title: PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli.
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.
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Area, Tianjin, 300308, China.
authors.forename: Yilan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Maohua
authors.initials: M
authors.lastname: Yang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Daojiang
authors.initials: D
authors.lastname: Yan
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Wanwan
authors.initials: W
authors.lastname: Cheng
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center of Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800, Lyngby, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Thygesen
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Ruonan
authors.initials: R
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Xing
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Area, Tianjin, 300308, China.
authors.forename: Qinhong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Area, Tianjin, 300308, China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D016133:Polymerase Chain Reaction; D020080:Tandem Repeat Sequences / Q000235:genetics