Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli
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.
KeywordsChemical 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.
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