Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum
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BIOTECHNOLOGY METHODS - ORIGINAL PAPER
Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1‑RecE/T system in Corynebacterium glutamicum Nannan Zhao1,2 · Lu Li1,2 · Guangjuan Luo1,2 · Shan Xie1,2 · Ying Lin1,2 · Shuangyan Han1,2 · Yuanyuan Huang1,2,3,4 · Suiping Zheng1,2 Received: 19 January 2020 / Accepted: 23 August 2020 © Society for Industrial Microbiology and Biotechnology 2020
Abstract 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/Cpf1RecT 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. Keywords Corynebacterium glutamicum · CRISPR/Cpf1 · Multiplex gene editing · Large DNA fragment deletion
Introduction Multiplex gene simultaneous editing is an essential strategy for biotechnology research that can be achieved by robust CRISPR technology. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats and associated Nannan Zhao and Lu Li These authors contributed equally to this work Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02304-5) contains supplementary material, which is available to authorized users. * Yuanyuan Huang [email protected] * Suiping Zheng [email protected] Extended author information available on the last page of the article
proteins) system, which consists of a combination of Cas effector proteins and CRISPR RNA [9], is a simple, versatile and efficient genome editing technology [1]. Currently, the
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