There is an important need to develop new therapeutic tools to modulate the gene content of microbiomes. A potential strategy for microbiome engineering relies on the delivery of genetic payloads by conjugative plasmids. Yet, the introduction of large DNA molecules in conjugative plasmids can be challenging. Here, we describe the Double Recombinase Operated Insertion of DNA (DROID), an efficient method to assemble large DNA molecules without introducing antibiotic resistance genes or other unwanted sequences in the final construct. We exemplify this method by demonstrating that the Bxb1 integrase and FLP recombinase can be used successively to stably insert a relatively large DNA cargo consisting of a CRISPR-Cas9 system in a conjugative plasmid. We further show that the resulting CRISPR-Cas9 mobilization system was able to cure a multi-copy antibiotic resistance plasmid in a target bacterium. In addition to its utility for DNA payload integration in conjugative plasmids, the DROID method could readily be adapted to a multitude of other applications that require the manipulation of large DNA molecules.

中文翻译：

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通过双重重组酶操作的DNA（DROID）插入组装可动员的大型遗传货物。

非常需要开发新的治疗工具来调节微生物组的基因含量。微生物组工程学的潜在策略依赖于通过接合质粒传递遗传有效载荷。然而，在结合质粒中引入大的DNA分子可能是具有挑战性的。在这里，我们描述了DNA的双重重组酶操作插入（DROID），这是一种组装大型DNA分子的有效方法，无需在最终构建体中引入抗生素抗性基因或其他不需要的序列。我们通过证明Bxb1整合酶和FLP重组酶可以连续用于将由CRISPR-Cas9系统组成的相对较大的DNA货物稳定地插入结合质粒中来例证这种方法。我们进一步表明，所得的CRISPR-Cas9动员系统能够在靶细菌中治愈多拷贝抗生素抗性质粒。除了可用于将DNA有效载荷整合到结合质粒中外，DROID方法还可以轻松适应需要操纵大型DNA分子的众多其他应用。