Team:SDU-Denmark/Results
Results
Results
Assembly of Cas plasmids
Genes for the Cas12a and Cas13a proteins were assembled through PCR and ligated into a backbone. Cas13a and Cas12a are proteins that require sgRNAs to enable their functions as RNase and DNase, respectively. Successful insertion of cas13a and cas12a into biobrick BBa_B1006 (Backbone) was observed on a 1% agarose gel (Figure 1). Backbone was shown, both undigested and digested, using two of the restriction enzymes used in the ligation process. The plasmid digested only with EcoRI was used to show the size of the linear plasmid, while digestion on both EcoRI+SpeI was used together to show the expected length of the plasmid backbone when cas13a or cas12a is cut out. As seen from the restriction of the BBa_B1006 backbone with EcoRI and SpeI no part was cleaved out as expected.
Furthermore, cas13a or cas12a inserted into the backbone is also seen in Figure 1. It can be observed that the band for undigested plasmid is noticeably longer in size than the backbone without an inserted gene. Figure 1 lanes 6 and 9 show the cas-plasmids digested with EcoRI. This shows a single band at 6000 bp, indicating cas13a or cas12a was inserted into the plasmid. For both cas13a and cas12a, there are two bands each at 2000 bp and 4000 bp for the plasmids digested with both EcoRI+SpeI. The band at ~2000 bp corresponds to the band of the backbone that was digested with EcoRI+SpeI, showing cas13a or cas12a being cut out. Likewise, the band at 4000 bp shows the expected length of the gene being approx. 4000 bp.
In conclusion, the cas13a and cas12a genes have both been inserted into their respective backbones, part BBa_B1006, as seen in Figure 1.
Cas protein purification and tag removal
After successful transformations of both Cas12a and Cas13a into E. coli ER2566. The SOP for protein purification with His-tag was followed.
Both transformants were grown to OD600≈0.7 in LB-media with chloramphenicol (50 µg/mL). At OD600≈0.7 both cultures were induced with IPTG 1mM for ~3.5 hours, to then reach anOD600≈1.4. Both cultures were harvested, sonicated, and resuspended in TMN and anion-buffer corresponding to the concentration of ions in wash-buffer.
The following applies both for Cas12a and Cas13a illustrated in Figures 2 and 3 respectively. 1 mL Ni-NTA Agarose (Qiagen) per culture was prepared. Ni-NTA Agarose was incubated with the lysed cells over-night (O/N, approx. 10 hrs). After the incubation, the mixtures were washed (lane 4), pre-pre eluted (lane 5), pre-eluted (lane 6), and eluted (lane 7-12) from the chromatography column.
The elution was made in fractions of ≈ 1 mL. The protein concentration was estimated with Bradford assay . Concentrations for Cas12a (Figure 2) at a range 55-90 ng/mL for fraction 8-11 and Cas13a (Figure 3) at a range 50-80 ng/mL for fraction 8-11 were achieved. Lane 2 is lysate before IPTG-induction and lane 3 is lysate after IPTG-induction.
The first lane displays Cas13a above 140 kDa as expected. The second lane displays cas13a after incubation with SUMO protease following the SUMO-protease assay from Invitrogen. The SUMO-6Xhis-tag hasa molecular weight of 5 kDa. In the second lane, a band is seen at around 140 kDa, fitting relatively well with the cleaving of SUMO-6Xhis-tag, isolation a Cas13a with the molecular weight of 148,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”Gel: Bis-Tris 4-12% Running buffer:MOPS.
The 6xHis-SUMO-tag was cleaved off using the SUMO protease assay from Invitrogen. Successful cleaving was achieved only for Cas13a (Figure 4).
In-vitro transcription
RNA targets and sgRNAs were produced through in-vitro transcription. Products of this were run on a 13% polyacrylamide gel and were shown to all have length of around 64 bases, as seen by UV shadowing (results not shown).
Cas13a function
We tested the ability of Cas13a to become activated through complementary binding between sgRNA and the RNA biomarker. This was analyzed with the use of ribosomal RNA (rRNA) and agarose gel electrophoresis, where the rRNA is used as a reporter since active Cas13a collaterally cleaves ssRNA leaving it as small fragments.
Purified Cas13a protein (BBa_K3602020) and the in-vitro transcribed rs6983267-G SNP gRNA (BBa_K3602004) were mixed for co-incubation in 10 minutes at 37°C, to ensure the formation of Cas13a-sgRNA complex. Different concentrations of sgRNA were used (1.7 ng/ µL, 0.43 ng/µL, 0.043 ng/µL, and 0.0043 ng/µL) to examine the dependency of sgRNA on Cas13a activity. After co-incubation, the matching target RNA (BBa_K3602007) and rRNA were added to a concentration of 0.43 ng/µL and 20 ng/µL respectively. Then these were incubated at 37°C for the collateral cleavage to proceed. Samples were taken out after 15 min, 40 min and 2 hours and put on ice until being examined with agarose gel electrophoresis. The different time steps were included to get an idea of the RNase activity time.
When comparing to the negative control (lane 2) in Figure 5, it is seen that all rRNA is degraded to some extent in all other lanes. The rRNA in the positive control (lane 1) is totally degraded thereby leaving no signal. For the samples with Cas13a, sgRNA and target it is observed that the more time the samples were incubated the more rRNA is degraded. This indicates that the prolonged time gives Cas13a more time to degrade rRNA or that the rRNA is degraded because of the heat. Within the different time intervals, it is generally observed that the rRNA in the samples with 0.043 ng/µL sgRNA are most degraded ones. However, it is seen that the 0.0043 ng/µL samples are the less degraded ones besides from the negative control. This indicates, that for Cas13a to be fully activated an amount greater than 0.0043 ng/µL is needed. Thus, Cas13a is dependent on sgRNA binding upon binding to target and then activation.
To be able to conclude if Cas13a is also dependent on the target sequence and thus if Cas13a is only active in the presence of a matching target sequence, a new experiment was set up. In this experiment, the rs6983267-G target RNA and the matching sgRNA was also used. The experiment included two samples with Cas13a, target sequences, and 0.043 ng/µL or 0.0043 ng/µL sgRNA. In this experiment, five controls were included to properly ensure that the rRNA degradation was caused by collateral cleavage from Cas13a. The controls include a positive control with RNase A and four negative controls only with Cas13a (lane 2), sgRNA+target (lane 3), Cas13a+sgRNA (lane 4), and nuclease-free water (lane 7). Then the samples were incubated at 37°C and aliquots were put on ice after 20 mins and 1 hour before running on the gel electrophoresis.
The experiment in Figure 6 showed that upon addition of target RNA to the sgRNA-Cas complex solution, collateral cleavage was induced. This can be observed as a smear in lane 5 and 6 thereby indicating degradation of rRNA. It is possible to see some degraded rRNA in all samples. This is assumed to be because the samples were incubated at 37°C for 40 mins as RNA is easily degraded when heated. Lane 4 is without target and was therefore expected to be negative as the Cas protein should not be activated without the matching target. As more RNA is degraded in lane 4 than lane 2 it is expected that Cas13a has intrinsic collateral RNase activity when bound to the sgRNA. The sgRNA might induce conformational change activating the protein. In our PMT this would lead to false positives and it should therefore be optimized. Optimization includes shortening the heating time and optimizing the sgRNA. Heating time can easily be shortened as the results in Figure 5 show collateral cleavage of Cas13a even after 15 mins. By optimizing the sgRNA, the conformational changes might be avoided thus the sgRNA-Cas13a complex is only activated in presence of a target. However, as it is possible to see that lane 5 and 6 are more degraded than the rest, Cas13a depends on sgRNA and target for full activation.
Cas12a function
To test for the activation and enzymatic activity of Cas12a, we used our Cas13a plasmid as a reporter, both in its plasmid form, but also linearized form through digestion with EcoRI. Cas12a only cleaves ssDNA nonspecifically, so we boiled the dsDNA from the plasmid for 10 minutes at 95°C and cooled the sample down directly on ice water to maintain the integrity of the now ssDNA. After producing the ssDNA, we used a 2% agarose gel to check for enzymatic activity under different conditions.
Here we tested for Cas12a activity using the target sequence (BBa_K36002012) and sgRNAs that we designed for the rs16901979 C;A genotype and measured this through the degree of degraded DNA in the gel. As shown in Figure 7, controls were made consisting of dsDNA+DNase (lane 1), dsDNA without DNase (lane 2), DNA digested on an EcoRI restriction site (lane 3), and ssDNA (lane 4). Two different incubation times were used to illustrate how fast the endonuclease activity is and different concentrations of gRNA were also used, namely 1µM and 2µM sgRNA (lanes 5-16).
It can be observed in lanes 9-12 that Cas12a was only activated when ssDNA was used as a reporter together with 2µM sgRNA and target DNA. No activity can be seen with only 1µM concentration of gRNA. A no-target control was made with 2µM sgRNA found on lanes 13 and 14, which showed no change in ssDNA levels compared to the control with only ssDNA.
Through the ssDNA, we were also able to observe collateral activity of Cas12a, which can cut only if there is a match between the gRNA and dsDNA target sequence. Based on the controls, it can be concluded that it was the target sequence that induced collateral ssDNA cleavage, and not due to foreign DNases.
RNase inactivation and reintroduction
We tested whether it was possible to inhibit naturally occurring RNases in urine and saliva with proteinase K. This inhibition is especially important for PMT, as RNases would cleave the RNA reporter, giving false positive readouts. Firstly, inhibition of RNases was tested with the RNase Alert kit from ThermoFisher [1]. The kit contains a fluorophore quenched by a quencher attached by an RNA strand. If the strand is cut by an RNase, a signal will be emitted. A saliva and urine sample were each incubated for three hours with proteinase K at a concentration of 400 µg/mL. Afterwards, 5 µL of RNase Alert was added to a 45 µL sample. After 30 min incubation, fluorescence was scanned using the Typhoon FLA 9500 with excitation at 490 nm and emission at 520 nm.
As the tests show in Figure 8, a 3-hour treatment was effective in removing RNase activity from the samples, as seen from the clear decrease in intensity of the spots. However, some level of RNase activity was still present in the saliva, so a sample of saliva was incubated O/N at 55°C with proteinase K. As seen in Figure 8, the sample did not emit any light after O/N incubation and extensive vortexing, meaning that all RNase activity was halted.
The treated urine and saliva had 1 mM phenylmethylsulfonyl fluoride (PMSF) added to inhibit proteinase K. To ensure that proteinase K activity had been properly neutralized, untreated urine or saliva was reintroduced to the samples. The solution was then incubated for three additional hours and analyzed with RNase Alert. As seen in Figure 9, the inhibition of proteinase K was successful, as the RNase Alert shows a clear presence of active RNases.
Flow strip
After testing the inhibition of RNases with RNase Alert, we also tested it with the flow strip read-out as this read-out was preferred for the PMT. Samples of urine and saliva were treated with proteinase K and PMSF prior to testing, and were then reintroduced with fresh urine or saliva. For each step, an aliquot was taken, and all were tested on flow strips in the end. The result can be seen in Figure 8.
As seen in Figure 10, the strips with only urine or saliva are positive, which shows the problem that RNases are already present in the samples. For the urine samples, it is seen that when treated with proteinase K the strip is negative denoting that RNases in urine was effectively inhibited. Treatment with PMSF still results in a negative test, and when urine is reintroduced the test is positive again. Thus, showing that the proteinase K was efficiently inhibited as the reintroduced RNases was still active. The reintroduction of urine reflects the introduction of Cas13a after inhibition of already present RNases.
For the strips with saliva it is seen that all strips are positive. The intensity of the test line is falling with treatment of proteinase K and strengthened when saliva is reintroduced. This indicates that 3-hours was not enough time to inhibit the RNases in saliva fully but that they are generally more inhibited when proteinase K is added.
