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Updated: Mar 23, 2026

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems
Published on: October 14, 2025
PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli
Yilan Liu1, Maohua Yang2, Jinjin Chen2
1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Area, Tianjin, 300308, China.
Abstract:
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.
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