Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1–7 nt at the 5′ end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the − 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

谷氨酸棒杆菌是必需的工业菌株，已被广泛用于生产各种增值产品。高效的多重基因编辑和大的DNA片段缺失是工业生物技术研究的基本策略。Cpf1是一个健壮且简单的基因组编辑工具，用于同时编辑多重基因。然而，尚无关于谷氨酸棒杆菌中通过CRISPR / Cpf1系统进行有效的多重基因编辑和大DNA片段缺失的研究的报道。在这里，我们通过优化CRISPR / Cpf1-RecT系统和谷氨酸棒杆菌中的CRISPR / Cpf1-RecET系统使用大染色体片段缺失策略，开发了一种多重基因编辑方法。ATCC14067。CRISPR / Cpf1-RecT系统对PAM序列TTTC，TTTG，GTTG或CTTC的精确编辑效率超过91.6％。由于PAM区域和原间隔物区域5'端的1-7 nt，可编辑的位点受到限制。PAM区域中的突变使− 6 nt区域的编辑效率从0提高到96.7％。使用crRNA阵列，可以通过CRISPR / Cpf1-RecT系统一步同时编辑两个和三个基因，同时编辑两个基因的效率为91.6％，但是同时编辑三个基因的效率低于10％。通过CRISPR / Cpf1-RecET系统，缺失1 kb的编辑效率为79.6％，长度为5 kb和20 kb的DNA片段缺失的编辑效率分别为91.3％和36.4％。谷氨酸棒杆菌基因组编辑可以进一步加速代谢工程的努力和基因组的进化。