Team:Lethbridge HS/Measurement
Measurement Overview
This year our team needed an assay to characterize the efficiency of our diagnostic tool. After looking into what previous teams (Munich 2017 and TU_Delft 2017) had done, we noticed that they used the commercially available RNase Alert . However, the total cost ($271 CAD)and the amount of material you are supplied in the kit (25 reactions) may not be cost-effective for some iGEM teams.
We chose to still work with fluorescence as our reporter for activity of our Cas13a enzyme. However, we chose to work with the RNA aptamer RNA Mango II ( Autor et al., 2018 ). We were graciously gifted a plasmid from the Kothe lab at the University of Lethbridge that encodes for the snoRNA snR30, but has been modified to contain RNA Mango II within a native stem loop of snR30. For a proof-of-concept, we designed our CRISPR RNA (crRNA) sequence for our in vitro studies to target the RNA Mango II sequence within the construct. RNA Mango II can be used in any sequence that iGEM teams need to have as a target as long as it is within a stem loop structure. RNA Mango II is able to fluoresce when it interacts with the dye Thiazole Orange. The cost of thiazole orange is less expensive than RNase Alert ($187 CAD for 1 g). You also only need to work with a final concentration of 100 nM thiazole orange in solution, making it cost $0.05 for a 60 μL reaction( Autor et al., 2018 ). Additionally, the use of an RNA aptamer simplifies this activity assay than what has been done in previous attempts. Lastly, we believe that this measurement assay can be adapted to test the activity of other RNA cleaving enzymes, making it useful for more iGEM teams than teams working on furthering our understanding and use of Cas13a.
Cas13a Activity Assay
Our team conducted a Cas13a activity assay to test the effectiveness of our enzyme. We incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA Mango to see the change in fluorescence of the dye thiazole orange. By analyzing our results, we can see that the fluorescence for the RNA Mango at the concentration 25nM sample decreased. This likely indicates that the Cas13a enzyme is cleaving. However, for higher concentrations, there was not a significant change in fluorescence. This may be due to having an insufficient amount of enzyme for proper cleaving to occur or our enzyme not being active enough to cleave larger amounts of RNA Mango in the same amount of time as the 25 nM sample. The sample with concentration 100 nM is excluded from these generalizations. We believe there may have been an error made in the reading. Another reason for the differences seen in the concentrations of RNA Mango could be caused by if our purified RNA is taking on multiple conformations that affect its ability to interact with thiazole orange. If the G Quadruplex is not forming properly, we would not see fluorescence. Graph C shows the results of our control experiment. Similar to the previously mentioned experiment we incubated 300 nM of Lbu Cas13a complexed with the crRNA with various concentrations of RNA, however this time we used RybA. This was a negative control experiment to test the specificity of our CRISPR Cas13a system. It seems that there was no significant change in fluorescence over time thereby indicating that no RybA was cleaved and our CRISPR Cas13a is specific. Since there was no added fluorescence molecule, it is also hard to interpret what any changes would be.
Figure 15. Determining activity of Lbu Cas13a by targeting snR30 containing RNA Mango II by detecting a loss of fluorescence. Excitation occurred at 510 nm and emission at 535 nm and scans were completed for 3 hours. Data was normalized by dividing by the negative control, which contained all components except for RNA. (A) Raw scans normalized to the negative control of snR30-RNA Mango II at various concentrations in complex with Lbu Cas13a and the crRNA (n=1). Controls are also shown of only snR30-RNA Mango II, the target molecule and Lbu Cas13a, and the target molecule and RNase A (n=2 +/- SD for controls). (B) Relative fluorescence at the maximum fluorescence of snR30-RNA Mango II at 72 minutes (n=1 for snR30-RNA Mango II at various concentrations, n=2 +/- SD for controls). (C) Controls for activity of Lbu Cas13a and crRNA. RybA was used as a specificity control for cleavage (n=2 +/- SD for all data shown).
To confirm the effectiveness of our Cas13a enzyme we ran before and after fluorescence scanning samples on a urea page. Additionally, this would allow us to better interpret our experimental controls. This confirmed our speculation that the 25 nM sample was indeed cleaved by Lbu Cas13a. For the other samples, no significant difference is seen between the before and after samples. For the 50 nM before sample, we believe there may have been a loading issue that affected the quantification results seen in table 1. Our controls seen in Figure 16 B and C show that there are no significant changes between before and after scanning the samples. This means that are assay is specific for our targeted molecule needing to be present to have enzyme activation. Cas13a will also not cleave our target molecule without the crRNA in the complex.
Figure 16. 10% Urea PAGEs of Lbu Cas13a activity assay components. (A) Left to right: lanes 1-3: empty; lane 4: RNA Mango 100 nM post-scan; lane 5: RNA Mango 100 nM pre-scan; lane 6: RNA Mango 75 nM post-scan; lane 7: RNA Mango 75 nM pre-scan; lane 8: RNA Mango 50 nM post-scan; lane 9: RNA Mango 50 nM pre-scan; lane 10: RNA Mango 25 nM post-scan; lane 11: RNA Mango 25 nM pre-scan; lane 12: RNA Mango + RNase A post-scan; lane 13: RNA Mango + RNase A pre-scan; lane 14: negative control (no target or control RNA) post-scan; lane 15: negative control (no target or control RNA) pre-scan. (B) Left to right: lane 1: RNase A + RybA pre-scan; lane 2: RNase A + RybA post-scan; lane 3: RybA 25 nM pre-scan; lane 4: RybA 25 nM post-scan; lane 5: RybA 50 nM pre-scan; lane 6: RybA 50 nM post-scan; lane 7: RybA 75 nM pre-scan; lane 8: RybA 75 nM post-scan; lane 9: RybA 100 nM pre-scan; lane 10: RybA 100 nM post-scan. (C) Left to right: lane 1: crRNA pre-scan; lane 2: crRNA post-scan; lane 3: RNA Mango pre-scan; lane 4: RNA Mango post-scan; lane 5: RybA pre-scan; lane 6: RybA post-scan; lane 7: Cas13a Lbu pre-scan; lane 8: Cas13a Lbu post-scan; lane 9: Cas13a Lbu + crRNA pre-scan; lane 10: Cas13a Lbu + crRNA post-scan; lane 11: Cas13a Lbu + RNA Mango pre-scan; lane 12: Cas13a Lbu + RNA Mango post-scan; lane 13: Cas13a Lbu + RybA pre-scan; lane 14: Cas13a Lbu + RybA post-scan. Lane 15: empty.
Table 1. Quantification of figure 16 A lanes 4-11 of decrease in RNA Mango using ImageJ.
| Concentration of RNA Mango (nM) | 25 | 50 | 75 | 100 |
|---|---|---|---|---|
| Pre-scan | 49.34% | 20.8% | 45.3% | 50.0% |
| Post-scan | 1.32% | 79.2% | 54.5% | 50.0% |
References
- Autor, A., Jeng S, C.Y., Cawte A., D., Abdolahzadeh, A., Galli, A., Panchapakesan, S.S.S., Rueda, D., Ryckelynck, M., and Unrau, P.J. (2018). Fluorogenic RNA Mango Aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun 9(1): 656.