On this page you can find the reasoning and structure of our experimental workflow. You can also find all protocols we worked with attached under each section. Have fun :)
To be able to detect extremely low amounts of DNA in a sample, the diagnostic tool would need to contain a DNA amplification step in addition to the detection mechanism. Our research showed that Recombinase Polymerase Amplification (RPA) would work best for our purpose. RPA is fast, works at a constant low temperature, and is therefore very well suited for a test that relies on as few resources as possible. [1]
We first confirmed that the symmetrical RPA works for our target sequence, HPV16. Following this success, we proceeded to test it with asymmetrical RPA. In contrast to symmetrical RPA which produces double-stranded DNA (dsDNA), the asymmetrical RPA results in single-stranded DNA (ssDNA). ssDNA would later be required for our final diagnostic test, if we were to use DNA hybridization as the detection method. In contrast, symmetrical RPA is used in combination with CRISPR-Cas12a as a detection method, because the CRISPR-Cas12a enzyme binds to dsDNA. The experiments with asymmetrical RPA yielded excellent results as well.
We then demonstrated that RPA also works at room temperature. This is an important feature for future at-home testing, since users cannot easily perform incubations at higher temperatures.
Next, we investigated whether RPA can be performed directly on a paper strip. The symmetrical RPA on the strip experiment worked on the first attempt. Unfortunately, asymmetrical RPA on a strip did not work on our first try and still needs optimization. We now aim to optimize the conditions for asymmetrical RPA on paper.
To assure specificity and a clear test result, we explored the use of Cas12a as a detection mechanism for DNA target sequences.
To do so, we expressed and purified the LbCas12a protein ourselves with the assistance of Christelle Chanez from the Jinek Laboratory at the University of Zurich. The expression vectors were provided by the Jinek Laboratory.
A test expression was conducted to determine the best suitable E. Coli strain for our vector (pDS113). Then a large-scale expression was conducted in Rosetta2 cells and the protein was purified. In parallel an alternative vector (pDS115) was expressed in Rosetta2 cells and then later purified. See Engineering Cycles for more information on this decision.
CRISPR-Cas12a is among the most widely used and established detection approaches for STD diagnostics [2] . We found many publications describing its use, which is why we initially planned to rely on CRISPR-Cas for our final test. However, as our project progressed, we developed the idea of DNA hybridization, which turned out to be better suited for our circumstances (see below). Still, to be able to compare the novel method to the conventional approach, we decided to test this method as well. We began with a standard Cas12a assay in a reaction tube, where we aimed to detect a clear color change by eye. For this we used the protein derived from the pDS113 vector. After confirming that this setup worked successfully, we also performed additional Cas experiments to provide quantitative measurements for the Dry Lab, helping them verify their models. Finally, we moved on to test CRISPR-Cas on a paper strip using the Milenia Biotec HybriDetect kit. This last Cas experiment also worked and gave us clear results, rounding off our Cas12a tests.
As an alternative detection method for our project we came up with the novel idea of using DNA hybridization. While CRISPR-Cas is very specific, it requires proteins, guide RNAs, and complex reaction conditions. This makes it more expensive and sensitive to temperature and storage changes [3] . In contrast, DNA hybridization only needs a DNA probe, which is very stable and cheap to synthesize [4]. For these reasons we concluded that DNA hybridization would be the preferred detection method for a paper-based home test, given it is comparably sensitive and specific. To be able to be conclusive on this hypothesis, we proceeded to experiment with both methods. We have shown that single DNA hybridization (DNA hybridization with one binding) works for products of symmetrical, asymmetrical and even isothermal RPA products from the pathogen sequence of HPV16. We also designed a second probe with a different fluorophore to test double hybridization, meaning that two probes bind at the same time, while each of them can be detected at different wavelengths. Whereas the first double DNA hybridization attempt did not work, we successfully confirmed that the new probe is specific. After several more trials, we finally achieved double DNA hybridization using two different oligos, each labeled with a distinct fluorophore, on both symmetrical and asymmetrical RPA products of the HPV16 L1 gene. The detection of both signals marked an important milestone in our project. As a next step, we aimed to transfer the entire setup onto a lateral flow strip, which worked on the first attempt. This brings us one step closer to developing a paper-based home test.
To ensure compatibility we conducted experiments on the detection of the probes with the product of the RPA experiments as a substrate. We did not have the resources to test the direct interaction of the methods. However, the literature indicates that RPA and CRISPR-Cas proteins do not inhibit each other [5] . There has been no research conducted on the combination of RPA and DNA hybridization, but there is no reason to believe that the presence of the additional protein might interfere with the hybridization. Our idea of the test design separates the amplification and the hybridization spatially, connecting them by microfluidic pathways. This should avoid premature binding of the scarce DNA probe before sufficient amplification ensures a visible band.
The experiments described were all conducted with the L1 sequence of HPV16. This proof of principle would need to be extended by repeating the experiments with the additional sequences. However, the different sequences should not influence the outcome of the experiments, as long as the gRNA and hybridization elements are designed accordingly (this includes verifying specificity and secondary structures).