A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne <i>mcr-1</i> in Escherichia coli

Abstract

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the <i>mcr-1</i> 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 <i>mcr-1</i> gene facilitated by IS<i>Apl1-</i>formed transposon Tn<i>6330</i> and mediated rapid dispersion among <i>Enterobacteriaceae</i> species. Here, we developed a CRISPR/Cas9 system flanked by IS<i>Apl1</i> in a suicide plasmid capable of exerting sequence-specific curing against the <i>mcr-1</i>-bearing plasmid and killing the strain with chromosome-borne <i>mcr-1</i>. The constructed IS<i>Apl1</i>-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne <i>mcr-1</i> or directly eradicated the bacteria harboring chromosome-borne <i>mcr-1</i> by introducing an exogenous CRISPR/Cas9 targeting the <i>mcr-1</i> gene. This method is highly efficient in removing the <i>mcr-1</i> gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous <i>mcr-1</i> 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 <i>mcr-1</i> resistance among clinical pathogens.