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Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum

  • Biotechnology Methods - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1–7 nt at the 5′ end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the − 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

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Acknowledgements

All the authors are thankful for the financial support of the National Natural Science Foundation of China (Grant No. 31671840), the National Key R&D Program of China (Grant No. 2018YFA0901700) and the China Postdoctoral Science Foundation (Grant No. 2019M661676).

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Author notes

  1. Nannan Zhao and Lu Li These authors contributed equally to this work

Authors and Affiliations

  1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, People’s Republic of China

    Nannan Zhao, Lu Li, Guangjuan Luo, Shan Xie, Ying Lin, Shuangyan Han, Yuanyuan Huang & Suiping Zheng

  2. Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, People’s Republic of China

    Nannan Zhao, Lu Li, Guangjuan Luo, Shan Xie, Ying Lin, Shuangyan Han, Yuanyuan Huang & Suiping Zheng

  3. School of Physical Science and Technology, Shanghai Tech University, Shanghai, 201210, People’s Republic of China

    Yuanyuan Huang

  4. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, 200032, People’s Republic of China

    Yuanyuan Huang

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  1. Nannan Zhao
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  2. Lu Li
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Correspondence to Yuanyuan Huang or Suiping Zheng.

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Zhao, N., Li, L., Luo, G. et al. Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 47, 599–608 (2020). https://doi.org/10.1007/s10295-020-02304-5

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  • DOI: https://doi.org/10.1007/s10295-020-02304-5

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