Using mouse embryonic stem cells and established protocols I was able to derive a near pure population (>~90%) of somatic spinal motor neurons. These were then subjected to the FIREWACh procedure to identify more than a thousand regulatory elements active within the neuronal population. Gene ontology analysis of these elements found that they resided near genes important to neuronal development. This is consistent with the notion that active regulatory elements govern gene expression important to cell type specific functions, for example in this case synapse formation, rather than processes important to all cells such as RNA synthesis pathways. It further provides evidence that FIREWACh is applicable to a variety of cell types. A list of the genomic location of these elements will be uploaded to the Gene Expression Omnibus and made publically available.
The second aim of this proposal was to adapt FIRE-WACh into an approach that could be used to quantify the activity of CRMs. The strategy I adopted involved two major modifications: firstly, rather than use lentiviral vectors to deliver the reporter construct as used in FIRE-WACh I aimed to use a transgenic approach. This new approach took advantage of the rapid advances in CRISPR technology to enable homologous recombination to specifically target CRM reporters to a specific location within the genome. Here, as with any new technology, there were significant challenges. Firstly, the efficiency of homologous recombination I achieved was
considerably less than that that had been reported in the literature. However using several different approaches I was able to optimize first the design of the guide RNA which directs the CRISPR molecular machinery to the correct genomic location. Secondly I optimized the conditions that allow homologous recombination by using small synthetic molecules that inhibit non-homologous end repair and promote homologous recombination.
Unfortunately,even under optimized conditions the efficiency of the method is insufficient to provide the genome wide coverage initially hoped for. Luckily rapid advances in the field of functional genomics have provided alternate approaches such as STARR-seq that can be used in lieu of the adapted FIREWACh approach. These experiments therefore have revealed new challenges in the development of functional genomic approaches that require future study.
Lastly, the development of the stem cell based model of RTT was largely successful. Here, I utilized the ability of embryonic stem cells to develop in functional neurons. First, using CRISPR again, I generated mouse embryonic stem cells that carry a deletion of the MeCP2 allele. These cells were then differentiated to neurons that phenocopy neurons of RTT patients. That is, the neurons generated from the stem cells lacked extensive dendritic arborization, had smaller cell bodies (somas), and had lower electrical activity. Three key characteristics found in the neurons of RTT patients. However, due to the limitations of my ROQ-WACh approach I was unable to quantify the impact of MeCP2 mutations on the activity of CRMs in these cells.
