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 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.

Global Epidemiology and Genetic Environment of mcr genes: A One Health Systematic Review of Current and Emerging Trends

“…The CRISPR-Cas9 system was designed to target IS Apl1, which eliminated mcr-1 harbouring plasmids, and in the case of chromosomally encoded mcr-1 , cell death was seen. Similar results were achieved 170 , where recipient cells further acquired immunity against the acquisition of the exogenous mcr-1 containing plasmid 172 . These studies show that CRISPR-Cas9 system is an efficient tool for plasmid curing and to sensitise clinical isolates to antibiotics in vitro 170, 171 .…”

“…Wang et al (2019a) 171 used the same concept but developed the tool to remove both mcr-1 -harbouring plasmids and MDR plasmids present in recipient cells using partial sequences of the targeted plasmids. He et al (2021) 172 used the tool to eliminate both chromosomal and plasmid-borne mcr-1 genes in E. coli. The CRISPR-Cas9 system was designed to target IS Apl1, which eliminated mcr-1 harbouring plasmids, and in the case of chromosomally encoded mcr-1 , cell death was seen.…”

Global epidemiology, genetic environment, risk factors and therapeutic prospects of mcr genes: A current and emerging update

“…A phylogenetic analysis of all MCR proteins shows that MCR-1, MCR-2, MCR-6 and Neisseria LptA were part of a parallel evolutionary event for functional acquisition of colistin resistance during some environmental selection pressure, i.e., the intensive use of colistin in animals feed (Sun et al, 2017b;Wang et al, 2017;Islam et al, 2020). Thereafter, MCR-3, MCR-4, MCR-7, and MCR-9 have similar amino acid sequences He et al, 2021), and further, are closely related at a structural level (Carroll et al, 2019). However, MCR-5, MCR-8 and MCR-10 have a low identity to the other MCR proteins, though a sequence alignment shows that all ten MCR proteins each encode the conserved active residues, six cysteine residues and each is capable of LPS modifications (Borowiak et al, 2017;Wang et al, 2018b;Carroll et al, 2019).…”

Targeted Elimination of blaNDM-5 Gene in Escherichia coli by Conjugative CRISPR-Cas9 System

“… 10−13 The mobile genetic elements (MGE)-associated CRISPR-Cas9 may serve as a therapeutic approach to control the dissemination of antibiotic resistance among clinical pathogens. 14 , 15 These works support that CRISPR-Cas9 system can solve the problem of multi-drug resistance in clinical and environmental settings. However, concerning the CRISPR-Cas9 system in clinical application, the delivery method must be developed and optimized.…”

“… 13 , 40 In addition to plasmid elimination, the CRISPR-Cas9 could further provide immunity in E. coli against the acquisition of plasmid-mediated ARGs. 14 , 34 …”