Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria

Area of Science:

• Molecular Biology
• Microbiology
• Genetic Engineering

Background:

• Antimicrobial resistance (AMR) is a critical global health issue.
• CRISPR-Cas systems offer potential for gene-curing to combat AMR.
• Efficient delivery systems are needed for broad application of CRISPR-Cas AMR strategies.

Purpose of the Study:

• To design and construct a novel, broad-host-range gene delivery plasmid for CRISPR-Cas systems.
• To enable efficient gene-curing of antimicrobial resistance genes in diverse bacteria.
• To evaluate the efficacy of the new system against clinically relevant resistance genes.

Main Methods:

• Construction of a novel plasmid, pQ-mini, using an Inc.Q plasmid backbone.
• Integration of the CRISPR-Cas12f system into the pQ-mini plasmid.
• Evaluation of gene transfer efficiency and conjugation in various bacterial species, including Enterobacterales.
• Assessment of plasmid-borne antimicrobial resistance gene (mcr-1, blaKPC) curing efficiency.

Main Results:

• The pQ-mini plasmid demonstrated efficient genetic element transfer across a broad bacterial host range.
• Conjugation efficiency of pQ-mini surpassed that of a common pMB1-like plasmid, especially in Enterobacterales.
• pQ-mini successfully delivered the CRISPR-Cas12f system to resistant strains.
• High curing efficiencies for mcr-1 and blaKPC genes were achieved, comparable to existing systems.

Conclusions:

• pQ-mini is an effective broad-host-range functional gene delivery vector.
• The pQ-mini/CRISPR-Cas12f system shows significant potential for broad-host delivery of antimicrobial strategies.
• This technology represents a promising novel tool to combat the escalating threat of antimicrobial resistance.