PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

Yilan Liu; Maohua Yang; Daojiang Yan; Wanwan Cheng; Anders Thygesen; Ruonan Chen; Jianmin Xing; Qinhong Wang; Yanhe Ma; Mark Isalan

doi:10.1371/journal.pone.0149762

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

Yilan Liu, Maohua Yang, Daojiang Yan, Wanwan Cheng, Anders Thygesen, Ruonan Chen, Jianmin Xing, Qinhong Wang, Yanhe Ma, Mark Isalan (Editor)

Department of Chemical and Biochemical Engineering

Chinese Academy of Sciences

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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.

Original language	English
Journal	P L o S One
Volume	11
Issue number	3
Pages (from-to)	e0149762
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0149762
Publication status	Published - 2016

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Copyright: © 2016 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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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. ",

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AU - Liu, Yilan

AU - Yang, Maohua

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AU - Cheng, Wanwan

AU - Thygesen, Anders

AU - Chen, Ruonan

AU - Xing, Jianmin

AU - Wang, Qinhong

AU - Ma, Yanhe

A2 - Isalan, Mark

N1 - Copyright: © 2016 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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N2 - 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.

AB - 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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SN - 1932-6203

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PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

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