State 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, China.
Genomics, Center for Molecular and Cellular Bioengineering, Biotechnology Center, Technische Universität Dresden, Dresden, Germany.
Nat Protoc. 2024 Nov;19(11):3360-3388. doi: 10.1038/s41596-024-01016-9. Epub 2024 Jul 15.
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.
无缝定点突变是研究蛋白质功能、调节酶催化活性和多次修饰遗传元件的重要技术，因为它可以在目标位点插入、删除或替换核苷酸、DNA 片段甚至整个基因，而不会引入任何不需要的变化。为了在具有重复序列的大型质粒和细菌人工染色体 (BAC) 中方便进行无缝定点突变，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的不想要的重组，以高准确性实现了正确重组体的回收。RedEx 很容易在大型多功能聚酮合酶基因簇中实现超过 80%的无缝 DNA 插入和缺失的准确性，由于模块中编码几乎相同酶的重复 DNA 序列数量众多，这些基因簇是最难的目标之一。在这里，我们通过详细描述 BAC 载体中的无缝定点突变来介绍 RedEx 方法。总的来说，该过程包括三个部分：(1) 通过重组酶将包含所需突变的 RedEx 盒与两侧带有独特限制位点和 20 个碱基重叠序列的选择-抗选择标记插入到目标位点，(2) 通过限制消化去除 BAC 中的选择-抗选择标记，(3) 通过外切酶介导的体外 DNA 退火使线性 BAC 环化。该方案可以在 3 周内完成，将使具有 DNA 克隆经验的研究人员能够掌握无缝定点突变技术，从而加速他们的研究。
