A CRISPR-Cas9 mediated technology for controlling the bla_NDM-1 gene

To construct a single-plasmid clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) single-plasmid system integrated with conjugative transfer elements, evaluate its ability to eliminate antibiotic resistance genes (ARG) harbored by bacteria, and explore the feasibility of containing the dissemination of ARG via conjugative transfer.

A CRISPR-Cas9 plasmid vector targeting the bla_NDM-1 gene was designed, and the oriT-traJ conjugative transfer module was integrated into the vector backbone. Electrotransformation and conjugative transfer assays were performed to assess the elimination efficiency of this system against bla_NDM-1. Antibiotic susceptibility assays were conducted to verify the restoration of antibiotic susceptibility in edited bacterial strains.

The elimination efficiency of the established CRISPR-Cas9 single-plasmid system was 97.9%, 87.5% and 56.3% against bla_NDM-1 located on the chromosome, plasmid, and dual location respectively. PCR results demonstrated that the system was still able to eliminate target genes after conjugative transfer and efficiently reversed the antibiotic resistance of recipient bacteria.

This system can eliminate the bla_NDM-1 gene and restore bacterial susceptibility to meropenem. Conjugative transfer delivery mediated by the single-plasmid system provides a novel tool for eradicating ARG and restoring the susceptibility of recipient bacteria to antibiotics.