期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 9, 页码 2502-2507出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1520040113
关键词
genome engineering; synthetic biology; recombineering; off-target effects; methyl-directed mismatch repair
资金
- European Research Council
- Wellcome Trust
- Lendulet Program of the Hungarian Academy of Scienc
- Hungarian Scientific Research Fund [OTKA PD 109572, OTKA PD 106231]
- Hungarian Academy of Sciences Postdoctoral Fellowship Program [SZ-039/2013]
- Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences
- Boehringer Ingelheim Fonds
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.
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