Originally, our team knew that we wanted to use Cas13a to target and cleave HIV RNA. However, as a therapeutic, we had to consider how Cas13a would be delivered into human body cells. To account for this, we first proposed a three-fold fusion protein design with the DNA that encodes for each protein formed into a plasmid. This original design, named Unit 0, intended for Cas13a to serve as the cargo being transported, Diphtheria Toxin (DTA) to serve as the facilitator for endocytosis, and CD4 monoclonal antibody (OKT4) to serve as our ligand that binds to the CD4 receptors on helper T cells. The OKT4 and DTA were on the C-terminus of the Cas13a. According to our understanding, the receptor-ligand binding of OKT4 and the CD4 receptor of the Helper T cell would trigger the endocytosis mechanism of the DTA, successfully transporting the Cas13a protein into the HIV-infected Helper T cell.
In experiments, we HiFi assembled the different parts of the fusion protein plasmid, using a plasmid backbone with Cas13a. This fully assembled plasmid with Cas13a, DTA, and OKT4 was transformed into Rosetta, BL21, and W3110 cells, and grown in triplicates to determine which cell variety was most efficient in growing the protein. Rosetta was determined to be the most efficient, but with one problem: the OKT4 was getting deleted from the transcription process, following assembly of our DTA-OKT4 insert into our Cas plasmid. Our theory as to why the OKT4 was not present when the uptaken plasmid was sequenced is due to the linkers in for the insert. The linkers were repeats of a nucleotide sequence, so the Rosetta cells could have possibly counted that as unnecessary scaffolding and cut out the OKT4 insert entirely.
Unfortunately, the Unit 0 design itself was also vastly flawed. It is more optimal for the Cas13a to be on the N terminus and the OKT4 on the C terminus, but due to the misaligned ORFs at the C terminus of the Cas13a as well as the issue of multiple STOP codons that interfered with assembly, we designed an insert to put OKT4 on the N terminus. This underlined the novelty of our work as a fusion protein had never been constructed with the targeting domain on the N terminus.
However, two issues arised. First, without binding, the OKT4 cannot work with the DTA to facilitate endocytosis, and the Cas13a protein will stay outside of the cell. Secondly, the OKT4 was not a ligand. It is a receptor, so it was not capable of serving as a ligand for CD4. The entire design would, effectively, not work, even if it was able to be fully expressed, which it was not. Only the Cas13a and DTA were translated into protein and frozen at -80C until used for in-vitro protein purification and testing purposes, since there was no mechanism to get it into a cell.
In an attempt to ameliorate the lack of a ligand in plasmid design, we looked at the mechanism of action of HIV, which shows that HIV itself actually uses a ligand to bind to CD4 receptors on Helper T cells, called gp120, a glycoprotein. We recreated our plasmid design to now include Cas13a, DTA, and gp120. Multiple attempts to ligate the gp120 dna sequence to the Cas13a C terminus failed, so we instead used the N terminus, which worked. We transformed the now formed Cas13a + DTA + gp120 plasmid into competent Rosetta cells, and incubated them overnight at 18 degrees Celsius with 0.1 mM IPTG, 100 µg/mL Ampicilin, and 20% glucose diluted to 0.2%, all in LB Broth at 250 RPM. Due to the nature of bacteria, they are unable to synthesize glycoproteins, so the resulting protein was Cas13a linked to DTA, meaning that another attempt to synthesize the full protein complex for in vivo testing was unsuccessful. We utilized the resulting Cas13a+DTA protein for in vitro testing to evaluate if our novel fusion protein can successfully cleave HIV ssRNA, and aimed to use a cell-free system to fully create our Cas13a+DTA+gp120 fusion protein without the restrictions of prokaryotic cells or maintenance of mammalian cell synthesis.
Our main takeaways for future teams to consider are that:
In the works… stay tuned for Paris!
Our goal was to generate a stable HEK293T (Human Embryonic Kidney Cell) landing pad cell line expressing both the CD4 receptor with gRNA attachment cassette and HIV-mimic cassette using FLP recombinase integration. Despite multiple transfection attempts under varying conditions, stable integration was not achieved.
We initially tested a range of FLP:CD4 plasmid ratios (5:2, 2:5, 1:6, and 1:1) to determine optimal efficiency and whether CD4 will be transfected and integrated into the landing pad by flip recombinase. However, no EGFP fluorescence was observed in the positive control wells, suggesting transfection failure. Even after optimizing Lipofectamine conditions, utilizing a lower concentration of antibiotic selection pressure, transferring the cells to a bigger surface area, and adjusting cell transfection confluency; all cells failed to survive hygromycin selection.
To assess CD4 expression independent of FLP recombination, we performed a transient transfection, utilizing both Lipofectamine and CaCl₂ transfection, of the CD4 cassette without FLP recombinase. Cells were cultured for 48 hours post-transfection, lysed, and analyzed via Western blot to verify CD4 protein expression.
Our first Western blot did not show detectable CD4 bands, likely due to low transfection efficiency (the fluorescence in the positive controls were very minimal) and/or a low amount of protein being loaded into the SDS-Page gel. After troubleshooting with optimizing our lysis buffer and re-running transfection, we repeated the experiment.
The second Western blot successfully showed bands, however location of bands were significantly higher than the expected ~55 kDa, exact band size can’t be determined. Possible explanations for this can be non-specific antibody binding and/or incomplete denaturation. Further testing, such as using a denaturing control or performing an antibody validation experiment, will be necessary to confirm whether the bands correspond to CD4 or another product.
Figure 1a: Rapid scan 1.
Figure 1b: Optimal results.
Figure 2: Rapid scan 2.