Energy-assisted CRISPR cleavage and probiotic vesicle signaling platform: Microbiome reprogramming for homeostasis

Pathogenic infections drive microbial dysbiosis and persistent inflammation, posing therapeutic challenges due to difficulties in precise pathogen eradication and microbiome restoration. Although CRISPR-based therapeutics enable pathogen-specific antibacterial targeting, their effectiveness in treating pathogenic infections is constrained by difficulties in navigating complex microbial ecosystems, penetrating pathogenic barriers, sustaining energy-intensive intracellular cleavage, and, critically, restoring microbial balance after pathogen clearance. Here, we engineer a probiotic vesicle-synergized CRISPR platform by encapsulating gtfB -targeting CRISPR plasmids within hybrid extracellular vesicles from probiotics and pathogenic Streptococcus mutans . The pathogen-derived vesicle component enables targeted uptake by S. mutans , facilitating intracellular cleavage of the virulence gene gtfB . Vesicle-carried endogenous adenosine triphosphate (ATP) boosts CRISPR activity, amplifying targeted DNA cleavage for potent and selective pathogen elimination. Probiotic-derived vesicle components further remodel quorum-sensing networks and immunity, restoring microbial homeostasis. This probiotic vesicle-based strategy integrates ATP-enhanced CRISPR cleavage with microbiome and immune modulation, offering a next-generation therapeutic paradigm for microbiome-associated diseases.