(a) E. coli WM 3064 carrying a pIS26-CRISPR/Cas9 plasmid targeting the mcr-1 gene conjugated with E. coli 001, E. coli 004 and E. coli 005 and the transposition efficiency was calculated. (b) Time course characterization of the Tn: IS26-CRISPR/Cas9 plasmid-curing system. Plasmid curing of pEc001, pUC19-mcr-1 and pUC19-IS26mcr-1 containing the mcr-1 gene. Plating on antibiotic LB agar plates was performed at 4, 8, 12 and 24 h after induction. The genotype of each plasmid-cured strain was confirmed by PCR using specific primers and the AR phenotype was confirmed using selective plates. The curing efficiency was calculated as PCR negative per 50 single colonies. Error bars represent the results from at least three biological replicates. After plasmid curing, Tn: IS26-CRISPR/Cas9 remained in the bacterial host as an immune system, and could block plasmid acquisition. (c) The Tn: IS26-CRISPR/Cas9 targeting the mcr-1 gene remained in E. coli 004 and E. coli 005 (resulting strains were ΔE. coli 004 and ΔE. coli 005). The resulting strain ΔE. coli 004 was used as a recipient and conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002 and E. coli WM3064: pEc003, then the conjugation efficiency was calculated. (d) After plasmid curing, the resulting strains ΔE. coli 001, ΔE. coli 002 and ΔE. coli 003 with integrated Tn: IS26-CRISPR/Cas9 targeting replication genes IncX4, IncI2 and IncHI2, respectively, were also used as recipients conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002, E. coli WM3064: pEc003, E. coli WM3064: pEc006 and E. coli WM3064: pEc007. The conjugation efficiency was calculated. (e) After plasmid curing, the resulting strains ΔE. coli 006 and ΔE. coli 007 with integrated Tn: IS26-CRISPR/Cas9 targeting bla_KPC-2 and bla_NDM-5 genes were also used as recipients conjugated with E. coli WM3064: pEc001 and E. coli WM3064: pEc002. Error bars represent conjugation results from at least three biological replicates and P < 0.01 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Mean ± SEM, **P < 0.01, P values were calculated using the Student’s t-test. Red asterisks denote the absence of detectable transconjugant colonies.