Experiments with 3 Exon Dual Fluorescence Reporter

After designing our 2-exon and 3-exon mKate HBG reporter, which only showed mKate fluorescence knockdown, we wanted to see a color change between when our system is present and absent. We designed two 3-exon reporters, one of which is what we called the Dual Fluorescent Reporter.

3-exon Dual Fluorescent Reporter

In order to fully demonstrate the functionality of our guide system, we designed a reporter that, instead of showing an increase or decrease of a single color, fluoresced a different color in the presence of our system. Our reporter contains a fluorescent conserved region, HBG Intron two, a yellow fluorescence exon, HBG Intron one, and a blue fluorescence exon. The Yellow Fluorescent Protein (YFP) and the Blue Fluorescent Protein (BFP) used in our lab have a 200 bp conserved region between them, which was ordered a g-block with complementary sticky ends to HBG Intron 2. The YFP and BFP only differ by approximately 50 bp in their non-conserved regions. In order to design a g-block that contained both YFP and BFP, the third base of every amino acid was wobbled in the YFP gene. (The wobble base was chosen based on human codon usage). The second g-block also included HBG Intron one between the two fluorescent exons, and complementary sticky ends with HBG Intron two.

If our guide system is not present in the cell, the conserved region, YFP and BFP will be spliced together. During translation, the stop codon at the end of YFP will cause the ribosome to fall off, resulting in a complete YFP and therefore yellow fluorescence will be made.

When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the YFP exon. The conserved region and BFP will be spliced together, resulting in a complete BFP, and therefore blue fluorescence will be made.

Results

Verifying the construct

Reporter Titration

Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the 3-exon DF construct. We expected that in absence of our system, there would be an increase yellow fluorescence across the reporter concentrations, as YFP would be translated. In this case, blue fluorescence will be low, because the ribosome will fall off at the stop codon of YFP, before reaching BFP. If our system is present, we expect that levels of blue output will rise with the increase in reporter construct, because YFP will be skipped over, allowing BFP to be produced. These expected results are summarized below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of 3-exon DF reporter from 10 to 500 ng. We also transfected Guide3 with dCas13a, and ASO2+ with Ms2 at optimized amounts from earlier experiments. (For a detailed explanation of how to plan a mammalian transfection click here)

mKate Titration for 3-exon DF reporter

3-exon DF reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bins of each result.

We expected that the reporter would show no blue fluorescence in the control condition, but it in fact shows the same amount of yellow fluorescence as blue fluorescence in all conditions. Additionally,adding dCas13a and Ms2 have no effect on the color output.

Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was producing equal levels of blue and yellow in the control condition. '

Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.