A Transposon-Associated CRISPR/Cas9 System Specifically Eliminates both Chromosomal and Plasmid-Borne mcr-1 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 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 ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 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 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.

Keywords: CRISPR/Cas9; ISApl1; antibiotic resistance; mcr-1; transposon.

FIG 1

The pISApl1-CRISPR/Cas9 system for plasmid curing and bacterial killing. A suicide plasmid containing the ISApl1-formed transposon carrying CRISPR/Cas9 was transferred to recipient bacteria by biparental mating. Donor cell E. coli WM3064 contains a chromosomal copy of the RP4 transfer machinery used to mobilize the suicide plasmids. Once inside the recipient cell, The Tn::ISApl1-CRISPR/Cas9 was integrated into the plasmid or chromosome in the recipient strain. Selection on antibiotic plates lacking DAP eliminates the E. coli WM3064 donors and retains recipients with an integrated ISApl1-CRISPR/Cas9 library. The coexpression of sgRNA and Cas9 is capable of plasmid curing and bacterial killing.

FIG 2

Plasmid map of pISApl1-CRISPR/Cas9 and the transposon Tn::ISApl1/CRISPR/Cas9. (a) Plasmid pISApl1-CRISPR/Cas9 containing the cas9 gene with the promoter pLteto-1 (39) and the sgRNA with the synthetic J23119 promoter; F and R denote the locations of primers used for the sgRNA construct. (b) The Tn::ISApl1-CRISPR/Cas9, the cas9, and the tpm gene, along with sgRNA was flanked by two ISApl1 in the same orientation.

FIG 3

(a) Schematic of mobilizable ISApl1-CRISPR/Cas9-mediated plasmid curing and cell killing. (b) E. coli WM3064 donor cells possessing pISApl1-CRISPR/Cas9-mcr-1 or pISApl1-CRISPR/Cas9-IncX4 were mated at a donor/recipient ratio of 1:1 with strain E. coli CSZ4 harboring a plasmid-borne mcr-1 or strain E. coli MG1655-MCR in which mcr-1 is located on the chromosome. Cultures were plated on selective LB agar sodium tellurite (25 μg/ml) in the presence and absence of colistin (2 μg/ml). The mobilization of the pISApl1-CRISPR/Cas9 construct into cells containing the mcr-1 gene resulted in plasmid curing and cell death. Mean ± standard error (SE). Asterisks denote the absence of detectable transconjugant colonies.

FIG 4

(a) Schematic diagram of the immune system against foreign plasmids of E. coli C600 with ISApl1-CRISPR/Cas9 integrated into the chromosome. (b) The strain E. coli C600 with the chromosome bearing ISApl1-CRISPR/Cas9-ISApl1 and the parental strain E. coli C600 were used as recipients conjugated with donor strains E. coli CSZ4, S. enterica 19E0341, and K. pneumoniae YZ01, respectively. The bar chart represented the results when E. coli C600 was used as a recipient (positive control). The asterisks denote the chromosome-borne ISApl1-CRISPR/Cas9-ISApl1 strain used as recipient. Each experiment was performed in triplicate. Data points represent the mean values of three biological replicates with error bars showing standard deviation (SD).

FIG 5

Characterization of escape mutants that tolerated transformation of the pISApl1-CRISPR/Cas9 construct. (a) The CRISPR/Cas9 region of the escape mutants was amplified by a high-fidelity enzyme and followed by sanger sequencing. (b, c) The results show that 510 or 511 deletions at the same sgRNA area led to pISApl1-CRISPR/Cas9 inactivation in the successful transformants.