A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species
A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species
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DOI：
10.1073/pnas.1520040113
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发表时间：
2016-03-01
影响因子：
11.1
通讯作者：
Pal, Csaba
中科院分区：
文献类型：
--
作者：
Nyerges, Akos;Csoergo, Balint;Pal, Csaba
目前可用的多重细菌基因组工程工具针对一些实验室模型菌株进行了优化，需要对宿主菌株进行广泛的事先修饰，并导致大量脱靶修饰的积累。我们的工作以先前开发的多重自动化基因组工程 (MAGE) 为基础，在单一框架中解决了这些问题。使用甲基定向错配修复（MMR）系统的显性失活突变蛋白，我们实现了大肠杆菌中DNA修复的瞬时抑制，这对于有效的寡核苷酸整合是必要的。通过将所有必要的组件集成到广泛宿主载体中，我们开发了一个新的工作流程，我们称之为 pORTMAGE。它允许有效修饰多个基因座，而无需任何可观察到的脱靶诱变和对宿主基因组的事先修饰。由于细菌 MMR 系统的保守性，pORTMAGE 同时允许在其他生物技术和临床相关细菌物种中进行基因组编辑和突变文库生成。最后，我们应用 pORTMAGE 研究了肠道沙门氏菌和大肠杆菌中的一组抗生素耐药性突变。尽管两个物种之间存在超过 1 亿年的差异，但突变效应总体上仍然保守。总之，pORTMAGE 质粒的单次转化使感兴趣的细菌物种成为基因组工程的有效宿主。这些进步为生物技术和治疗应用铺平了道路。最后，pORTMAGE 可以系统地比较多种细菌物种的突变效应和上位性。
Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.