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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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Paperstrips
After the implementation of the working principle of the Cas13a to detect different targets in the plate reader, we decided to go further and do the same experiment by adding the samples to the glass fiber filter paper. We used overnight BSA treated glass fiber filter paper, which was then dried for 20-30 mins at 70C before pipetting the reaction mix. For this, we 3D printed a 96 well plate with 2 upper and lower parts which was suited for placing the paper in between the parts.
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Graph #1
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Graph #1
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The first figure shown above shows that even in the paper, we can observe the increasing amount of fluorescence with the increasing target concentration. The second linear graph which is time plot showing the amount of fluorescence released, also shows that we can the Cas13a system on the paper to detect the fluorescence activity. Both graphs above thus prove that the Cas13a can be used as an efficient system that can be integrated into the paper for the simple lab on chip detection of viral and bacterial infections.
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Hardware
We also designed a simple fluorescence reader with an exchangeable paper-based chip during our project, targeting the areas which do not access to advanced and space consuming machines. We basically tried it out with the Fluorescein in the beginning to see how far could we go with our detection limit. Then we worked on optimizing the device conditions and we could measure concentrations down to 100 nM. Finally, we tried our experimental setting of plate reader on to the fluorescence detector that we assembled along with the positive control containing RNase A and negative control containing RNase inhibitor only.
From the given graph, it is clearly visible that we can measure the fluorescence activity of the RNaseAlert substrate over time in the presence of Cas13a using our simple fluorescence detector with a paper based chip. This also shows that our CascAID system has can be developed further for a wide scale production.
Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The
positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are
connected with lines for the convenience of the eye. Error bars represent the measurement uncertainties of the detector.
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Variety targets
We tested different targets apart from E.coli 16s RNA. Our other targets were gram positive B. subtilis, Noro virus and the Hepatitis C virus.
E. coli
In case of E.coli , we took the 16s RNA as our target RNA. We first worked with a part of the 16s RNA sequence ( 161 bp) which was then in vitro transcribed and was used for our assays. Later, we also did the extraction of the RNA from the E.coli overnight culture and performed the same experiments. The results are visible under section in vivo and proof of principle.
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B. subtilis
For B. subtilis , we did not perform any in vitro transcription. We basically extracted the RNA from the B. subtilis culture and then did the RNaseAlert assay. We had a low-quality RNA after the extraction and the reason could be that since it is a gram-positive spore forming bacteria, the RNA extraction didn’t work so well. However, the results from the RNaseAlert assay are as convincing as in case of E. coli . As seen in the results on the right, although the quality of the extracted RNA is not so good, one can use the Cas13a to detect the RNA present in the extracted sample.
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Graph #1
Graph #1
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Graph #1
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Noro virus
For Noro virus, we took a part of the sequence from its 5’ UTR and designed the crRNA accordingly. The results from the RNaseAlert assay were very promising and showed that Cas13a can be effectively used to detect the viral RNA as well. Since Noro virus is the cause of a very common viral gastroenteritis, Cas13a could be a good option for the detection of this viral infection and this also opens the possibility of using Cas13a as the detection system for other viral infections. As shown in the graph on the left above, the fluorescence activity increases with the increase in the concentration of the viral target.
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HCV
For the HCV virus, we also took the part of the sequence from its 5’UTR and designed the crRNA accordingly. Also, the kinetics from the RNaseAlert assay using the HCV viral target as shown in the graph below showed the same effect as in case of the Noro virus assay. This again shows that we can detect even viruses like HCV with the help of this simple fluorescence assay.
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Comparison of the fluorescence kinetics of different targets
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The following graph shows a comparative plot with all the different targets we used apart from the E.coli and we can see how the kinetics of the fluorescence changes with the increase in the target concentration. This pattern of increase in the activity of Cas13a is similar in all kinds of targets being used in our experiments, hence promoting the fact that Cas13a is a universal system that can be basically used for any RNA targets.
