Biotechnology Letters

, Volume 41, Issue 2, pp 293–303 | Cite as

Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli

  • Dongchang SunEmail author
  • Lin Wang
  • Xudan Mao
  • Mingyue Fei
  • Yiyang Chen
  • Minjia Shen
  • Juanping Qiu
Original Reserach Paper



To develop a convenient chemical transformation mediated CRISPR/Cas9 (CT-CRISPR/Cas9) system for genome editing in Escherichia coli.


Here, we have constructed a CT-CRISPR/Cas9 system, which can precisely edit bacterial genome (replacing, deleting, inserting or point mutating a target gene) through chemical transformation. Compared with the traditional electroporation mediated CRISPR/Cas9 (ET-CRISPR/Cas9) system, genome editing with the CT-CRISPR/Cas9 system is much cheaper and simpler. In the CT-CRISPR/Cas9 system, we observed efficient genome editing on LB-agar plates. The CT-CRISPR/Cas9 system has successfully modified the target gene with the editing template flanked by short homologous DNA fragments (~ 50 bp) which were designed in primers. We used the lab-made CaCl2 solution to perform the CT-CRISPR/Cas9 experiment and successfully edited the genome of E. coli. Potential application of the CT-CRISPR/Cas9 system in high-throughput genome editing was evaluated in two E. coli strains by using a multiwell plate.


Our work provides a simple and cheap genome-editing method, that is expected to be widely applied as a routine genetic engineering method.


Chemical transformation CRISPR/Cas9 Escherichia coli Genome editing 



We acknowledge Dr. Sheng Yang and Dr. Junjie Yang for kindly donating plasmids pCas and pTargetF-pMB1. This research was supported by National Natural Science Foundation of China under Grant No. 31670084 and Zhejiang Provincial Natural Science Foundation of China under Grant No. LY16C010003.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2639_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1870 kb)


  1. Barrangou R, Marraffini LA (2014) CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell 54:234–244. CrossRefGoogle Scholar
  2. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. CrossRefGoogle Scholar
  3. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964. CrossRefGoogle Scholar
  4. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. CrossRefGoogle Scholar
  5. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645. CrossRefGoogle Scholar
  6. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356:438–442. CrossRefGoogle Scholar
  7. Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2:924–932. CrossRefGoogle Scholar
  8. Heidari R, Shaw DM, Elger BS (2017) CRISPR and the rebirth of synthetic biology. Sci Eng Ethics 23:351–363. CrossRefGoogle Scholar
  9. Heo MJ, Jung HM, Um J, Lee SW, Oh MK (2017) Controlling citrate synthase expression by CRISPR/Cas9 genome editing for n-butanol production in Escherichia coli ACS. Synth Biol 6:182–189. CrossRefGoogle Scholar
  10. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. CrossRefGoogle Scholar
  11. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514. CrossRefGoogle Scholar
  12. Komor AC, Badran AH, Liu DR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168:20–36. CrossRefGoogle Scholar
  13. Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng 31:13–21. CrossRefGoogle Scholar
  14. Marraffini LA (2015) CRISPR-Cas immunity in prokaryotes. Nature 526:55–61. CrossRefGoogle Scholar
  15. Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322:1843–1845. CrossRefGoogle Scholar
  16. Mojica FJ, Montoliu L (2016) On the origin of CRISPR-Cas technology: from prokaryotes to mammals. Trends Microbiol 24:811–820. CrossRefGoogle Scholar
  17. Pyne ME, Moo-Young M, Chung DA, Chou CP (2015) Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli. Appl Environ Microbiol 81:5103–5114. CrossRefGoogle Scholar
  18. Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem 82:237–266. CrossRefGoogle Scholar
  19. Su T, Liu F, Gu P, Jin H, Chang Y, Wang Q, Liang Q, Qi Q (2016) A CRISPR-Cas9 assisted non-homologous end-joining strategy for one-step engineering of bacterial genome. Sci Rep 6:37895. CrossRefGoogle Scholar
  20. Sun D (2016) Two different routes for double-stranded DNA transfer in natural and artificial transformation of Escherichia coli. Biochem Biophys Res Commun 471:213–218. CrossRefGoogle Scholar
  21. Sun D (2018) Pull in and push out: mechanisms of horizontal gene transfer in bacteria. Front Microbiol. Google Scholar
  22. Sun D, Zhang Y, Mei Y, Jiang H, Xie Z, Liu H, Chen X, Shen P (2006) Escherichia coli is naturally transformable in a novel transformation system. FEMS Microbiol Lett 265:249–255. CrossRefGoogle Scholar
  23. Sun D, Zhang X, Wang L, Prudhomme M, Xie Z, Martin B, Claverys JP (2009) Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli. J Bacteriol 191:713–719. CrossRefGoogle Scholar
  24. Sun D, Wang B, Zhu L, Chen M, Zhan L (2013) Block and boost DNA transfer: opposite roles of OmpA in natural and artificial transformation of Escherichia coli. PLoS ONE 8:e59019. CrossRefGoogle Scholar
  25. Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227–264. CrossRefGoogle Scholar
  26. Wright AV, Nunez JK, Doudna JA (2016) Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164:29–44. CrossRefGoogle Scholar
  27. Zerbini F, Zanella I, Fraccascia D, Konig E, Irene C, Frattini LF, Tomasi M, Fantappie L, Ganfini L, Caproni E, Parri M, Grandi A, Grandi G (2017) Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli. Microb Cell Fact 16:68. CrossRefGoogle Scholar
  28. Zhang Y, Shi C, Yu J, Ren J, Sun D (2012) RpoS regulates a novel type of plasmid DNA transfer in Escherichia coli. PLoS ONE 7:e33514. CrossRefGoogle Scholar
  29. Zhao D, Yuan S, Xiong B, Sun H, Ye L, Li J, Zhang X, Bi C (2016) Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15:205. CrossRefGoogle Scholar
  30. Zhu X, Zhao D, Qiu H, Fan F, Man S, Bi C, Zhang X (2017) The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway. Metab Eng 43:37–45. CrossRefGoogle Scholar

Copyright information

© Springer Media B.V. 2018

Authors and Affiliations

  • Dongchang Sun
    • 1
    Email author
  • Lin Wang
    • 1
  • Xudan Mao
    • 1
  • Mingyue Fei
    • 1
  • Yiyang Chen
    • 1
  • Minjia Shen
    • 1
  • Juanping Qiu
    • 1
  1. 1.College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouPeople’s Republic of China

Personalised recommendations