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Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD

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Abstract

Objectives

Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application.

Results

In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that.

Conclusion

The present study reported a novel genome manipulation method for C. glutamicum.

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Funding

The authors would like to acknowledge financial support of the National Natural Science Foundation of China (No. 31871247), Central Public-interest Scientific Institution Basal Research Fund (No. Y2019PT10, Y2020XK18) and Chinese Academy of Agricultural Sciences grants (No. CAAS-Y2019YJ07-03).

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Authors and Affiliations

  1. Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China

    Fa-Yu Yang, Nan Wei, Zhi-Hao Zhang, Mi Wang, Ying-Chun Liu, Li-Fang Zhang & Feng Gu

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  1. Fa-Yu Yang
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  2. Nan Wei
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  3. Zhi-Hao Zhang
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  4. Mi Wang
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  7. Feng Gu
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Correspondence to Feng Gu.

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Yang, FY., Wei, N., Zhang, ZH. et al. Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD. Biotechnol Lett 43, 2273–2281 (2021). https://doi.org/10.1007/s10529-021-03195-x

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  • DOI: https://doi.org/10.1007/s10529-021-03195-x

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