ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes
ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes
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DOI：
10.1128/mbio.01467-18
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发表时间：
2018-11-01
期刊：
影响因子：
6.4
通讯作者：
Sassetti, Christopher M.
中科院分区：
文献类型：
--
作者：
Murphy, Kenan C.;Nelson, Samantha J.;Sassetti, Christopher M.
将两种高效的重组系统结合起来，产生了一种通用的染色体工程方法，该方法不需要制备双链DNA(DsDNA)重组底物。一种合成的“靶向寡核苷酸”通过噬菌体Che9c RECT退火酶介导的同源重组进入染色体。该寡核苷酸含有定向Bxb1整合酶(Int)的位点特异性重组位点，它允许同时整合包含同源重组位点和可选择标记的“有效载荷质粒”。将靶向寡核苷酸和有效载荷共转化到RECT和Int表达菌株，一步筛选出耐药的同源重组子。可重复使用的靶标非依赖性有效载荷质粒库可用于产生基因敲除、启动子替换或C-末端标签。这个新的系统被称为Orbit(“寡核苷酸介导的重组工程，然后是Bxb1整合酶靶向”)，非常适合于创建由细菌染色体中的大量缺失、插入或融合组成的文库。我们通过构建在结核分枝杆菌和耻垢分枝杆菌中100多个基因的插入或缺失来证明这种拖放策略的有效性。本文描述了一种用于耻垢分枝杆菌和结核分枝杆菌的系统。通过将噬菌体Bxb1 attP位点的单链DNA(SsDNA)版本整合到寡核苷酸中，并将其与携带attB位点和药物选择标记的非复制质粒共电穿孔，我们显示了染色体attP位点的形成和质粒在一次转化中的整合。不需要靶标特异性dsDNA底物。该系统将使研究分枝杆菌疾病(包括结核病)的研究人员能够轻松地产生多个突变体，用于分析毒力因素、识别新的药物靶标和开发新的疫苗。
Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacterium tuberculosis and M. smegmatis.IMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.