Abstract: CRISPR/Cas9 gene editing system has been widely used in the construction of Escherichia coli engineering strains. If a continuous gene editing-based CRISPR/Cas9 system can be constructed, the construction efficiency of E. coli engineering strains will be greatly improved. In this study, a donor plasmid pV4, with self-cutting function was constructed to optimize the existing double plasmid CRISPR/Cas9 gene editing system in the laboratory, and to realize the continuous knockout or integration of the gene. Three elements were added to the skeleton region of the original donor plasmid placZ: the N20-gRNA sequence of chloramphenicol gene(Cat) on the donor plasmid; Ptrc promoter; lacIq sequence. After gene editing with pV4 series plasmids(knockout or integration), these elements were induced by IPTG and with the help of Cas9 protein to achieve self-cutting of donor plasmids. The optimized pV4 series plasmids(knockout or integration) were used to engineer a strain to produce itaconic, and the result showed that this new CRISPR/Cas9 gene editing system can quickly perform continuous knockout or integration of genes, which is a broad prospect of construction and application of engineering strains.

Key words: CRISPR/Cas9, gene editing, Escherichia coli, self-cutting