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. ⁽²⁾ 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.

Our Project

This summer, our team will tackle three key features of this project: the incorporation of Cas9 into OMVs, the packaging 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

Our team will make use of an E.coli strain that is genetically engineered to hypervesiculate (JC8031). These bacteria will be transformed with a plasmid that allows the metabolic production of the Cas9 protein attached to amino acid sequences which have been found to be good candidates for periplasmic localization. To test for the presence of Cas9 in different cell compartments we will make use of fractionation - lysing only the outer membrane of the cell. Following the confirmation of Cas9 production, we will proceed by analyzing the contents of OMVs purified by the transformed strain.

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 in other contexts, 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 still chose electroporation as our method to get gRNA into the periplasm of the cell. Using the same hypervesicular strain of E. coli to maximize our chances of gRNA ending up in OMVs, we will electroporate the cells in a gRNA rich medium to introduce the gRNA into the periplasm of the cell. Then, when OMVs form, we expect to see gRNA in the vesicles as well (which can be tested by adding a fluorescent dye to the guide RNA). These OMVs will then fuse with the same target cell as the Cas9 OMVs.

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 interact with different types of cells, we will gain a better understanding of the concepts behind our experiment.

References

⁽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

⁽⁴⁾Li Ling, He Zhi-Yao, Wei Xia-Wei, Gao Guang-Ping, and Wei Yu-Quan. Human Gene Therapy. June 2015, 26(7): 452-462. https://doi.org/10.1089/hum.2015.069

⁽⁵⁾Roier, S., Zingl, F. G., Cakar, F., Durakovic, S., Kohl, P., Eichmann, T. O., . . . Schild, S. (2016). A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nature Communications, 7, 10515. doi:10.1038/ncomms10515

⁽⁶⁾Sjöström, A. E., Sandblad, L., Uhlin, B. E., & Wai, S. N. (2015). Membrane vesicle-mediated release of bacterial RNA. Scientific Reports, 5, 15329. doi:10.1038/srep15329