Difference between revisions of "Team:BostonU/Proof"

Phase 1 Results

The first phase of our project was to develop a library of digital parts. Our journey began with finding the guide RNA sequences we would use in our work. We generated over one thousand 20 base guide RNA sequences in silico using a random sequence generator.

It was critical these guide RNA sequences be orthogonal to the human genome because we wanted to test our system in HEK293FT cells and prevent off target activation in the host genome. To test for orthogonality, we entered the sequences into the CRISPR Optimized Design tool developed by the Feng Zhang lab. We selected the top 18 sequences, which had an orthogonality score of 98% or higher, to synthesize and use in our research.

We synthesized these guide RNA sequences through IDT and cloned each one into guide RNA expressing vectors and guide RNA operator pairs. To act as controls for our experiments, we also cloned the guide RNA sequences Tre and UAS into guide RNA expressing vectors and guide RNA operator pairs. These 20 initial operators expressed an iRFP gene.

We transiently transfected and ran through a flow cytometer our paired gRNA expression vectors and gRNA operator reporters and dCas9-VPR to test their expression behaviors. We wanted to see high activated states coupled with low basal expression, a true digital system. The screen of this can be seen below.

Based on this screen, we decided to proceed with guide 1, guide 3, guide 8, and guide 13 for all future experiments. Next, we needed to prove that our parts could drive a diverse library of genes of interest. To complete this, we replaced the iRFP in our guide RNA operator reporters with a GFP, a BFP, and an mRuby protein. The results from these test can be seen in the matrix below.

In all cases, there was low basal expression and strong activation, proving that our system not only behaves digitally, but that it could also do so over multiple genes.

Finally, our system needed to be mutually orthogonal to prevent significant undesired crosstalk. If there was off target gene activation between operators, then we could not transfect multiple operator-expression pairs and obtain predictable results. We performed an orthogonality transfection with the GFP driven operators from our library. The experiment was designed such that every guide RNA expressing vector would be paired with each guide RNA operator reporter vector. We predicted significant GFP fluorescence when the guide RNA expressing vector has the same guide RNA sequence as the guide RNA operator reporter vector, and no significant expression elsewhere. The results of the experiment can be seen in the matrix below.

The diagonal down the matrix demonstrates that there is mutual orthogonality between the guides in our system. With this matrix, we proved that we developed a digital parts library.

Our final test was to compare the relative strength of our parts to a CMV, a strong mammalian promoter, to determine the relative strength of our system. This would also allow for a better interpretation of where our parts fit into the grand scheme of synthetic biology. The graph below shows the results from this experiment.

While our parts expressed well, they did not have a higher expression than the CMV. This was not particularly surprising. But it did raise the question, would we ever be able to show higher activity with a minimal CMV in our system than with a full CMV?

Our next step was to expand to a functioning analog library...

Phase 2 Results

With our parts of our project well characterized, we began experimenting with achieving a variety of consistent expression levels with our dCAS9-VPR activator. Our first strategy was to attempt multimerizing the target operators, placing two and then three operators up stream of the gene of interest using two of our operators. During this experiment, we also tested the effect of spacing the target operators with a different number of intervening base pairs. We tested with 0, 3, 6, 12, and 24 intervening base pairs. The results of these experiments yielded a predictable, linear correlation between the number of operators and the expression level and the number of base pairs in the intervening sequences. The results can be found below:

There were some anomalies within this data set. The target operators with 0 and 12 base pair spacing had lower expression than expected. One explanation is that when the operators are at 0 and 12 base pairs apart, they are on the same side of the DNA helix and crowd each other out. Once we eliminated these results, were able to create a smooth progression in expression level across several levels of expression, as seen below.

Finally, we realized that while we could achieve a wide range of high expression levels, it is sometimes favorable to lower expression levels below the standard single operator expression level. In order to accomplish this, we mutated our operators, replacing a base pair at a time. We tested 20 versions of 2 of our operator plasmids. Each version had one of the twenty base pairs in the twenty base pair operator mutated, so that there was a single mismatch between the operator and the guide RNA attached to the dCAS9-VPR complex. By changing the location of the single mismatch, we were able to create another smooth curve of changing expression levels, this time lowering expression levels. The expression levels can be found in the graph below.

Phase 3 Results

Now that we could achieve a variety of expression levels, we began integrating our system into recombination based circuitry. The circuit we used could express a different gRNA for every combination of drugs introduced. Before integrating our multimerized plasmids, we first tested the circuit by making each gRNA correspond to a different fluorescent reporter. The data for this experiment can be found below. There were four possible combinations of two drugs, and each combination yielded a relatively high expression of its corresponding fluorescent gene.

Results

Results