UC Davis

The rapid development of genome editing technologies in the last couple of decades has opened the door for the creation of novel therapeutics and cures for genetic disorders that were once thought to be untreatable. In this dissertation, we explore the potential to develop these tools for the treatment of three genetic disorders, Neurofibromatosis type 1 (NF1), Syngap1, and 22q11.2 deletion syndrome (22q11.2 DS). Although these disorders are very different from one another in terms of gene targets and patient presentation, they share a common theme: they are all a type of haploinsufficiency where an epigenetic gene therapy may provide immense benefit to patients.Epigenetic editing using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) involves modifying the chemical modifications that control gene expression without altering the underlying DNA sequence. CRISPR is a powerful tool for editing the genome, which involves using Cas proteins and their ability to complex with RNA guides to localize to specific regions of the genome. The CRISPR system has been adapted to target epigenetic marks using modified versions of the Cas proteins and fusing it to an epigenetic enzyme that adds or removes chemical marks on DNA or histones, such as DNA methyltransferase or histone deacetylase, respectively. This modified Cas9 protein can be directed to specific regions of the genome using guide RNAs that are designed to target specific sequences. Alternatively, CRISPR can also be used to recruit proteins that activate or repress gene expression to specific regions of the genome. This involves fusing a modified Cas9 protein to a transcriptional activator or repressor protein respectively, which can then be targeted to specific regions of the genome using guide RNAs. Overall, epigenetic editing using CRISPR provides a powerful tool for understanding the role of epigenetic modifications in gene regulation and has potential therapeutic applications in treating diseases that involve aberrant epigenetic marks. In summary, for NF1, we utilized a duplex CRISPR repression system to downregulate disease specific genes, and for Syngap1 and 22q11.2 deletion syndrome, we utilized a CRISPR activation system to upregulate disease specific genes.