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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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Size exclusion purification
SEC purification Lbu plus gel
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SEC purification Lsh plus gel
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Affinity purification and Size exclusion purification of TEV protease
His purification TEV
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Gel #1
Gel #2
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Assays used for the experiments
For our experimental design, we used different fluorescence assays as stated below:
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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. 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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RNAase alert
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Lightbringer
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Clariostar
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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.
Figure (a) Structure of the Spinach aptamer in absence (yellow) and in presence (green) of DFHBI. (b) G-quadruplex motif of the Spinach aptamer in absence (yellow) and in presence (green) of DFHBI.
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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Proof of principle
To characterize key protein of our diagnostic device we conducted several experiments.
Firstly, we confirmed that Cas13a activity is target dependent. Despite the fact that Cas13a exhibits RNase activity in absence of target RNA, its activity in presence of target RNA is up to 8 times higher. However, this is true at low protein concentrations. At high concentrations of Cas13a presence of target RNA does not have significant effect on enzyme activity as depicted in the Figure 3. Secondly, we verified that enzyme is activated by crRNA. As Figure 4 (this is the only figure with old enzyme, so concentrations are completely off the values of enzyme purified and used later on) shows, enzyme is active only in the presence of crRNA. It can be seen the higher is the concentration of crRNA, the more of enzyme gets activated, which is in accordance with the first step of reaction --link to overall reaction equation--. Besides that, crRNA when forming a complex with Cas13a defines specificity of ribonuclease. This was confirmed by cross-reactivity experiment.
please place results of cross-reactivity experiment here
And most importantly we determined detection limit of Cas13a-crRNA complex by varying target RNA concentration. Figure 2 shows that target concentrations above two-digits in nanomolar range can be detected.
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Crosstalk experiments
To show that the Cas13a is highly specific for a particular target, we tested the CRISPR RNA designed for Noro virus with different targets, namely E. coli , HCV and Noro virus. As observed in the bar graph on the right, the Cas13a activity is visible only there is the presence of the target as Noro virus itself. Whereas in presence of other targets there is very low background fluorescence visible, which is also measurable when no target is present. The results observed showed that there is no crosstalk between the targets and that a particular crRNA is specific for one type of target RNA only. With this we can confirm that our system CascAID can be efficiently used to differentiate different viral and bacterial target RNAs.
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Bar graph
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In vivo (chemical lysis)
After the successful experiments with the in vitro transcribed target RNA from E.coli, we decided to extract the 16s RNA from the E.coli culture in the lab and to perform the same RNaseAlert assays with the extracted RNA. At first, we used the chemical lysis using SDS plus heat followed by phenol chloroform purification. However, there was presence of some RNases in the extracted RNA which led to higher fluorescence activity of the RNaseAlert in presence of Cas13a. Therefore, we took the extracted RNA and then conducted a serial dilution to use them for our experimental setup. The extracted target RNA was 40 x concentrated.
Graph
As shown in the graph above, the level of fluorescence activity increases with the increasing target concentration which again verifies the activity and the sensitivity of the Cas13a. Since the RNA was extracted directly from the E. coli culture, we can say that the Cas13a can be easily manipulated to be used not only for in vitro samples but also for in vivo samples, which makes it even more suitable for practical uses.
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