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.

The goal of phase 1 to establish a consistent method of gene activation through dCAS9-VPR. Using MIT's CRISPR optimization tool, we generated 20 sequences to be used as our gRNA and target site. The dCAS9-VPR complex, the target sequence and reporter gene, and the gRNA expression vectors were constructed and transfected into HEK293 cells. Another set of transfections took place simultaneously with the same materials minus the gRNA expression vector as a negative control. The fold increase between the basal level of expression from the control and the activated level of expression was then recorded. The results can be found below.

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