Description

---

The goal of Aim 2 is to redesign the switch-gRNA from Collins et al. (2021) to show specificity to c-Myc mRNA rather than an arbitrary sequence. First, we picked two different, specific trigger DNA sequences from c-Myc mRNA (T1 and T2). Then, with code from Collins et al. that specified the parameters of their switch-gRNA, we set the specificity of two switch-gRNA to T1 and T2 respectively. This generated two unique switch-gRNA DNA sequences (S1 and S2). Once we had our two switch and trigger DNA sequences, we engineered plasmids with them to create four different combinations: S1T1, S2T2, S1T2, and S2T1.

Experiments

---

We transformed the respective switch/trigger plasmids into E. coli that also contained a CRISPR plasmid and a GFP plasmid. We would expect switch-gRNA activation only when the switch and trigger correspond, such as S1T1 and S2T2, that would target CRISPR to the GFP plasmid resulting in a lack of fluorescence. When they don’t match, such as S1T2 and S2T1, we would expect inactive switch-gRNA, where CRISPR wouldn’t be targeted and the cells would fluoresce. By showing that there’s a lack of activation through GFP fluorescence when a non-matching trigger from c-Myc mRNA is present, we can conclude that our switch-gRNA is specific to only a specific section of c-Myc mRNA and no other. We expected to see GFP expression to be significantly higher in the switches with the incorrect trigger sequence.

Reference the Experiments Page to view a complete list of the protocols used for each aim.

Results

---

[Figure 2a] From our results we were able to observe a significant difference in GFP expression between the plasmids with the correct switch and trigger pairings versus the plasmids with incorrect pairings. When plated on agar plates and incubated overnight, differences in GFP expression are apparent as seen in Figure 2b. However, in order to obtain more quantitative data we performed a 9 hour incubation in LB-antibiotic solution while simultaneously performing plate readings every hour to measure GFP expression. After a 9 hour incubation, we observed that the switches with the incorrect trigger sequence were on average 8330.75 a.u. greater than the switches with the correct trigger sequence. The results of the full incubation can be seen above. From these results we are able to conclude that the switches are able to identify their correct c-myc sequence and are specific to that sequence.

---

[Figure 2b] For Aim 2, c-Myc is chosen because it is overexpressed in the majority of cancers. New switches were designed to identify specific modeled triggers of c-Myc mRNA using Benchling and NUPACK. Specifically two new switches (S1 and S2) and respectively matching triggers (T1 and T2). We used the 1229 plasmid as the backbone, cutting out the old switch and trigger with a restriction digest and then isolating the backbone using a gel extract. Then, we created four plasmids with combinations of switches and triggers with a ligation or Gibson assembly:(S1T1, S1T2, S2T1, S2T2)

We then cultured these cells to effectively clone our plasmid and miniprepped them to isolate our new plasmids, allowing insertion into other cells.

---

[Figure 2c] These plate readings/images demonstrate the transformed cells with four different combinations of switch and triggers. The results of this show the specificity of our switch to a particular trigger, and that a sequence of c-Myc RNA can be set as the trigger. While we cannot conclude we could detect a section from a full, naturally-occurring c-Myc mRNA, we can conclude that detection of isolated sections of c-Myc mRNA is possible.

We found that the plasmids with corresponding switch and trigger sequences correctly suppress GFP expression.