CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

TLDR

Corynebacterium glutamicum is a key industrial metabolite producer that is difficult to genetically engineer, and the Streptococcus pyogenes CRISPR‑Cas9 system cannot be introduced into it. The authors present a Francisella novicida CRISPR‑Cpf1–based genome‑editing method for C. glutamicum. The method employs Fn Cpf1 and CRISPR RNA together with single‑stranded DNA recombineering to introduce precise edits.

Abstract

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 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.