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Updated: Oct 16, 2025
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD
Fa-Yu Yang1, Nan Wei1, Zhi-Hao Zhang1
1Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
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.
