首页|CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli
CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli
CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli
遗传学生物工程学分子生物学
Wang Jialei,Zhang Xueli,Huo Yi-Xin,Wang Xueqin,Ding Tingting,Huang Chaoyong,Wang Jingge,Bi Changhao,Ma Xiaoyan,Zhu Lin.CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli[EB/OL].(2025-03-28)[2025-09-24].https://www.biorxiv.org/content/10.1101/605246.点此复制
Abstract
Unlike eukaryotes, bacteria are less proficient in homologous recombination (HR) and non-homologous end joining (NHEJ). All existing genomic editing methods for Escherichia coli rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in chromosomal engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient knocking-out of E. coli genomic sequence of up to 83 kb. Moreover, the positive rate and editing efficiency is independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9 complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.
SignificanceThe application in prokaryotes is difficult because of the weak homologous recombination and non-homologous end joining systems. E. coli, as the most-used prokaryote in metabolic engineering, has no NHEJ system. All existing genomic editing methods for E. coli rely on exogenous HR or NHEJ systems to repair double-strand breaks introduced by CRISPR/Cas9. In this report, we firstly demonstrate that the weak and previously ignored end-joining mechanism in E. coli can be used for efficient large-scale genetic engineering assisted by CRISPR/Cas9. Our efforts greatly simplify the genomic editing procedure of E. coli and provide an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.
Escherichia coligenetic engineeringCRISPR/Cas9end-joiningLarge fragment deletion
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