CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli

  • Chaoyong Huang
  • Tingting Ding
  • Jingge Wang
  • Xueqin Wang
  • Liwei Guo
  • Jialei Wang
  • Lin Zhu
  • Changhao Bi
  • Xueli Zhang
  • Xiaoyan Ma
  • Yi-Xin HuoEmail author
Applied genetics and molecular biotechnology


Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. 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 genetic 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 deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA 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.


Escherichia coli Genetic engineering CRISPR-Cas9 End-joining Large-fragment deletion 


Funding information

The work finished in Beijing Institute of Technology was supported by the National Key R&D Program of China (grant No. 2017YFD0201400), the National Natural Science Foundation of China (grant No. 21676026), and Fundamental Research Funds for the Central Universities.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

253_2019_10104_MOESM1_ESM.pdf (8 mb)
ESM 1 (PDF 7.95 MB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Molecular Medicine and Biotherapy, School of Life SciencesBeijing Institute of TechnologyBeijingPeople’s Republic of China
  2. 2.UCLA (Suzhou) Institute for Technology AdvancementSuzhouPeople’s Republic of China
  3. 3.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinPeople’s Republic of China

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