Abstract: At present, type Ⅱ CRISPR/Cas9 tools are widely used in genome editing. The use of CRISPR/Cas9 genome editing technology to edit the Escherichia coli genome can be used in the research of metabolic engineering and bacterial drug resistance. Using Cas9 nickase（Cas9 n） tool to modify host cell genes could reduce the toxicity of double-strand breaks to the host. However, currently there are few studies on the Escherichia coli genome editing using CRISPR/Cas9 n, which hinders the application of Cas9 D10 A or Cas9 H840 A nickase for Escherichia coli genome editing. In this study, on the basis of the previously reported dual-plasmid Cas9 gene editing system（pCas/pTarget） in bacteria, we systematically studied the effectiveness and efficiency of Cas9 D10 A or Cas9 H840 A nickase on the editing of the endogenous sseA gene in Escherichia coli MG1655 strain. The pCas-D10 A and pCas-H840 A plasmids were constructed by molecular mutation methods. Next, the MG1655 strain stably expressing the pCas plasmid was transformed with a plasmid carrying the repair template pTargetT-△sseA（993 bp）, and the efficiency of gene editing were determined by colony PCR and DNA sequencing methods. The function of the knocked out of sseA gene in MG1655 strain was verified by the biochemical method of lead acetate test stripe. Meanwhile, the pTargetT-△sseA plasmid lack of repair template was transformed into the MG1655 strain expressing of pCas, and the editing efficiency was resolved by counting the number of survived colony in the plate. Regardless of the presence or absence of the repair template, both Cas9 D10 A and Cas9 H840 A nickase could edit the endogenous gene sseA in Escherichia coli. The editing efficiency of Cas9 D10 A or Cas9 H840 A was found to be similar, but both of them are lower than that of Cas9-WT. Compared with the wild-type Cas9, Cas9 D10 A or Cas9 H840 A has much less bacterial toxicity when editing the Escherichia coli genome. This research provides new research basis and ideas for the development of genome editing technology in Escherichia coli.