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Results
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Bacterial targets used for the experiments
Escherichia coli
We took 16s rRNA of the E. coli as our target RNA. Since 16s rRNA is highly conserved in all bacterial species and can used as a well characterized site for our cleavage assays. It can also be easily extracted from bacterial cultures. For our experiments, we used only a part of the 16s rRNA since the whole 16s rRNA is too large to be transcribed (1500 bp). For this particular target RNA sequence we took, we designed the crRNA and in vitro transcribed the crRNA and the target RNA in our lab. We also performed RNA extraction using chemical lysis and heat lysis for the E. coli samples. Although the chemical lysis gave us good quality and detectable concentration of the RNA, the heat lysis didn’t work so well. There was always some cellular residues, RNases present in the sample due to which the fluorescence activity in the cleavage assay was way higher than the positive controls.
16s rRNA part used for the experiment
Figure 1: Gel picture showing the our 16s rRNA partial sequence used for our experiments
Figure 2: Urea gel picture of the different crRNAs
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Bacillus subtilis
We also focused on trying out our experiment with other target RNAs and for this we chose the gram positive Bacillus subtilis since it is widely used in microbiological research. Plus we wanted to see if one can detect the difference between the 16s rRNAs of B. subtilis and E. coli . For B. subtilis , we did not perform any in vitro transcription, rather we directly used the bacterial culture for the RNA extraction. However, we did encounter some problems due to the spore forming nature of the Bacillus subtilis . Also, the quality of the extracted RNA was not so good and there were some cellular residues apart from the RNA which caused some problems during the assay.
crRNA designed for the Bacillus subtilis 16s RNA
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Viral targets used for the experiments
Noro virus
Noro virus originally called Norwalk virus, of the family Caliciviridae, is one of the major cause of viral gastroenteritis in humans and it affects patients of all age groups. It is also the cause of high rate of deaths and is associated with hospital infections. For our experiments, we took the 5’ UTR of the Noro virus and also did in vitro transcription to get the target RNA and the crRNA. The 5’ UTR of the viruses are very specific to each individual virus so one can use this part to design the crRNA and detect different viral RNAs using the Cas13a system.
crRNA designed for the Noro virus
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Hepatitis C virus
HCV is a small single stranded RNA virus of family Flaviviridae which is the major cause of the Hepatitis C and liver cancer. Common setting for transmission of HCV is also intra-hospital (nosocomial) transmission, when practices of hygiene and sterilization are not correctly followed in the clinic. There are no vaccines for HCV virus. For our experiments, we took the 5’ UTR of the HCV virus and also did in vitro transcription to get the target RNA and the crRNA.
crRNA designed for the HCV virus
Gel picture
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Cas13a strains used for the experiments
The genus Leptotrichia was one of the first microorganisms to be drawn and described by the Antoni van Leeuwenhoek. The generic name was first used in 1879 for filamentous organisms found in the human mouth. We used the following strains of Cas13a for our experiments.
- Leptotrichia buccalis (referred as Lbu in our experiments)
- Leptotrichia wadei (referred as Lwa in our experiments)
- Leptotrichia shahii (referred as Lsh in our experiments)
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We expressed our His-tagged proteins in E. coli strains and purified them using a Äkta purification system or Ni-NTA agarose. To cleave off the His-SUMO or His-MBP tags from Cas13a proteins, we incubated them with the SUMO or TEV protease (BBa_K2323002) during dialysis overnight, respectively. In some cases, we reloaded the cleaved protein solution again on Ni-NTA agarose to get rid of the thereby binding His-tag. For higher purity, we loaded the proteins on a size exclusion column. Protein purity was always checked by SDS PAGE.
Both the Cas13a Lbu and Lwa are the central component of our diagnostic platform. The TEV Protease is part of our idea to the Intein-Extein readout, but apart from that, served as molecular tool for cleaving off the protein tags. So far, we managed to express and purify all three mentioned Cas13a proteins and the TEV protease as you can see in following chromatograms and SDS gels.
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Cas13a 3D structure
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Äkta purification
His purification Äkta graph Lbu plus gel
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His purification Äkta graph Lbu plus gel
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Nickel NTA purification of Lwa
HCV is a small single stranded RNA virus of family Flaviviridae which is the major cause of the Hepatitis C and liver cancer. Common setting for transmission of HCV is also intra-hospital (nosocomial) transmission, when practices of hygiene and sterilization are not correctly followed in the clinic. There are no vaccines for HCV virus. For our experiments, we took the 5’ UTR of the HCV virus and also did in vitro transcription to get the target RNA and the crRNA.
Lwa gel from ni nta
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Affinity purification and size exclusion purification of TEV protease
SEC purification Lbu plus gel
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SEC purification Lsh plus gel
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Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.
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We wanted to start our project by showing that Cas13a's collateral activity could be used to detect the presence of specific RNA. For this, we used the RNAse alert system, as done in a recent publication11, to detect RNA digestion. In this assay, the presence of RNAse-like activity is detected by an increase in green fluorescence. Our experiments yielded a convincing proof-of-principle which we went on to model to determine the theoretical detection limit of our system. Moreover, CascAID can be used to detect a wide spectrum of pathogens, as our experiments with gram-positive and viral targets suggested.
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Cas13a can be used to detect specific RNA sequences.
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Picture of the Thermocycler
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For RNA extraction from the samples we tested three methods: extraction with silica beads, extraction with silica membrane and heat lysis. We custom-built an affordable thermocycler for signal amplification by RT-PCR to improve the detection limit. We explored recombinase polymerase amplification (RPA), an isothermal amplification procedure, to use over more conventional PCR methods as its simplicity makes it the more attractive option.
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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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AeBlue: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
activity. After digestion, the interaction between the two strands is too weak to hold the dimer and it
decays. We can then use the DNA-strand as template to translate the chromoprotein aeBlue.
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Intein-Extein: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
to regulate the protease's diffusion and use it to cleave a TEV tag separating the intein regions of a
modified chromophore. After the first cleavage, the intein segment excises itself13, bringing together the
halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
read-out.
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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 state14.
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 linker- RNA which hybridizes with both thiolated DNA strands, form aggregates, changing the color from red to blue. The design of the linker-RNA 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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Software
To help facilitate the design of crRNA, the sequences that give CascAID its specificity, we developed a
software tool that checks crRNA for unwanted secondary structures. This gives valuable insight on
whether the sequence is suited to use with Cas13a or whether some modifications are needed.
Together with Team Delft's software tool which designs the corresponding crRNA based on the target,
we collaborated to develop a powerful tool that suggests crRNA sequences and checks their usability
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References
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- Link, Stephan, and Mostafa A. El-Sayed. "Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles." The Journal of Physical Chemistry B 103.21 (1999): 4212-4217.
- Zhao, W., Ali, M.M., Aguirre, S.D., Brook, M.A., and Li, Y. (2008). "Paper-based bioassays using gold nanoparticle colorimetric probes." Analytical Chemistry 80, 8431–8437.
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