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.⁽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?

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. OMVs have a wide variety of functions and, by pinching off from the cell, can move independently from the host and facilitate cell-cell communication in order to regulate bacterial colonies or remove toxic compounds.⁽³⁾

Our Project

To begin, 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 a FLAG tag, will signal 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. [Here we will somehow test the OMVs for the presence of Cas9 using a chemical or enzyme to test for activity, both when the OMVs are sealed, and when the OMVs are lysed. That way we know that the Cas9 is INSIDE the OMV, and not merely attached to the surface.] 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 protein will have been programed 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. [Here we are unsure whether or not we need to program the OMVs to target the new cells, or whether or not this will happen naturally. Also, once the Cas9 makes it to the periplasm of the new cell, we are not sure how this will cross the inner membrane into the cytoplasm.]

EDIT THIS SO IT SOUNDS LIKE WE KNOW WHAT WE'RE DOING MORE With regards to the gRNA of the Cas9 complex, we do not yet know where/if we are going to introduce it. The first option is the somehow create the gRNA and the Cas9 in the same cell and hope that it forms a complex before the Cas9 is secreted. However, this does not seem particularly efficient, and it will become difficult to test for this. The second option is to introduce the gRNA to the OMVs once the Cas9 is confirmed to be enclosed inside them. However, we do not know how to do this. Electroporation is not a viable option, because it will destroy most of the OMVs. The more likely candidate would be via chemical inoculation with CaCl2 or RbCl. The final option is to introduce it into the target cell separately from the Cas9. This could be done either by creating separate OMVs for the gRNA, or by transforming the target cells with the gRNA. (Electroporation? Chemical inoculation?).

One problem that must be overcome before this project is successful is the trouble of getting the gRNA across the inner membrane of the target bacteria. Even if we introduce the gRNA via OMVs, this will only put the RNA in the periplasm, with no method or reason for it to travel into the cytoplasm, and thus target the gene for antibiotic resistance.