Author:
Su Rui,Wang Ting,Bo Taidong,Cai Ningyun,Yuan Meng,Wu Chen,Jiang Hao,Peng Huadong,Chen Ning,Li Yanjun
Abstract
Abstract
Background
Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum.
Results
In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only.
Conclusions
We developed a RecET-assisted CRISPR–Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.
Funder
Natural Science Foundation of Henan province
European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement
National Key Research and Development Program of China
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
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