Grant: Project Grant

We propose to develop a safe-to-use, efficient and programmable conjugative probiotic (COP) as a highly innovative tool to precisely edit the microbiome and fight multidrug resistant pathogens. The E. coli Nissle 1917 probiotic strain, which has previously been shown to be clinically safe, is used as the COP chassis for our experiments. COP bacteria also include potent bacterial conjugation machineries for high-efficiency delivery of CRISPR-Cas9 systems into the microbiota. The CRISPR-Cas9 system can easily be programmed to cleave DNA simply by providing custom guide RNA (gRNA) spacer sequences that match the desired target sites. Targeting DNA sequences located in a bacterial chromosome leads to cell death by compromising genome integrity. The CRISPR-Cas9 system can also cure cells from plasmids carrying antibiotic resistance or virulence genes, thereby "disarming" potentially dangerous bacteria by restoring susceptibility to antibiotics. Unlike broad-spectrum antibiotics, the COP system is designed to be highly specific, with selectivity conferred by carefully chosen CRISPR-Cas9 target sequences and tuning of the host range through the selection of appropriate COP conjugative machineries. This new technology would also constitute a powerful microbiome manipulation tool for fundamental research, and to progressively advance towards clinical applications. Once established, this innovative method could have a significant impact on medicine by eventually offering a new tool to treat dysbiosis or as an alternative to antimicrobials for the prevention or treatment of many infectious diseases.