Our Project

Our team is investigating the utility of outer membrane vesicles (OMVs) in the delivery of Cas9 in vivo. All Gram-negative bacteria produce OMVs and use them to deliver toxins, communicate with other bacteria, mediate membrane composition, and extract materials such as metal ions from their environment. OMVs have been successfully engineered to carry heterologous proteins,¹ which makes them an attractive candidate for systems that require the delivery of a functional protein or complex to recipient cells. One such system is the application of CRISPR-Cas9 to treating antibiotic-resistant bacterial infections. At the moment the most effective methods of delivering Cas9 to target cells are either limited to DNA delivery (bacteriophage delivery² and hydrodynamic injection³) or require extensive vector engineering in order to facilitate controlled delivery and uptake (lipid-mediated tranfection⁴). We hope to use signal peptides and protein fusions to direct Cas9 protein into OMVs so that the functional complex can be delivered to and act on the resistance genes in antibiotic-resistant bacteria without extensive lipid modification to facilitate uptake.