Team:ZJUT China B/Proof Of Concept
Proof of concept
LwaCas13a and PsmCas13b cleavage base preference
Figure1. the cleavage base preference of LwaCas13a (Raw results)
Note: The "negative" refers to groups without adding targets and "experiment" refers to groups with targets; AC, AU, GA,UC corresponds to different fluorescent reporters
Figure 2. The cleavage base preference of PsmCas13b (Raw results)
Note: The "negative" refers to groups without adding targets and "Psm" refers to groups with targets ; AC, AU, GA,UC corresponds to different fluorescent reporters
Analysis
We investigated the cleavage base preference of LwaCas13a and PsmCas13b in a single system.
As for LwaCas13a protein, the results suggest that the protein could cleave AU and AC oligonucleotide even though there is no target in the detecting system which we called "the leakage activity of Cas13 protein ". Obviously, the LwaCas13a protein exhibits more "leakage" upon AC while the leakage upon GA and UC are quite slight. When we added target sequence in the detecting system, the cleavage of AC obviously increased (the RFU increased 2.04 times at 30min) but the background subtracted fluorescence of AU reporter increased in a cliff like manner (the RFU increased 13. 89 times at 30min), which means that when the target was added, the protein's activity to cleave AU was activated significantly. Due to the preference appears only when the target was added, we defined this as the cleavage base preference of LwaCas13a protein, or more specifically, "the activating associated cleavage base preference " of the protein.
And as for PsmCas13b protein, we found that the PsmCas13b protein shows the cleavage upon AC, AU and GA while it only shows tiny cleavage activity upon UC. Interestingly, the cleavage activity of the PsmCas13b upon AC, AU and GA are different. As for GA, Psm exhibits target dependent cleavage activity. That's to say, only when the target is added, will the cleavage activity of protein be activated ( Assume that leakage cutting is not considered ) . As for AU and AC, the PsmCas13b protein shows no obvious difference between systems with target and without target. The results suggest that both LwaCas13a and PsmCas13b have the cleavage base preference upon specific oligonucleotides, Lwa is AU while Psm is GA. Thus, our concept could be achieved in a separate system.
Normalized result
Because we need to obtain fluorescence values of different colors, we use different excitation and emission wavelengths for detection. However, under different excitation emission wavelengths, the values of blank background are quite different, which makes the fluorescence values of different colors can not be compared, and the blank background fluorescence may lead to false positive detection results. Therefore, we normalized the obtained fluorescence data. As shown in the Table 2 , we multiply the fluorescence value of each detection wavelength by the corresponding coefficient, so that their blank fluorescence values are controlled the same. Thus, a new fluorescence kinetic curve is obtained, as shown in Figure 3 .
Table1 Raw fluorescence result of LwaCas13a
Table2 Normalized fluorescence result of LwaCas13a
As shown in Figure 3 , after normalizing the fluorescence value, we can see that Lwa protein shows obvious cleavage preference upon AU. Meanwhile, the leakage cleavage of LwaCas13a protein upon AC can hardly affect the detection results. As can be seen from Figure 4 , normalizing weakens the influence of nonspecific cutting of Psm upon AC and AU on the detection results, thus reflects the cutting preference of GA. In summary, we have proved that the cleavage preference of Lwa is AU, and that of Psm is GA.
Device
Due to the high sensitivity, out device can efficiently detect the concentration of different fluorescein simultaneously and a kinetic curve can be drawn based on the resulting data. Please click >>Engineering<<.
Conclusions
By adding four fluorescent probes into the detecting system, we verified the cleavage preference of proteins LwaCas13a and PsmCas13b. Different from what we expected, the Cas13 protein is able to cleave all base nucleotide. However, significant base preference can be observed when the target sequence is added. By normalizing the data, we confirmed that the cleavage prefere of Lwa is AU, while that of Psm is GA. However, according to the data, we need to set up a blank control group to reduce the probability of false positive and make the detection results more accurate because of the obvious leakage and cleavage of Psm, which can guide the design of the device to achieve our multivirus detecting goal. With this result, we demonstrate that our concept of multivirus detection could be realized on Lwa and Psm proteins. However, due to time constraints, we only proved the specificity of Lwa and Psm proteins. We put the specificity verification of CcaCas13b and LbaCas13a in our future steps.
For more details on experiments data, please click >>Results<<
Next step
Future for Wet lab
1. Improvement of the sample processing plan and complete the modeling;
2. Revision of the expression and purification process of LbaCas13a and CcaCas13b to explore the reasons for the low activity of LbaCas13a and the unsuccessful purification of CcaCas13b;
3. Enzyme digestion of four expressed cas13 proteins;
4. Verification of the cas13 protein activity after enzyme digestion and comparison with that of non digested protein to explore the effect of tag on protein activity;
5. Verification of the cleavage base preference of CcaCas13b and LbaCas13a protein;
6. Specificity verification of four Cas13 proteins in one pot;
7. Exploration of the detection limits of LbaCas13a, PsmCas13b and CcaCas13b;
8. Determination of the protective effect of TDPs on the activity of lyophilized Cas 13 proteins.
Future for Device
1. Although our device can detect the slight change of fluorescence, the cleavage of Cas13 protein is quite different from our expectation: only the corresponding reporter is cut. In fact, cas13 protein cleaves the reporter in different degrees when the target is negative or positive. This is likely to result in false positive results. Therefore, the original design of the detection device before and after the self time control is not feasible, so we need to add a negative control sample plate, and use its difference to correct it.
2. Considering the characteristics of Cas protein, we can't freeze-dry it with the reporter directly. In the process of lypholyzation, a large number of reporters will be cleaved.
3. We also hope that the sample processing microfluidic chip and the detection chip can be combined into one to make a disposable detection chip with higher integration, smaller size, cheaper and more convenient.
Future for HP
1. Find and contact experts in protein expression and purification to solve the problem of the purification process, which led to low activity of LbaCas13a and CcaCas13b part.
2. Contact with the Environmental Protection Bureau to solve the problem of medical waste recycling.
3. Contact relevant experts to find out how to run the virus database.
4. Find cooperation to build and operate a comprehensive database and contact with government agency to learn the regulations for the data uploading.
Future for Modeling
1. The Cas13 protein also exhibits strong RNase activity when the target is negative. The possible reason is that the Cas13 protein is excessive. We hope that in the future, mathematical modeling can be used to predict the optimal concentration of Cas13 protein. Under this concentration, the RNase activity in the absence of the target can be minimized while the target detection sensitivity can be relatively high. This will help the wet lab more.
2. Modeling group can also cooperate with the device group to give better suggestions such as correcting the results. This can help us get more precise results.
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