Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, People's Republic of China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, People's Republic of China.
J Ind Microbiol Biotechnol. 2020 Aug;47(8):599-608. doi: 10.1007/s10295-020-02304-5. Epub 2020 Sep 2.
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.
谷氨酸棒杆菌是一种重要的工业菌株，已广泛应用于各种增值产品的生产。高效的多重基因编辑和大片段 DNA 缺失是工业生物技术研究的重要策略。Cpf1 是一种强大而简单的基因组编辑工具，可用于同时编辑多个基因。然而，尚未有关于 CRISPR/Cpf1 系统在谷氨酸棒杆菌中进行有效多重基因编辑和大片段 DNA 缺失的研究报道。在这里，我们通过优化 CRISPR/Cpf1-RecT 系统和使用 CRISPR/Cpf1-RecET 系统的大片段染色体缺失策略，开发了一种在谷氨酸棒杆菌 ATCC 14067 中的多重基因编辑方法。CRISPR/Cpf1-RecT 系统在 PAM 序列 TTTC、TTTG、GTTG 或 CTTC 时，具有超过 91.6%的精确编辑效率。由于 PAM 区域和原间隔区 5'端的 1-7 个核苷酸的限制，可编辑的位点是有限的。PAM 区域的突变将 -6 nt 区域的编辑效率从 0 提高到 96.7%。使用 crRNA 阵列，通过 CRISPR/Cpf1-RecT 系统可以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%，但同时编辑三个基因的效率低于 10%。1 kb 缺失的编辑效率为 79.6%，通过 CRISPR/Cpf1-RecET 系统，5 kb 和 20 kb 长度的 DNA 片段缺失的编辑效率分别达到 91.3%和 36.4%。这项研究为谷氨酸棒杆菌基因组编辑提供了一种高效、简单的工具，可以进一步加速代谢工程努力和基因组进化。
