ABSTRACT Repeat expansion diseases are a group of over 40 severe neurological disorders that are caused by an abnormal expansion of trinucleotide or hexanucleotide repeat sequences. While there are several proposed mechanisms of repeat expansion disease pathogenesis, it is widely accepted that RNA-gain-of-function toxicity is the major contributor to pathogenesis in several repeat expansion diseases. RNA-gain-of-function toxicity is characterized by repeat expansion RNA sequestering of RNA-binding proteins (RBPs) and causing global dysregulation in splicing events and cellular toxicity. Repeat expansion RNA is also known to form nuclear, phase-separated aggregates of expansion RNA and RBPs, also known as RNA foci. However, the role of RNA foci in RNA-gain-of-function toxicity is poorly understood. This is due to the fact that the only existing tools for studying RNA-gain-of-function toxicity rely on expression of expansion repeats and correlation of RNA foci formation with RBP sequestration and cellular toxicity. Several studies using the aforementioned methods have shown that expression of long, foci-forming repeats elicits higher cellular toxicity compared to expression of short, non-foci forming, repeats. However, it is unclear whether cellular toxicity is dependent on the formation of RNA foci to reach toxic levels of RBP sequestration, or whether the increase RBP binding sites of longer repeats is sufficient to reach toxic levels of RBP sequestration and the formation of RNA foci is merely a byproduct. By comparing cellular toxicity and levels of protein sequestration in cells that form foci, to cells that do not form foci but harbor the same length repeat RNA, we can directly assess whether RNA foci formation is required for cellular toxicity in repeat expansion diseases. To do this, inducible RNA foci formation is required. Recently, I have developed an approach that allows, for the first time, for chemogenetic controlled RNA aggregation. This technology presents the opportunity to determine whether RNA foci formation is responsible for repeat expansion disease pathogenesis. In this proposal, I will optimize my previously developed inducible RNA aggregation system to improve its usability as a tool to study RNA foci in repeat expansion disease. Then, I will use this system to determine whether RNA foci formation is responsible for increased levels of RBP sequestration and cellular toxicity in the repeat expansion disease C9orf72-type amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). My findings will determine whether disruption of RNA foci will directly provide therapeutic benefit for C9orf72-type ALS/FTD and potentially other repeat expansion diseases. In addition, publication of the first ever tool for chemogenetic control of RNA aggregation will pioneer an entirely new approach for studying RNA aggregation events in molecular biology and disease.