Project Overview

Talk about what a Cas9-CRISPR complex could do.

How can Cas9 be delivered to infected cells?

Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection. However, all of these methods merely work for in vitro delivery of Cas9, and not in vivo (delivery to cells in live organism). We need a delivery system that can transport this protein directly to infected cells.

Two common in vivo methods for Cas9 delivery are viral vectors and hydrodynamic injection. The latter of these two methods resulted in both liver and cardiovascular damage to mice, and therefore does not seem to be a viable delivery methods for humans. Bacteriophages as vectors, on the other hand, are efficient in their delivery and expression of genes, but fall short in their size limitations and their potential to harm the immune system of the host, or the potential of the host’s immune system to eradicate the phages. Therefore, there is a growing potential for the use of non-viral vectors for the in vivo delivery of Cas9. Thus, we decided that OMVs are the simplest, most viable option for non-viral lipid-based vectors.

What are OMVs?

An OMV, or outer membrane vesicle, is a spheroid made up of the outer membrane material of a Gram-negative bacterium and filled with periplasm (Schwechheimer). OMVs have a wide variety of functions and, by pinching off from the cell (Figure 2, from Berleman), can move independently from the host and facilitate cell-cell communication in order to regulate bacterial colonies or remove toxic compounds.