Our approach is based on random genetic modification of hundreds of millions of target cells, such that expression of genes in the vicinity of these random mutations is induced. The law of large numbers thus results in the right, sought-after gene to be randomly induced in a very small subset of cells (Figure 1-p). These are identified by genetically encoded reporters, inducing fluorescence of a specific colour. These cells are selected, grown into clonal population and the mutations read and mapped onto the genome, thus identifying the correct gene.
We have successfully developed a bioinformatics analysis pipeline capable of comprehensive analysis of genome-wide insertional datasets produced by our screening methods. We demonstrated its use not only in our existing (control) dataset, but by deploying this custom method to a new screening library as well.
We have made significant progress in developing the necessary screening environment to successfully finish Objective 2. This required the building of a novel, upgraded mechanical stimulation device with improved throughput and higher reproducibility. To this end, we have designed a device based on the concept of an iris shutter, and molded custom PDMS dishes to fit this device. We have identified the most suitable cell line for this screening and are currently progressing with genome-wide screening.
Objective 3 was diverted to an alternative as delineated in our Risk Management Plan. This was due to limited availability of the compound, OH-alpha-Sanshool, due to limitations in shipment during the pandemic. We therefore conducted a screen to identify novel receptors for noxious cold. Our screen has clearly and unequivocally re-identified known temperature sensor molecules TRPM8 and TRPA1 in the cold temperature range. Importantly, our results indicate that at least one, but likely more novel cold receptors were also identified by our efforts. We are currently in the process of validating these targets and testing their physiological relevance. Overall, we have successfully demonstrated proof of concept for our novel platform technology by successfully identifying novel target molecules. A PCT patent application has been filed on the basis of this, as a first step to commercial exploitation of our results.
