Cas9 Delivery

The Cas9 endonuclease, when co-localized with a single guide RNA, can generate a double strand break (DSB) at a specified location. Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection. ⁽²⁾However, these methods work excursively 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 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.⁽²⁾ 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?

Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition has 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 removing toxic compounds and regulating 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.

Our Project

This summer, our team will tackle three key features of this project: the incorporation of Cas9 into OMVs, the inclusion of guide RNAs into separate OMVs and finally the fusion of these vesicles with target cells and distribution of their contents.

Delivery of Cas9 to target cell

To deliver Cas9 using an OMV, our team is going to start with a particular strain of E. coli (JC8031) that is genetically engineered to be hyper vesiculating (constantly creating OMVs from its outer membrane). These bacteria will be introduced to a plasmid that will allow the cell to metabolically create the Cas9 protein and, using signalling peptides, will encourage the secretion of Cas9 through the inner membrane and into the periplasm. Here we will take a sample and test for the existence of Cas9 in the periplasm using fractionation– lysing only the outer membrane of the cell. Once we have confirmed that the Cas9 has been introduced into the periplasm, the Cas9 protein will exit the cell via an OMV.

The team will then separate the OMVs from the original cells using ultracentrifugation. Then, we will take a sample, lyse the cells, and test for Cas9 using a western blot. Once the presence of Cas9 in the OMVs has been confirmed, the OMVs will be introduced to a strain of E. coli (DH5-Alpha) with a gene for antibiotic resistance-- the same gene that the Cas9-CRISPR complex will have been programmed to cut. When introduced, the OMVs will fuse with the target cell and the Cas9 will be introduced to the periplasm of the antibiotic resistant bacteria.

Delivery of gRNA to target cell

To introduce the gRNA to OMVs, the team looked at a few different methods: Electroporation, chemical induction, and cholesterol-binding. Electroporation has proven to be a viable method for introducing synthetic RNA into periplasm, but cell death is a possibility.⁽⁶⁾ However, due to very few protocols on chemically inducing gRNA uptake and introducing gRNA through cholesterol-binding, we chose electroporation as our method to get gRNA into the periplasm of the cell. We will be using the same hypervesicular strain of E. coli to maximize our chances of gRNA ending up in OMVs. ( NOT FINISHED, NEED TO READ UP ABOUT THIS ).

Fusion of OMVs to target cell

Our team wants to determine exactly how OMVs fuse to cells and what types of cells accept OMVs. To do this, we will put CFSE (a hydrophobic dye) inside the vesicles. With this dye and a microscope, we will be able to observe the OMV as it fuses to the cell and confirm that the contents of the OMV end up in the target cell's periplasm. Additionally, we can test the OMV's ability to fuse with different types of bacteria such as Gram-positive and Gram-negative. By observing how OMVs fuse and how they interact with different types of cells, we will gain a better understanding of the concepts behind our experiment.