Liu Yilan, Yang Maohua, Chen Jinjin, Yan Daojiang, Cheng Wanwan, Wang Yanyan, Thygesen Anders, Chen Ruonan, Xing Jianmin, Wang Qinhong, Ma Yanhe
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Area, Tianjin, 300308, China.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
PLoS One. 2016 Mar 28;11(3):e0149762. doi: 10.1371/journal.pone.0149762. eCollection 2016.
Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.
效率和保真度是基因组编辑工具箱的关键障碍。在本研究中，开发了一种基于PCR的串联重复序列辅助基因组编辑（TRAGE）方法，该方法具有高效率和保真度。TRAGE的设计基于通过复制叉重新激活修复自发双链断裂（DSB）的机制。首先，在Red酶的辅助下，将侧翼为串联重复序列的cat-sacB盒整合到染色体的靶位点。然后，对于cat-sacB盒的切除，仅需要继代培养。所开发的方法成功应用于使用PCR产物无缝删除、替换和插入靶向基因。研究了不同操作（包括蔗糖添加时间、在含蔗糖的LB中传代次数和接种阶段）对效率的影响。按照我们推荐的程序，可以在48小时内高效实现cat-sacB盒的无缝切除。我们相信，所开发的方法在大肠杆菌的无缝基因组编辑方面具有巨大潜力。
