School of Life Sciences, Shanghai University, No. 99 Shangda Road, Shanghai 200444, China; School of Economics and Management, Tongji University, No. 1239 Siping Road, Shanghai 200092, China; Institute of Logistics Science and Engineering, Shanghai Maritime University, 1550 Haigang Avenue, Shanghai 201306, China.
School of Life Sciences, Shanghai University, No. 99 Shangda Road, Shanghai 200444, China; Department of Biology, Georgia State University, Atlanta, GA 30303, United States of America.
Int J Biol Macromol. 2024 Jun;270(Pt 2):132431. doi: 10.1016/j.ijbiomac.2024.132431. Epub 2024 May 15.
Escherichia coli has emerged as an important host for the production of biopharmaceuticals or other industrially relevant molecules. An efficient gene editing tool is indispensable for ensuring high production levels and optimal release of target products. However, in Escherichia coli, the CRISPR-Cas9 system has been shown to achieve gene modifications with relatively low frequency. Large-scale PCR screening is required, hindering the identification of positive clones. The beta protein, which weakly binds to single-stranded DNA but tightly associates with complementary strand annealing products, offers a promising solution to this issue. In the present study, we describe a targeted and continuous gene editing strategy for the Escherichia coli genome. This strategy involves the coexpression of the beta protein alongside the CRISPR-Cas9 system, enabling a variety of genome modifications such as gene deletion and insertion with an efficiency exceeding 80 %. The integrity of beta proteins is essential for the CRISPR-Cas9/Beta-based gene editing system. In this work, the deletion of either the N- or C-terminal domain significantly impaired system efficiency. Overall, our findings established the CRISPR-Cas9/Beta system as a suitable gene editing tool for various applications in Escherichia coli.
大肠杆菌已成为生产生物制药或其他工业相关分子的重要宿主。为了确保高生产水平和目标产物的最佳释放，高效的基因编辑工具是必不可少的。然而，在大肠杆菌中，CRISPR-Cas9 系统实现基因修饰的频率相对较低。需要进行大规模的 PCR 筛选，这阻碍了阳性克隆的鉴定。β蛋白与单链 DNA 弱结合，但与互补链退火产物紧密结合，为解决这一问题提供了一个有前途的解决方案。在本研究中，我们描述了一种针对大肠杆菌基因组的靶向和连续基因编辑策略。该策略涉及β蛋白与 CRISPR-Cas9 系统的共表达，可实现多种基因组修饰，如基因缺失和插入，效率超过 80%。β蛋白的完整性对于 CRISPR-Cas9/β 为基础的基因编辑系统至关重要。在这项工作中，N 端或 C 端结构域的缺失显著降低了系统效率。总的来说，我们的研究结果确立了 CRISPR-Cas9/β 系统作为大肠杆菌中各种应用的合适基因编辑工具。
