CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

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 Fn Cpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3 N +4 or 3 N +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. Corynebacterium glutamicum is an important industrial microbe, however it has proven difficult to genetically engineer using Cas9 from Streptococcus pyogenes . Here the authors report effective genome engineering of the bacterium using Cpf1 from Francisella novicida .