A CRISPR Activation Screen Reveals a Cilia Disassembly Pathway Mutated in Focal Cortical Dysplasia
Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Breslow, David
Abstract
Primary cilia are assembled by an array of proteins whose disruption underlies ciliopathies. However, the critical process of cilium disassembly, its regulation during cell growth and differentiation, and how it contributes to disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of cilia biogenesis, we outline a pathway of novel factors that enact cilia disassembly. Genes include the neurodegeneration-linked NADase SARM1, Ryanodine Receptor calcium channels, and RhoA, which we place in a functional pathway for cilia disassembly. We demonstrate that this pathway is activated in response to mitogenic signaling and a top screen hit, the GPCR F2r. Pathway hyperactivation induces cilia loss, while its pharmacologic inhibition blocks cilia disassembly and promotes ectopic ciliation under non-permissive conditions. Strikingly, several pathway components are somatically mutated in focal cortical dysplasia (FCD), a neuronal disorder characterized by disorganization and dysfunction of cortical neurons, leading to intractable epilepsy. Consistent with aberrant cilia disassembly as a prevalent pathomechanism in this neurological disease, we demonstrate that FCD-associated SARM1 and RhoA patient mutations induce cilia loss. Intriguingly, Sarm1 inhibition restores cilia in cells with FCD-associated mutations, raising the prospect of a potential therapeutic strategy. Collectively, this work identifies a novel pathway for regulated cilia disassembly, implicates aberrant activation of this pathway as the basis of a new class of ciliopathy, and demonstrates the power of CRISPRa screening to provide new insights into cell biology and into diseases caused by somatic, activating mutations.
Recommended Citation
Elliott, Shane, "A CRISPR Activation Screen Reveals a Cilia Disassembly Pathway Mutated in Focal Cortical Dysplasia" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1194.
https://elischolar.library.yale.edu/gsas_dissertations/1194
