Using genome editing of induced pluripotent stem cells to investigate the genetics of neuronal network function.

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Neural differentiation and the formation of neuronal networks is a finely balanced process essential for cognitive function that relies on stringent regulation of gene expression. This project will combine the powerful new tool of genome editing by Cripsr/Cas9 with the ability of human induced pluripotent stem (iPS) cells to differentiate to neurons to investigate the role of gene expression in neuronal differentiation. Mutations causally linked to brain cancer and to neurodevelopmental disorders in genes that control transcription, RNA processing and protein synthesis, demonstrate the importance of the regulation of gene expression in the nervous system. Defects caused by mutation of gene expression regulators are normally complex and reflect the roles of the target genes controlled by the regulators. We have recently identified critical regulators of protein synthesis and target genes which are involved in synapse activity and the formation of the neuronal cytoskeleton, processes essential for the normal function of neuronal networks [1-3]. The project will examine the role of these genes in neuronal differentiation. The genes will be disrupted in human iPS cells using Crispr/Cas9 technology. iPS cells can be differentiated to pluripotent neural progenitor cells and further to specific neuron types able to form networks. By comparing neurons derived from isogenic cell lines that are identical except for the Crispr/Cas9 -induced mutation, this approach allows to identify the role of single genes. Network formation will be followed by analysing the development of neuronal structures and properties by a combination of high resolution microscopy, gene expression analysis by quantitative PCR, Western blotting and ribosome profiling, and the analysis of electrophysiological properties of the neurons. The application of these powerful new tools will provide fundamental insight into the genetics of neuronal network formation.The successful candidate will be trained in iPS cell culture and differentiation, genome editing using Crispr/Cas9 technology, the analysis of gene expression using quantitative PCR based approaches, ribosome profiling or Western blotting, and advanced microscopy techniques, and electrophysiology. Training will be provided by the participating supervisors in Aberdeen and Edinburgh as appropriate.