CRISPR/Cas9 Assisted Multiplex Genome Editing Technique in Escherichia coli
CRISPR/Cas9 Assisted Multiplex Genome Editing Technique in Escherichia coli
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DOI：
10.1002/biot.201700604
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发表时间：
2018-09-01
影响因子：
4.7
通讯作者：
Bi, Changhao
中科院分区：
文献类型：
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作者：
Feng, Xu;Zhao, Dongdong;Bi, Changhao
染色体定点修饰的基因组编辑是生物学研究中最重要的技术之一。虽然传统技术通常一次只处理一个基因组位点，但为了发展微生物细胞工厂，往往需要修改多个基因组靶点。因此，有必要开发同时编辑多个基因座的技术。在这项工作中，作者发展了一种CRISPR/Cas9辅助的大肠杆菌多重基因组编辑(CMGE)技术。这种编辑方法将所有功能部分组装成可复制的质粒，并使用严格的可诱导表达系统来控制Cas9基因的表达，即将转化与编辑过程解耦，以提高编辑效率。设计了一种模块化组装策略，以便于构建复杂的多gRNA质粒。利用该技术，两个和三个基因座的修改效率分别为100%和88.3%，四个基因座的编辑效率超过30%，这是目前报道的最好的结果。虽然CMGE策略是在模式生物中发展起来的，但它也可以适用于其他原核细胞。这是一种精心设计和图文并茂的技术，没有特殊要求，可以很容易地被任何生物实验室使用。
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.