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Project Design
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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 distributive, 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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Cas13a, Purification and Characterization
We chose Cas13a as it, as RNA-guided RNAse, could practically be optimised to detect any sequence of interest, this way allowing customization of device for any pathogen of interest. Also it is highly specific and sensitive, which enables detection even when sequence of interest is only present in traces in sample.
Our plan was to investigate proteins of the Cas13a family from different organisms - Leptotrichia buccalis (Lbu), Leptotrichia shahii (Lsh) and Leptotrichia wadei (Lwa) - and choose the one that performs best. Lbu and Lsh were available at AddGene, while we cloned and codon optimised Lwa ourselves. We therefore used two different E. coli strains for protein expression. We used BL21 Star for optimised plasmid and for not optimised ones, we chose Rosetta strain which is a BL21 derivate that provides tRNAs for codons that are usually not present in E. coli. In order to prevent the large Cas-protein to form inclusion bodies we expressed it together with SUMO (Small Ubiquitin-like Modifier) or MBA (Multiple Banded Antigen) proteins, which force Cas13a to stay in solution. We opted for Ni-NTA purification followed by size exclusion, with the His-tag attached to SUMO/MBA. To determine the purity of protein and see if there are differences between investigated proteins we used SDS-PAGE.
For characterisation of Cas13a-Proteins we used the RNaseAlert assay, a cleavage assay in which Cas13a is activated through the presence of appropriate crRNA and target RNA. We transcribed target RNA in vitro to accelerate the process and then screened for optimum conditions. Using this assay for different target sequences from Noro Virus, Hepatitis C Virus and Bacillus subtilis we investigated the specificity of the Cas13a activity.
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Readout
CascAID utilizes the collateral RNase activity of Cas13a to produce a highly amplified signal. The purpose of this module was to translate the cleavage of RNA into a readout that is easy to measure and interpret by the user. We identified this part as a key challenge of the project, so we opted for a redundant project design and tried different approaches.
For our first prototype and the characterisation of Cas13a we used RNase Alert, which is a commercially available fluorescence beacon for monitoring RNase activity. Given the appropriate equipment, fluorescence can be measured down to low concentrations, which is very practical for laboratory work. Due to the lack of a portable, affordable and sufficiently sensitive fluorescent detector, our hardware team built our own device, as described in the following section.
However, the most practical readout would be a colorimetric assay, which produces a test result readable by the human eye. We tested three alternative methods for this purpose.
Our first approach exploits the optical properties of gold nanoparticles (AuNPs), as it is already successfully used in other diagnostic tests. Due to the phenomenon of Localized Surface Plasmon Resonance, AuNPs appear in a different color when they are aggregated, rather than in free solution. We planned to connect this effect to the cleavage of RNA by Cas13a by aggregating AuNPs with ‘linker RNA’. More precisely, we labelled AuNPs with thiolated DNA and used complementary linker RNA to cause their aggregation. Once the RNAse domain of Cas13a becomes active, the linker RNA will be cleaved, causing disintegration of the AuNP-aggregates, which will result in a visible color change. To make linker RNA prone to Cas13-mediated cleavage we inserted an U-rich segment, which we varied in length as the plasmonic effect is strongly distance dependent.
Our second option was creating an intein-extein construct, where separated intein regions are coding chromophore and extein cleavage is regulated through TEV cleaving site. We bound TEV protease to a RNA linker to make its diffusion dependant on Cas13a RNAse activity. Upon its release extein will be cleaved and inteins brought together. Only when inteins together the chromophore can be produced for a colourful positive read out.
For the third possibility, we investigated the aeBlue protein. The design involved creating a RNA/DNA dimer, which melts to liberate the single stranded DNA as a primer, once RNA is digested by Cas13a's collateral activity. After decay of the RNA, the DNA oligo would activate expression of the aeBlue chromophore, by completing the T7 promoter. Design of this complex required detailed tuning of free energies and melting temperatures, which we performed in silico using NUPACK. The design was confirmed and optimized by native PAGE using a labelled version of the DNA oligo.
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Hardware and Paper Fluidics
We developed an open source fluorescence detector as a portable solution for fluorescence based readout. With the rise of 3D printing and microcontroller-based DIY electronics, the open source philosophy has spread from software engineering into the world of hardware. By providing detailed documentations and CAD-drawings of our hardware, we want to enable other engineers and hobbyists to improve our designs, democratize access to the system and empower the user to self-educate about the working principle of our hardware.
We chose to perform our detection reaction on filter paper, which enables us to freeze-dry the mixture for long-time stability, and use very small reagent volumes per test. To enable transport of the sample-containing fluid to the areas containing the detection mixture, we used a technique called paper-fluidics. Thereto, we hacked our regular office printer, to pattern the paper with hydrophobic wax. Defined areas were left uncoated in order to form hydrophilic channels that promote fluid transport by capillary forces. This technique enabled us to rapidly prototype and test different channel geometries for optimal flow, with very short iteration cycles of about one hour.
After first tests, we recognized that bleached cellulose paper limits the resolution of fluorescence readout due to its auto-fluorescence. This led us to integrate glass microfiber filter paper into the paper strip in the respective detection areas, which shows little to no auto-fluorescence. Further, we used adhesive tape to seal the paper strip to avoid RNase contamination and keep the glass microfiber filter paper in place.
In a similar way, we prototyped our sample preparation module, consisting of a fluid control unit, a heating system and a fluidic chip. For the fluidic chip we used 3D-printed masters, rather than photo-lithography, to achieve faster prototyping cycles to optimise our design, while being open to
later miniaturization. The heating system is controlled by a microcontroller, and for the fluid control unit we chose to build valves driven with the pressure from a bike tire to direct flow without the need of carrying pressurized air. The design of our hardware components allows their integration in a single portable device.
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