Journal
BIOTECHNOLOGY JOURNAL
Volume 13, Issue 9, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201700604
Keywords
CRISPR; Cas9; E; coli; multiplex genome editing; multiplex gRNA
Funding
- National High Technology Research and Development Program of China [2015AA020202]
- Tianjin Key Technology R&D program of Tianjin Municipal Science and Technology Commission [14ZCZDSY00067]
- Key deployment project of the Chinese Academy of Sciences [ZDRW-ZS-2016-3]
- Novo Nordisk - Chinese Academy of Sciences (NN-CAS) Research Fund [NNCAS-2015-2]
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Genome editing for site-specific chromosome modification is one of the most significant techniques in biological research. While conventional techniques usually deal with one genomic locus at a time, multiple genomic targets are often required to be modified to develop microbial cell factories. Thus, it is necessary to develop techniques for simultaneous editing of multiple loci. In this work, the authors develop a CRISPR/Cas9 assisted multiplex genome editing (CMGE) technique in Escherichia coli. With this editing method, all functional parts are assembled into replicable plasmids, and stringent inducible expression systems are used to control Cas9 gene expression, which is to decouple transformation from editing process to increase editing efficiency. A modular assembly strategy is designed to enable construction of the complex multi-gRNA plasmid. With this technique, two and three loci are able to be modified with 100% and 88.3% efficiencies, while four loci can be edited with more than 30%, which are the best results reported. Although developed in model organism, the strategy of CMGE can be adapted to other prokaryotic cells. This is a well designed and illustrated technique with no special requirement, can be used by any biological lab easily.
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