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CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

Author

Listed:
  • Yu Jiang

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Fenghui Qian

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Junjie Yang

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Yingmiao Liu

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology
    Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM)
    College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University)

  • Feng Dong

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Chongmao Xu

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Bingbing Sun

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology
    School of Pharmacy, Shanghai Jiaotong University)

  • Biao Chen

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology)

  • Xiaoshu Xu

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences)

  • Yan Li

    (State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences)

  • Renxiao Wang

    (State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences)

  • Sheng Yang

    (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
    Shanghai Research and Development Center of Industrial Biotechnology
    Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM))

Abstract

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86–100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.

Suggested Citation

  • Yu Jiang & Fenghui Qian & Junjie Yang & Yingmiao Liu & Feng Dong & Chongmao Xu & Bingbing Sun & Biao Chen & Xiaoshu Xu & Yan Li & Renxiao Wang & Sheng Yang, 2017. "CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum," Nature Communications, Nature, vol. 8(1), pages 1-11, August.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms15179
    DOI: 10.1038/ncomms15179
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    Cited by:

    1. Jiao Liu & Moshi Liu & Tuo Shi & Guannan Sun & Ning Gao & Xiaojia Zhao & Xuan Guo & Xiaomeng Ni & Qianqian Yuan & Jinhui Feng & Zhemin Liu & Yanmei Guo & Jiuzhou Chen & Yu Wang & Ping Zheng & Jibin Su, 2022. "CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    2. Daphne Collias & Elena Vialetto & Jiaqi Yu & Khoa Co & Éva d. H. Almási & Ann-Sophie Rüttiger & Tatjana Achmedov & Till Strowig & Chase L. Beisel, 2023. "Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria," Nature Communications, Nature, vol. 14(1), pages 1-12, December.

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