Our engineering success came from the initial proof of concept. Our team has spent over 3 months researching everything to know about CRISPR systems and how the Cas enzyme works. Specifically, we were concerned about the activation mechanism of the RNase activity since there were not many articles directly referring to the proposed mechanism, or much information from protocols and how it works. Given the complexity of the system, we decided to start with a proof of concept experiment to showcase our theories from compiled research.
We designed our components based on a combination of multiple papers found for CRISPR-Cas13 systems. The miRNA used was miRNA 424-5p, which was previously sequenced in a research article. The crRNA was designed to be a complement to both the miRNA and the Cas13 enzyme. It has a section that makes a stem-loop structure to bind to the Cas13 as well as a section that base pairs with the miRNA. The theory is that the crRNA binds to the Cas13 as a precursor to the activation of RNase activity. When the miRNA is added to the system, it base pairs with the crRNA to activate the Cas13 enzyme. This enzyme will then begin cutting in the system, which is a goal for our product later on.
We ordered miRNA-424-5p and the specific crRNA from the design phase above. We also ordered Cas13a for our initial proof of concept experiment. Using multiple protocols and research, we developed a protocol that binds the Cas13 to the crRNA in an incubation step. It then adds miRNA and allows it to bind to the crRNA for the activation of RNase activity, also in an incubation step.
In order to test if the proof of concept is sound, we would use gel electrophoresis to compare pure miRNA and the results from the CRISPR system to see if the miRNA was cut. If the gel shows many small bands in the CRISPR column and one band in the miRNA column, it can be concluded that the proof of concept is valid. The CRISPR system will cut samples when miRNA is present. A follow-up test would be added where a random RNA that is not similar to the miRNA is put in a sample with Cas13 and the crRNA and compared to the designed CRISPR system. Both samples would be put into a gel again to be compared to the uncut miRNA and RNA. We would expect the designed system to show cut miRNA and the sample with the random RNA to show uncut RNA bands.
The gel electrophoresis showed only a band from the ladder. We learned that a 1% agarose gel created “holes” too big that allowed our 22 nucleotide-long chain to flow through and disassociate into the buffer solution. After further discussion, it was concluded that a new method is needed to validate that miRNA is cut by Cas13. The new method includes a protocol for gel electrophoresis, but this one is specifically meant for RNA. This then would allow the team to ensure that the miRNA is cut into small fragments when in a solution with the Cas13 enzyme and crRNA. Our 4% agarose gel electrophoresis was a step in the correct direction, as we were able to see bands in the gel. The samples however were not denatured which caused an appearance of double bands for one component. The next gel would add a heat denaturation step in order to avoid a secondary structure formation. We also learned that we would want to add more lanes for the individual components as well using a RNA ladder for size measurements. The next gel was able to give us more information, however we were unable to read half of it. Some of this may be due to too much ladder and running the gel for too long so the EtBr ran off. We were able to modify this error by adding more EtBr to the cathode side to prevent it from running off of the gel.