Graph
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Variety proteins
We tested variety of Cas13a proteins originating from different species of Leptotrichia and tested their efficiency to detect target RNAs. Both the Cas13a Lbu and the Cas13a Lwa show that this system can be efficiently used in the detection of the variety of target RNAs. We could not properly purify the Cas13a Lsh, which is the reason why there is no activity of Cas13a in case of Lsh.
Lbu results
Cas13a Lbu results: All of the results presented above were done with the Cas13a Lbu. Since the purity of the Cas13a Lbu was the best in our case, we did most of our experiments with this protein.
Lwa results
The Lwa protein also showed the same potential results as in case of Lbu. We were able to purify it only in the later phase of our project, so we didn’t use it for a variety of targets. The following graphs show the activity of the Cas13a Lwa with 16s rRNA of E. coli . The graphs below give us similar results as compared to the Cas13a Lbu . In conclusion we can say that the Cas13a proteins from different Leprotrichia species have similar cleavage activity.
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Graph 1
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Graph 2
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Lsh results
As mentioned earlier we faced some problems during the purification of the Cas13a from the Lsh. However, we did the RNaseAlert assay with the Elution of the Cas13a Lsh protein we had but in the results we see no activity of the protein as shown in figure on the right.
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Graph 1
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Aptamer
For more validation of our assay, we also tried the spinach aptamer for our experiments in place of RNaseAlert . The spinach aptamer releases green fluorescence when bound with the DFHBI and when the Cas13a is activated, it cleaves the spinach and DFHBI and thus the fluorescence activity decreases with time.
Graph
As shown in the figure, we see that the fluorescence activity of the spinach aptamer is decreasing with the increasing concentration of the target RNA. With this result, we can assure that the Cas13a detection can also be integrated and used in other aptamer systems effectively.
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In vivo (heat lysis)
To test if we can run the cleavage assay on different bacterial targets with simple heat lysis only, we heated the E.coli culture to 95 C for 10 mins and used the subsequent lysis product for the RNaseAlert assay. We did see that the Cas13a can effectively detect the target RNA and activate itself to successively release the fluorescence. However, we observed very high activity of the Cas13a in these experiments, which can be due to the fact that all the cellular components are still present in the lysis product and there can be some additional Rnases present in the lysate give rise to the activity of the Cas13a.
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Graph
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Graph
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TDP
During the characterization of the TDPs, Team TU Delft found that whenever their Cas13a was dried in combination with a TDP of the type CAHS, the resuspended Cas13a would start cleaving RNA even in absence of the target RNA, hence losing its specificity. We therefore did the characterization of the TDPs they provided with our Cas13a (Lbu) and repeated the exact same drying experiment with the CAHS protein, in order to corroborate our results and to find the cause of such an unexpected result. As observed in figure 1, we also got the same unexpected results when Cas13a was tried with CAHS TDP.
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Lyophilized samples
We also tried lyophilizing our reaction mix on the glass fiber filter paper to see if we can make the reaction mix portable in a paperstrip. For this, we pipetted the reaction mix on the paper pieces, freezed them at -80 C and then vacuum dried the paper pieces for 2-3 hours. Then we added the targets to the lyophilized paper and used it to detect the fluorescence activity in the plate reader. As shown in the figure below, we see that the Cas13a reaction mix can be used as lyophilized paper strips, however we still need to optimize the system for effectiveness. We think that it’s the process of lyophilisation that needs to be improved to make the Cas13a stable and to make the enzymatic system long lasting on the paper strips.
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Graph
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Graph
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General control
For getting affirmation about the activation of the Cas13a only in the presence of CRISPR RNA and the target RNA, we did different control assays. The assays had one or the other part of the reaction mix missing. i.e. either no crRNA, no Cas13a, no target RNA and additionally also had positive, negative controls and an assay with all reaction mix components. Thus, this made it possible for us to compare and validate the activity of the Cas13a as shown in the figure below. |
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