We spoke with Prof. Dr. Lucas Pelkmans, the Principle Investigator of the Pelkmans Group at UZH. The goal in talking to him was to gather feedback on our approach back in the beginning of May and discuss a solution for visualization of the detection results. We expected that his insights would refine our project approach and experimental designs.
We compared output systems from CRISPR diagnostics, which typically rely on quencher-fluorophore probes. On one hand this fluorescence is practical for proof-of-concept, by measuring with plate readers or cheap UV lights. On the other hand colorimetric outputs, like those in pregnancy or COVID-19 lateral flow tests, would be more user-friendly. Enzyme-based approaches (e.g. LacZ) are possible but technically complex. A promising alternative is to design an unquenching system that triggers a secondary reaction, producing a visible color change and combining the sensitivity of CRISPR cleavage with an accessible readout.
Dr. Pelkmans suggested Rolling Circle Amplification as an alternative to CRISPR/Cas detection. RCA works by aligning a target sequence to immobilized oligonucleotides, ligase then closes gaps to form a circular DNA template. The circular sequence undergoes massive amplification (~1000x), creating strong signal output. A downstream color-based detection system could bind either directly to the amplified sequence or to a designed secondary reporter. RCA allows for multiplexing: multiple pathogen-specific probes can be immobilized in stripes, enabling simultaneous detection of different STIs in a single assay.
RCA as a possible alternative approach to detection would allow easier multiplexing and amplified visual output.
We explored the possibility of replacing CRISPR-Cas with RCA, by researching its stability, primer design requirements, optimal incubation temperature and detection output. Additionally we discussed it with PhD student Cheng-Han (find his inputs in Page x). Although this approach would have been very interesting, as RCA is not commonly used in diagnostics, we ultimately decided not to pursue it. The limited literature and the additional time needed to optimize buffers made it too risky within our project scope. Instead, we found DNA hybridization to be more closely aligned with our goals. In parallel, we further investigated colorimetric read-outs for CRISPR-Cas and selected this method.