.等人，《核酸研究》42，e37（2014）：https://doi.org/10.1093/nar/gkt1339Song，C.等人。

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核酸研究48，e130（2020）：https://doi.org/10.1093/nar/gkaa956Zhang，Y。等人，Nat。Genet。20123-128（1998）：https://doi.org/10.1038/2417Muyrers，J.P.P.等人，EMBO Rep.1239–243（2000）：https://doi.org/10.1093/embo-reports/kvd049Bird，A.W.等人，《自然方法》9103-109（2012）：https://doi.org/10.1038/nmeth.1803Supplementary信息补充信息补充说明1-4.报告摘要权利和许可原告性质或其许可人（例如。

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et al. Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method..

等人。使用RedEx方法在复杂克隆的DNA序列中进行无缝定点诱变。。

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