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Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli

  • Yanjun Li
  • Fangqing Yan
  • Heyun Wu
  • Guoliang Li
  • Yakun Han
  • Qian Ma
  • Xiaoguang Fan
  • Chenglin Zhang
  • Qingyang Xu
  • Xixian XieEmail author
  • Ning ChenEmail author
Metabolic Engineering and Synthetic Biology - Original Paper
  • 220 Downloads

Abstract

Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3–4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.

Keywords

Chromosomal integration Large DNA fragment CRISPR/Cas9 Protospacer Escherichia coli 

Notes

Acknowledgements

We would like to thank Prof. Tao Chen of Tianjin University for providing plasmids used in the CRISPR/Cas9 system. This work was financially supported by National High Technology Research and Development Program (2015AA021003), National Natural Science Foundation of China (31500026, 31700037), China Postdoctoral Science Foundation funded project (2016M601269, 2017M61170) and Tianjin Key Technology R & D program of Tianjin Municipal Science and Technology Commission (17YFZCSY01050).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10295_2018_2114_MOESM1_ESM.docx (55 kb)
Supplementary material 1 (DOCX 55 kb)

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Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Yanjun Li
    • 1
    • 2
    • 3
  • Fangqing Yan
    • 1
  • Heyun Wu
    • 1
  • Guoliang Li
    • 1
  • Yakun Han
    • 1
  • Qian Ma
    • 1
    • 2
    • 3
  • Xiaoguang Fan
    • 1
    • 2
    • 3
  • Chenglin Zhang
    • 1
    • 2
    • 3
  • Qingyang Xu
    • 1
    • 2
    • 3
  • Xixian Xie
    • 1
    • 2
    • 3
    Email author
  • Ning Chen
    • 1
    • 2
    • 3
    Email author
  1. 1.College of BiotechnologyTianjin University of Science and TechnologyTianjinChina
  2. 2.National and Local United Engineering Lab of Metabolic Control Fermentation TechnologyTianjin University of Science and TechnologyTianjinChina
  3. 3.Key Laboratory of Industrial Fermentation Microbiology, Ministry of EducationTianjin University of Science and TechnologyTianjinChina

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