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We started our iGEM journey with brainstorming sessions resulting in many great ideas. The best of these ideas were framed into written project proposals by teams of two to five students and, after a constructive peer-review process, presented to the whole team. The final two ideas were subjected to SWAT-analysis, after which we settled on CascAID, our CRISPR-based diagnostic device for point-of-care testing.
Our project has both an experimental and an applied side and we quickly realized that our construct was quite complex, consisting of many pieces that had to work together. We therefore split our project into parts that could be investigated seperately. First, the infectious pathogens had to be lysed and their target sequences amplified to yield an amount of target RNA that is detectable by Cas13a. Then, Cas13a had to be purified and characterized in the lab. Next, we had to figure out how to make the signal visible to the user. Finally, everything had to be framed into a portable and user-friendly format, by designing appropriate hardware.
We therefore formed sub-teams who focused their work on these modules. A high degree of modularization requires a high level of cooperativity between the sub-teams, but also provides an increased flexibility in design and better possibilities for customization. Another advantage is that many ideas can be developed and tested in parallel, which speeds up the develpment process.
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RNA Extraction and Amplification
The goal of this module is to obtain a detectable amount of target RNA from a sample of E. coli cells, which we used as a dummy target. The extraction method should be distributable, efficient and not require the use of hazardous chemicals. Therefore, the first step was to test common lysis techniques differing in the required equipment and use of chemicals. We compared their efficiencies by agarose gel electrophoresis.
The standard approach for RNA extraction, Guanidinium-Phenol-Chloroform extraction, using chaotropic salts as a lytic agent, requires purification of the lysis product and involves use of toxic chemicals. Another commonly used lysis process: incubation at high temperature with detergents such as SDS, gave good results, but again, requires separation of SDS from the RNA afterwards. For RNA purification purposes we investigated silica based procedures, as a less harmful solution. However, a point-of-care device has to be robust and reliable. In this regard, a simple design is considered superior to complex ones. We therefore abandoned chemical lysis methods that require purification and chose a combination of heat lysis followed by isothermal PCR.
Recombinase Polymerase Amplification (RPA), as an isothermal alternative to PCR, is conducted at 37°C without the need for thermocycling and therefore reduces the requirements and costs of the accompanying hardware significantly. Since Cas13a targets RNA rather than DNA, we coupled RPA to in-vitro transcription (TX). Since both reactions take place at same temperature of 37 °C, this can be done in a one-pot reaction. To render the RPA-TX distributable, we lyophilized the enzymes on filter paper, which, when sealed in a tight container, stabilizes the assay for storage.
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