Abstract
Objectives
To develop a convenient chemical transformation mediated CRISPR/Cas9 (CT-CRISPR/Cas9) system for genome editing in Escherichia coli.
Results
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.
Conclusions
Our work provides a simple and cheap genome-editing method, that is expected to be widely applied as a routine genetic engineering method.
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Acknowledgements
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.
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Sun, D., Wang, L., Mao, X. et al. Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli. Biotechnol Lett 41, 293–303 (2019). https://doi.org/10.1007/s10529-018-02639-1
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DOI: https://doi.org/10.1007/s10529-018-02639-1