Team:Lethbridge HS/Demonstrate
Overview
This year our project consisted of two systems: a diagnostic tool for detecting pathogenic bacteria and a therapeutic for direct targeting of pathogenic bacteria as an alternative to traditional antibiotics. We were able to test our diagnostic tool in vitro using one Cas13a protein (Lbu). Please refer to our Results page for the production of the components.
For the therapeutic portion of our project, we were unable to complete our planned triple transformation (fluorescent protein, crRNA, Cas13a). However, we decided to move forward with a control experiment involving a dual plasmid transformation (fluorescent protein and crRNA) and using lysed cells that had overexpressed our Cas13a protein. This would indicate to us if our Cas13a would be activated outside of the cell (not in complex with the crRNA) and if we would need an encapsulation component. Please refer to our Results page for other work we did for characterizing components for this system.
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. Please visit our measurement page to see our further analysis of this assay as being beneficial for future iGEM teams to characterize any RNA cleaving enzymes they are working with!
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% |
In vivo Dual Plasmid Fluorescence Assay
We completed a dual plasmid transformation. To confirm that we had both plasmids present we completed a digest test because PCR amplification from the colony was not working. This should separate our construct from the plasmid confirming that both parts are present. In figure 20, only linearized cuts are seen. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. In figure 21, only linearized cuts are seen near the 3000 bp mark. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. The second band at approximately 2000 bp is plasmid backbone, while the 1000 bp mark is the GFP insert. The crRNA insert (expected size is ~260 bp) is likely not seen in the gel due to having to run the gel longer to get better resolution for the upper bands.
Initially, our team had planned on doing a triple plasmid transformation of our target fluorescent protein (GFP), crRNA, and Cas13a to test if our system would work in vivo . Additionally, we wanted to have RFP as the fluorescent protein to serve as a specificity control instead of transforming the plasmid containing GFP. We were unsuccessful in getting all three plasmids to transform, but did succeed in getting the fluorescent proteins and crRNA containing plasmids to transform together as seen in figures 20 and 21.
Figure 20. 1% agarose of restriction digests using PstI and EcoRI from Escherichia coli BL21(DE3) cells containing plasmids for RFP and either Lbu crRNA or Lwa crRNA. Left to right: lane 1: 1 kb ladder; lane 2: Lwa crRNA colony 1; lane 3: Lwa crRNA colony 2; lane 4: Lwa crRNA colony 3; lane 5: Lbu crRNA colony 1; lane 6: Lbu crRNA colony 2; lane 7: Lbu crRNA colony 3; lanes 8-13: empty.
Figure 21. 1% agarose of restriction digests using PstI and EcoRI from Escherichia coli BL21(DE3) cells containing plasmids for GFP and Lbu crRNA, Lwa crRNA, Lba crRNA, or Lsh crRNA. Left to right: lane 1: Lwa crRNA colony 1; lane 2: Lwa crRNA colony 2; lane 3: Lwa crRNA colony 3; lane 4: Lwa crRNA colony 4; lane 5: Lsh crRNA colony 2; lane 6: Lba crRNA colony 1; lane 7: Lba crRNA colony 2; lane 8: Lba crRNA colony 3; lane 9: Lbu crRNA colony 1; lane 10: Lbu crRNA colony 2; lane 11: Lbu crRNA colony 3; lane 12: Lbu crRNA colony 4; lane 13: 1 kb ladder.
As an alternative experiment, our team grew cells that expressed the Lbu and Lwa Cas13a protein overnight. We also grew cells that expressed dual plasmids; GFP and crRNA Lwa; GFP and crRNA Lbu; RFP and crRNA Lwa; and RFP and crRNA Lbu. We then lysed the cells that expressed the Cas13a proteins using a French Press and clarified the lysate via centrifugation. Following this, our team pipetted in a 1:1 ratio of clarified cell lysate: fluorescent protein and crRNA into a 96 well plate. This allowed us to observe if there would be an effect from the CRISPR Cas13a system on the fluorescent proteins. We observed that in our optical density data, both dual plasmid systems for GFP and RFP had stunted growth in comparison to only E. coli cells expressing GFP or RFP or no plasmid (Figure 22C). Adding the lysate may have caused the death of the culture. We neglected to include replicates of the dual plasmid system without adding lysate to observe how that grew. This would be beneficial for any future experiments. Alternatively, there may have been some effect of the protein in the lysate on the GFP fluorescence (Figure 22B). However, we are unsure of the specificity due to the potential of the RFP not folding correctly in vivo as demonstrated by the substantial standard deviation seen in our replicates (Figure 22A).
Figure 22. In vivo fluorescence assay of E. coli BL21(DE3) cells containing fluorescent protein and crRNA plasmids and E. coli Rosetta(DE3) cell lysate of overexpressed Cas13a proteins. This assay was conducted with 3 biological replicates and 3 technical replicates. (A) Fluorescence of E. coli cells containing only an RFP expressing plasmid, or dual plasmid expression of RFP and crRNAs from Lwa and Lbu that target GFP and the respective cell lysate containing the appropriate Cas13a. RFP excitation was at 558 nm and emission at 583 nm. (B) Fluorescence of E. coli cells containing only a GFP expressing plasmid, or dual plasmid expression of GFP and crRNAs from Lwa and Lbu that target GFP and the respective cell lysate containing the appropriate Cas13a. GFP excitation was at 475 nm and emission at 508 nm. (C) Optical density of E. coli cells expressing GFP, RFP, dual plasmid systems mentioned previously, or only E. coli BL21(DE3) cells with absorbance measured at 600 nm. (D) Relative fluorescence at maximum excitation at 81 minutes. GFP excitation was at 475 nm and emission at 508 nm and RFP excitation was at 558 nm and emission at 583 nm.
Future Directions
This year, we were able to successfully show that our diagnostic tool functions as expected in vitro. The next steps for us would be to optimize the concentration of Cas13a required to cleave our target RNA using the assay we designed. After this, we would need to test the functionality of our system in a lyophilized form. Additionally, we need to work on developing a method for sample purification that would minimize the chances of RNase contamination or designing a construct that incorporates unnatural nucleotides.
The therapeutic portion of our system will require more design and experimental work. We believe it would be easier to succeed with our triple plasmid transformations if our Cas13a constructs only contained a T7 promoter, ribosomal binding site, and the coding sequence as the purification tags add excess nucleotides. After confirming all three plasmids have been transformed, we would need to repeat a modified version of our control experiment. In this form, we would only have to grow cells on one day, and after induction grow cells in a 96 well plate to monitor growth and fluorescence. A loss of colour would indicate that our system is able to work. Note that it will still be important to still have a non-target fluorescent protein as a specificity control. Lastly, we would need to work more on the design of a phagemid system for delivery to see if this the most feasible method for how this system is envisioned to work.