RNAse conclusion
These results suggest that proteinase K is responsible for inhibition of RNase in urine after 3 hours at 55°C and that the activity of proteinase K can be stopped again after the treatment before addition of Cas13a. Three hours for a home test is a long time and optimization of this step should be done in the future. Optimization includes changing of temperature and time and possibly look more into RNase inhibitors to investigate the possibilities of a better solution.
Inhibition of RNases in saliva is seen when proteinase K treatment is prolonged overnight. This time span is not ideal for a home test and we therefore introduced the use of Cas12a instead of Cas13a when testing saliva. Cas12a cleaves single-stranded DNA rather than RNA and the read-out will not be affected by RNases. Cas12a will be affected by DNases instead and the inactivation of these prior to introduction of Cas12a also has to be investigated.
As seen on the flow strip results, it is possible to detect whether the RNases are active on the Universal Lateral Flow strips [2]. However, the negative tests are seen to show signal at both lines, while the positive tests show signal at one line. In this set-up, it is not a problem since it is still possible to see the difference between the two, as all or most of the RNA reporters are cleaved by the RNases leaving only a signal in the test line of the positive strips. In other experiments, this might not be the case as the RNases might not always cleave all RNA reporters. Therefore, the flow strip read-out needs further optimization. A visible T-line in the negative test is a common problem when combining flow strips with CRISPR/Cas detection. Therefore, many different strategies for eliminating the T-line are possible, as discussed lated in the "Future Perspectives" section [3].
Future perspectives
Finished products mostly require a great amount of work, and even though our team has been working hard from the beginning of our journey, there is still much work left before PROSTATUS is on the market.
Here you can find a list of things that still needs to be done to realize PROSTATUS . Some of this could very well be solved by a new iGEM team:
As of now, our flowstrips give 2 lines on negative sample because of extra gold particles that bind non-specifically to the FAM reporter. The goal is to optimize the flowstrip, so that only one line will appear. This could be done by changing the amount and quality of RNA reporter, increasing the viscosity of the buffer, and/or changing the ratio and dilution of Cas and gRNA.
The biomarkers our team has focused on are biomarkers that are mostly present in Caucasian people. Design of biomarkers, that are more present in other ethnicities and developing the test to other areas of the world, would be optimal.
When the flowstrips have been optimized the test should be performed on urine from healthy men and women, women with breast cancer, men with benign cancer, men with malignant prostate cancer. All of these should be performed on samples from people with multiple ethnicities.
An application for access to a urine biobank was sent into Palle Osther at “Sygehus Lillebælt”. However, time constraints kept us from gaining access to patient urine samples. The full application can be found here.
To commercialize our product the different biological components will need to be compressed in an inactive pill form, that will become activated when it comes into contact with the sample fluid.
When the whole biological process has been optimized in the lab, the test should be performed in PROSTATUS hardware to ensure that the materials do not interfere with any of the biological processes.
The test step and hardware components will have to be tested by the end-user. This could be done by observing the end-user implementation with the finalized product, to see whether the end-user can interact with the product the same way we expect them to. A round of interviews would also be an option to hear about the end-users opinions and possible suggestions.
This can also be done by focus group or pre-launch approach.
If the design is intuitive, the component has to go through a design team for manufacturing and assembly process, where the components will have to be standardized and simplified to ensure low production cost.
Before the final product can be launched, it will have to be certified and approved by medical institutions. Moreover, the intellectual property rights (IPR) will have to be protected by either patenting, trade secrets, etc. to minimize new entrance threats. Furthermore, the estimated supply chain in the business model canvas will have to be confirmed by forming production and distribution partnerships. Lastly, a marketing strategy should be created and an entry mode should be considered.
References
[1] ThermoFisher. RNaseAlertTMLab test Kit. From https://www.thermofisher.com/order/catalog/product/4479768#/4479768[2] Milenia biotec. Hybridetect - Universal Lateral Flow Assay Kit. From https://www.milenia-biotec.com/en/product/hybridetect/
[3] Breitbach, A (2020). Lateral Flow Readout for CRISPR/Cas-based detection strategies. From https://www.milenia-biotec.com/en/tips-lateral-flow-readouts-crispr-cas-strategies/
