The global spread of antimicrobial-resistant bacteria has been one of the most severe threat to public health. The emergence of mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by IS Apl1- formed transposon Tn 6330 and mediated rapid dispersion among Enterobacteriaceae species. Here we developed a CRISPR-Cas9 system flanked by IS Apl1 in a suicide plasmid capable of exerting the sequence-specific curing against mcr-1 bearing plasmid and killing the strain with chromosomal-borne mcr-1 . The constructed IS Apl1 -carried CRISPR-Cas9 system either restored the sensitivity to colistin of strains with plasmid-borne mcr-1 or directly eradicated the bacteria harbored the chromosomal-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting mcr-1 gene. This method is highly efficient in removing mcr-1 gene from Escherichia coli and thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with the immunity against the acquisition of the exogenous mcr-1- containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.

Objectives In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes. Methods A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts. Results This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids. Conclusions The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.