High-efficiency CRISPR-Cas9 delivery by engineered bacteria for precise elimination of antibiotic resistant Enterobactericeae

Bacterial resistance to antibiotics threatens our ability to treat infectious diseases, spurring a growing interest in alternative antimicrobial technologies. One attractive strategy is to use bacterial conjugation to deliver CRISPR-cas9 systems programmed to precisely target and eliminate antibiotic resistant bacteria. However, the success of this approach heavily depends on high in situ DNA transfer rates. Here, we repurposed conjugative plasmid TP114 as a high efficiency delivery vehicle for CRISPR-cas9 in the gut microbiota. A single dose of this system was able to eliminate 98% of targeted antibiotic resistant Escherichia coli in the mouse digestive tract while leaving an otherwise isogenic population and the rest of the microbiota essentially untouched. Through accelerated laboratory evolution, we next generated an improved system that killed >99.9% of the targeted antibiotic resistant population in situ. Taken together, these results demonstrate the potential of CRISPR-Cas9 delivery by engineered bacteria as a promising strategy to fight antibiotic resistance.