OBJECTIVE To utilize the CRISPR/Cas12f1 system to target and clear the mcr-10.1 resistance plasmid, restore bacterial sensitivity to colistin, and provide a new approach for the prevention and control of mcr-10.1 resistance genes.

METHODS A high-copy resistance plasmid pSEVA551-EGFP-mcr-10.1 containing mcr-10.1 and a model resistant bacterium DH5 were constructed. A total of 79 mcr-10.1 target spacers were designed following the protospacer adjacent motif (PAM) principle, and the three optimal targets were selected to construct recombinant CRISPR plasmids. The experimental group was established by heat-shock transformation of three recombinant CRISPR plasmids into the pmcr-10.1 model resistant bacteria. The control group was established by transforming the empty vector plasmid pCas12f1 into the pmcr-10.1 model resistant bacterium, and agarose gel electrophoresis, quantitative polymerase chain reaction (qPCR) and antimicrobial susceptibility testing (E-test) were conducted. The resistant plasmid pSEVA551-EGFP-mcr-10.1 was transformed into the control group containing the empty vector pCas12f1 plasmid, and into the experimental group containing three sets of recombinant CRISPR plasmids with targets, to carry out plasmid horizontal transfer block experiments. Data and graphs were processed with Graphpad Prism 10.1. 2.

RESULTS Colony PCR showed that the clearance rate of mcr-10.1 in the three CRISPR plasmid experimental groups was 100.00%. qPCR indicated that the target clearance efficiency could reach up to 92.00% within 12 hours, and the target clearance rate exceeded 90.00% within 24 hours, with the highest clearance rate reaching 99.22%, and the optimal target was selected. Antimicrobial susceptibility testing showed that the colistin minimum inhibitory concentration (MIC) value in the experimental group decreased from 5.33 μg/ml in the control group to 0.67-0.92 μg/ml, indicating the restoration of sensitivity. In the resistant plasmid transfer experiment, the plasmid transfer amount in the experimental group was reduced by 607-1 679 times compared to the control group.

CONCLUSIONS The CRISPR/Cas12f1 system can efficiently clear the mcr-10.1 resistance gene, reverse bacterial resistance phenotypes and block the transfer of resistance plasmids. Due to its small size and ease of delivery, it provides an efficient and feasible new strategy for the prevention and control of colistin resistance.