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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 SWOT-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 separately. First, Cas13a had to be purified and characterized in the lab. Then, the chosen pathogens had to be lysed and their target sequences amplified to yield an amount of target RNA that could be detectable by Cas13a. 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 development 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 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 the use of toxic chemicals. Another commonly used lysis process: incubation at high temperature with detergents such as SDS, gave good results, but again, required 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 the 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 an RNA-guided RNAse, could practically be optimized 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 optimized plasmid and for not optimized ones we chose the 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 chromatography, 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 characterization 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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Fluorescent read-outs
We used two well-established fluorescence assays to quantify Cas13a's activity:
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RNaseAlert Assay
This is a commercial kit readily available in the markets, which can be used for the detection of the RNase activity and sensitivity in real time. The RNaseAlert® QC System uses a novel RNA substrate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. In the absence of RNases, the physical proximity of the quencher dampens fluorescence from the Fluor to extremely low levels (RNaseAlert™ Lab Test Kit Instruction Manual). When RNases are present, however, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution. This causes the Fluor to emit a bright green signal when excited by light of the appropriate wavelength. Since the fluorescence of the RNaseAlert substrate increases over time when RNase activity is present, results can be easily monitored. For the detection and monitoring of the kinetics of the fluorescence, we used the plate readers in lab and our self-made fluorescence detector.
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Working principle of RNaseAlert Assay
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Structure of the spinach aptamer in absence(yellow) and presence(green) of DFHBI, b: G- quadruplex motif of the Spinach aptamer in absence(yellow) and in presence(green) of DFHBI
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Spinach Aptamer Assay
The spinach aptamer assay is based on a fluorophore DMHBI which was the first molecule against which a SELEX experiment was run. However, DFHBI was extracted from eGFP and it exhibits a higher extinction coefficient and lead to a brightness increase of eGFP. In 2012, Paige et al. developed the 24-2 aptamer, mostly known as Spinach due its green fluorescence when bound to DFHBI. The Spinach aptamer exclusively binds the deprotonated variant of eGFP (DFHBI) with a dissociation constant of Kd = 390nM. It increases the quantum yield of DFHBI from 0.0007 when free to 0.72 when bound to the aptamer.
The aptamer structure is elongated and it folds with two helical stems adjacent to the binding region, which exhibits a G-quadruplex pattern. The Spinach aptamer binds the DFHBI in a planar conformation. Hydrogen bonds are formed between the G-nucleotides and the fluorophore, and the aptamer changes its 3d-structure when bound to the DFHBI. In the absence of the fluorophore, the base triplet formed by the nucleotides A53-U29-A58 collapses on the G-quadruplex site. Spinach shifts the absorbance maximum of the DFHBI by approximately 60 nm comparing with the unbound form, from 405 nm to 469 nm. Spinach has been used for imaging protein and gene expression, and it has been also modified in order to be used as a sensor of biological reactions.
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Colorimetric read-outs
To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
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ssDNA: The scheme shows the basic idea of the ssDNA oligo based readout. Three different routes for a final signal readout have been thought of. All three readouts are based around the formation of a RNA/DNA dimer and the freeing of the DNA oligo by digestion of the RNA part. The RNA oligo has 2 poly-U loops, while bearing the complementary sequence for the small DNA oligo in between these poly-U loops.
The Cas13a should preferably digest the poly-U loops of the annealed RNA, which results in only three 6 base pair interactions being left between the RNA and the DNA oligo. This 6 bp connections between the RNA/DNA dimer should not be stable at room temperature and dissociate in a short time frame. This enables the release of the DNA oligo and therefore the interaction with another DNA template to generate one of the three thought of amplification methods. For this readout the RNA serves as an inhibitor strand, while the DNA oligo serves as the activator strand for another DNA template.
aeBlue.
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Working principle of AeBlue
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Working principle of Intein Extein
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Intein-Extein: The readout depends on an immobilized TEV protease, which gets released after aptamer cleavage by Cas13a. Once free, the TEV cleaves a linker within the 2 intein-extein-devices, that then form a fully functional beta-galactosidase. The active enzyme is then able to cleave 5-Brom-4-chlor-3-indoxyl-β-D-galactopyranosid, an artificial substrate which when cut forms a deep blue chromophore. This would allow for a visual readout, without the need of a detecting device.
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Gold nanoparticles: Other than in the other two colorimetric readouts, aeBlue and Intein-Extein, the only protein involved in the gold nanoparticle (AuNP)-readout is Cas13a, like in our RNase Alert readout. This reduces the necessary fine tuning of the biochemical circuit to a minimum, favoring high robustness of the readout. Due to the phenomenon of Localized Surface Plasmon Resonance, AuNPs appear in a distinct color, ranging from intense red to blue, black and colorless. This property depends on particle size, shape, the immediate environment, and -most critical for our purpose- aggregation state.
In our project we use AuNPs with a diameter of roughly 10 nm, giving them a bright red color in solution. Their small size and therefore high surface-to-volume ratio makes them ideal for functionalization with thiolated compounds, forming covalent Au-S bonds. The first step of our concept is to use these properties to functionalize AuNPs with either 5’- or 3’- thiolated DNA and, through addition of a RNA linker which hybridizes with both thiolated DNA strands, form aggregates, changing the color from red to blue. The design of the RNA linker includes an uracil-rich, single-stranded segment between the DNA-complementary termini, making it prone to Cas13a-mediated promiscuous cleavage.
It has been shown that, for purely DNA-based hybridization, AuNP aggregates can be spotted on filter paper, dried and severed by addition of a nuclease-containing solution, visible through diffusion of red AuNPs on the paper. Thus, the second part of our concept is to spot RNA-linked AuNPs on paper, dry them alongside the Cas13a mixture and detect specific target RNAs and resulting Cas13a activity with a simple change from blue to red.
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Working principle of Gold nanoparticles
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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 the 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 optimize 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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