RNA-triggered Cas12a2 antibacterial activity and resistance plasmid degradation

Antimicrobial resistance (AMR) necessitates programmable antibacterial strategies with novel mechanisms. Here, we evaluated an inducible intracellular CRISPR/Cas12a2 system in engineered Escherichia coli targeting a chloramphenicol-resistance (Chl) transcript. Cas12a2 activation significantly delayed bacterial growth and reduced recoverable colony-forming units (CFU) by 3-log10 in vitro, and reduced bacterial burdens in a mouse skin wound model. Transcriptomic profiling revealed upregulation of recA, a key mediator of the SOS response. We therefore designed a Chl-recA dual-targeting construct, which produced a greater growth delay than either single-targeting counterpart. The optimized dual-targeting system led to an approximately 4-log10 reduction in bacterial burden in vivo. Furthermore, exploratory qPCR of culture supernatants suggested a treatment-associated decrease in amplifiable plasmid DNA. Overall, this study provides proof-of-concept for resistance-transcript-guided Cas12a2 antibacterial activity and highlights the potential of co-targeting stress-associated transcripts to enhance programmable antimicrobials.