This summer, our team tackled three key features of this project: Cas9 expression, its packaging into OMVs and finally the fusion of these vesicles with target cells. To study these three areas we made use of an E.coli strain that is genetically engineered to hypervesiculate (JC8031). These bacteria were transformed with a plasmid that allows the metabolic production of the Cas9 protein attached to amino acid sequences which were identified as good candidates for periplasmic localization. To test for the presence of Cas9 in different cell compartments we made use of fractionation - lysing only the outer membrane of the cells.
Fusion proteins were employed for Cas9 export to to the periplasm of E.coli cells. We aimed to examine the possibility of exporting a large protein, saCas9 (~130kDa), through the Twin arginine translocation pathway (Tat), general secretory pathway (Sec) or both. Proteins that are able to fold in their 3D conformation in the cell cytoplasm are good candidates for Tat transport. The interaction between the signal peptide, which contains the Tat consensus motif with a characteristic twin arginine dipeptide, and Tat machinery makes protein export possible. Due to the size of the chosen protein, we also made use of signal peptides for Sec export. Proteins exported through Sec do not fold in the cytoplasm, where they are bound by SecB or other cytoplasmic chaperones until they move through the SecYEG cytoplasmic membrane translocon to the periplasmic space.
Cas9 selection and size considerations
For our project, we decided to use the CRISPR-Cas9 protein from the bacterium Staphylococcus aureus because of its smaller size relative to other Cas9 proteins. To compare the functionality and sizes of different CRISPR proteins, we designed experiments for constructs to test with cjCas9, from the bacterium Campylobacter jejuni. The size of cjCas9 is around ~130kDa. Due to time constraints, the experiments for the cjCas9 constructs were not completed. The PAM sequences for the two proteins are different, so in order to test the functionality of the proteins, the reporter gene functionality assay constructs had to be different.
(1). Isaac B Hilton, Anthony M D'Ippolito, Christopher M Vockley, Pratiksha I Thakore, Gregory E Crawford, Timothy E Redd (2015). Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33, 510–517 (2015) doi:10.1038/nbt.3199
(3) Paolo Natale, Thomas Brüserb, Arnold J.M.Driessena (2007). Review Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—Distinct translocases and mechanisms. Science Direct, 15(2), 347-354. doi:10.1111/1462-2920.1204