In order to make genes activate in response to certain signals, our system first needed a method to activate genes in general. We chose CRISPR/dCAS9-VPR as an activator. We chose dCAS9 due to its ease of use and its ability to target specific DNA sequences. dCAS9-VPR targets specific sequences by binding to specialized RNA. Part of this RNA (gRNA) contains 20 base pairs that will act as a guide, guiding the dCAS9 to the complimentary 20 base pairs found upstream of a gene one wishes target. This can be seen in the info-graphic below:
Once we were able to activate genes, we then expanded our system to activate genes to different levels, thereby achieving the graded analog expression level that we desired from our system. To accomplish this, we multermerized the 20 base pair target sequence, placing multiple copies of the target sequence upstream of the gene. By varying the number of copies, we were able to create a gradient of expression. The more target sequences we added, the more the gene was activated. This is illustrated in the image below:
Finally, once we completed phase one and two, we expanded our system once again. Using recombinase based circuits, we were able to control which gRNA was produced. gRNA 1 corresponded to a reporter with one target sequence, and gRNA 2 corresponded to the same gene but with two of the target sequence. Releasing gRNA 1 turned the gene on to a small degree, and gRNA two turned it on to a large degree. We then incorporated two more gRNA's flanked by two more recombinase recognition sites, allowing the system to have a smoother, four point analog expression level increase. Since the recombinase circuits that release the different gRNA is completely digital, (the recombinases are activated by the digital prescience or absence of a signal such as a hormone and once activated, are extremely efficient) the system was a merger of digital signals giving rise to different levels of analog gene expression, as stated in our goal. A diagram of these circuits can be found below:
