Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9

Mu En Chung, I. Hsin Yeh, Li Yu Sung, Meng Ying Wu, Yun Peng Chao, I-Son Ng, Yu Chen Hu

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172–183.

Original languageEnglish
Pages (from-to)172-183
Number of pages12
JournalBiotechnology and Bioengineering
Volume114
Issue number1
DOIs
Publication statusPublished - 2017 Jan 1

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Clustered Regularly Interspaced Short Palindromic Repeats
Chromosomes
Escherichia coli
DNA
Genes
Homologous Recombination
Metabolic Engineering
Lac Operon
Metabolic engineering
Cell Survival
Cell Death
Cell death
RNA
Cells
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Chung, M. E., Yeh, I. H., Sung, L. Y., Wu, M. Y., Chao, Y. P., Ng, I-S., & Hu, Y. C. (2017). Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9. Biotechnology and Bioengineering, 114(1), 172-183. https://doi.org/10.1002/bit.26056
Chung, Mu En ; Yeh, I. Hsin ; Sung, Li Yu ; Wu, Meng Ying ; Chao, Yun Peng ; Ng, I-Son ; Hu, Yu Chen. / Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9. In: Biotechnology and Bioengineering. 2017 ; Vol. 114, No. 1. pp. 172-183.
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Chung, ME, Yeh, IH, Sung, LY, Wu, MY, Chao, YP, Ng, I-S & Hu, YC 2017, 'Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9', Biotechnology and Bioengineering, vol. 114, no. 1, pp. 172-183. https://doi.org/10.1002/bit.26056

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9. / Chung, Mu En; Yeh, I. Hsin; Sung, Li Yu; Wu, Meng Ying; Chao, Yun Peng; Ng, I-Son; Hu, Yu Chen.

In: Biotechnology and Bioengineering, Vol. 114, No. 1, 01.01.2017, p. 172-183.

Research output: Contribution to journalArticle

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