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Metab Eng. 2018 May;47:200-210. doi: 10.1016/j.ymben.2018.02.016. Epub 2018 Mar 24.

MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.

Author information

1
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
2
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; School of Life Science, University of Science and Technology of China, Hefei 230026, China.
3
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: wangmeng@tib.cas.cn.
4
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; School of Life Science, University of Science and Technology of China, Hefei 230026, China. Electronic address: zheng_p@tib.cas.cn.
5
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China. Electronic address: sun_jb@tib.cas.cn.

Abstract

CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87.2% and 23.3%, respectively. In addition, MACBETH was applied to generate a combinatorial gene inactivation library for improving glutamate production, and pyk&ldhA double inactivation strain was found to improve glutamate production by 3-fold. Finally, MACBETH was automated with an integrated robotic system, which would enable us to generate thousands of rationally engineered strains per month for metabolic engineering of C. glutamicum. As a proof of concept demonstration, the automation platform was used to construct an arrayed genome-scale gene inactivation library of 94 transcription factors with 100% success rate. Therefore, MACBETH would be a powerful tool for multiplex and automated bacterial genome editing in future studies and industrial applications.

KEYWORDS:

CRISPR/Cas9; Corynebacterium glutamicum; Cytidine deaminase; Genome editing; Multiplex automated base editing

PMID:
29580925
DOI:
10.1016/j.ymben.2018.02.016
[Indexed for MEDLINE]

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