Epigenome Editing for Reprogramming Neuronal Cell Fate Specification

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The cell-type diversity in a multicellular organism is precisely defined through epigenetic regulation of an underlying DNA sequence. However, cell fate can be reprogrammed artificially with the proper extrinsic and intrinsic cues. This ability to manipulate cell-type specification has revolutionized the fields of regenerative medicine and disease modeling that aim to program cell phenotypes to better understand and provide therapy for human disease. Strategies for cell reprogramming commonly involve the overexpression of naturally occurring transcription factors to instruct the transcriptional codes of a new cell identity. However, this approach often results in a low efficiency of conversion with poor maturation and limited functional integration in vivo. Thus, our objective was to utilize tools in synthetic biology based on the CRISPR/Cas9 system to more precisely and accurately control endogenous gene expression towards applications in cell reprogramming. We first applied CRISPR/Cas9- based transcription factors to convert fibroblasts to neurons. This strategy entailed activating endogenous proneural genes within fibroblasts, rewriting the epigenetic signatures at the target loci and enabling stable autonomous expression of the target genes. Next, we systematically profiled the proneural activity of every human transcription factor with CRISPR activation pooled gRNA screens. Through these unbiased screens, we uncovered transcription factor combinations that increase conversion efficiency, influence subtype specification and improve synaptic maturation of in vitro-derived neurons. Finally, we applied the same CRISPR-based transcription factors to study mechanisms of gene regulation at the 15q11-13 imprinted locus implicated in the development of Prader-Will syndrome (PWS), a genetic neurobehavioral disorder. Through this work, we identified allele-specific regulatory elements that serve as candidate target sites for epigenetic therapy in PWS. Overall, we've developed a novel approach using tools in synthetic biology to improve the specification of neuronal cell types and to elucidate gene regulatory mechanisms in a neurobehavioral disorder.





Black, Joshua Benjamin (2019). Epigenome Editing for Reprogramming Neuronal Cell Fate Specification. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/19830.


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