5 years ago

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum
Fenghui Qian, Bingbing Sun, Xiaoshu Xu, Yingmiao Liu, Yan Li, Sheng Yang, Yu Jiang, Junjie Yang, Chongmao Xu, Feng Dong, Biao Chen, Renxiao Wang
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.

Publisher URL: http://www.nature.com/articles/ncomms15179

DOI: 10.1038/ncomms15179

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