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The clustered regularly interspaced short palindromic repeats(CRISPR)-associated nuclease 9(Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655.However,this system failed to manipulate the genome of E.coli BL21(DE3),owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain.In this study,we modified the pCas/pTargetF system by replacing the pro-moter of gRNA-pMB1 with a tightly regulated promoter PrhaB,changing the replicon of pCas to a nontemperature-sensitive replicon,adding the sacB gene into pCas,and replacing the original N20-specific sequence of pTargetF with ccdB gene.We call this updated system as pEcCas/pEcgRNA.We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA.We also confirmed that genome editing can successfully be performed in BL21(DE3)by pEcCas/pEcgRNA with high efficiency.The application of pEc-Cas/pEcgRNA was then expanded to the E.coli B strain BL21 StarTM(DE3),K-12 strains MG1655,DH5α,CGMCC3705,Nissle1917,W strain ATCC9637,and also another species of Enterobacteri-aceae,Tatumella citrea DSM13699,without any specific modifications.Finally,the plasmid curing process was optimized to shorten the time from～60h to～32h.The entire protocol(including plasmid construction,editing,electroporation and mutant verification,and plasmid elimination)took only～5.5days per round in the pEcCas/pEcgRNA system,whereas it took～7.5days in the pCas/pTargetF system.This study established a faster-acting genome editing tool that can be used in a wider range of E.coli strains and will also be useful for other Enterobacteriaceae species.