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What is the problem we are trying to solve?

Antibiotics are among the most frequently administered drugs in human medicine. However, overprescription and failure to complete the prescribed course have contributed to microbes becoming resistant. In 2014, the World Health Organization classified antibiotic resistance as a global epidemic, highlighting the need for a solution. Inactivating resistance genes via Cas9 nuclease-mediated cleavage has been shown to be an effective means of combating this epidemic; however, methods of in vivo delivery are currently limited.

Our proposed solution

Cas9, when bound to guide RNA, results in a versatile gene editing tool that gives rise to a wide range of potential applications. Through non-homologous end joining of the double stranded break, bases are added and subtracted knocking out the gene of interest. Scientists hope to use this system to battle infectious diseases and treat multi-drug resistant microorganisms.(1) Despite the promise behind CRISPR-Cas9, the shift from its use as a research tool to a therapeutic device poses many challenges such as undesirable host immune responses and cleavage in unwanted locations due to low system specificity. Our team is researching the use of Outer Membrane Vesicles (OMVs) as a Cas9 delivery system. We hypothesize that the successful packaging and delivery of Cas9, when combined with a specific guide RNA that defines the genomic target, could be used in a therapeutic setting to combat antibiotic resistance.

Cas9 delivery

Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection.(2) However, these methods work exclusively for in vitro delivery of Cas9, and not in vivo. Thus, an alternative delivery system to transport this protein to target cells is needed. Two common in vivo methods for Cas9 delivery are viral vectors and hydrodynamic injection. The latter of these two approaches resulted in both liver and cardiovascular damage to mice, and therefore does not seem viable for humans. Alternatively, bacteriophages have been effectively used for the delivery and expression of genes, but fall short in their size limitation and their potential to harm the host’s immune system. Thus, we believe that a non-viral approach is optimal and have selected OMVs as the simplest, most viable option for delivery of Cas9 as well as the guide RNA.

What are OMVs

Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition shows a high degree of similarity with the bacterial periplasm. OMVs pinch off from the outer cell membrane and move independently from the host cell, allowing them to serve numerous functions such as the removal of toxic compounds and regulation of bacterial colonies by facilitating cell-cell communication. (3) Because OMVs are innately produced by bacteria, they are excellent candidates for non-viral lipid-based delivery systems.


(1) Doerflinger, M., Forsyth, W., Ebert, G., Pellegrini, M., & Herold, M. (2016). CRISPR/Cas9-The ultimate weapon to battle infectious diseases? Cellular Microbiology, 19(2). doi:10.1111/cmi.12693

(2) Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology,13(10), 605-619. doi:10.1038/nrmicro3525

(3)Berleman, J., & Auer, M. (2012). The role of bacterial outer membrane vesicles for intra- and interspecies delivery. Environmental Microbiology, 15(2), 347-354. doi:10.1111/1462-2920.1204