Unveil the functional role of RNA spatial localization in neuronal function and neurodegeneration by developing novel CRISPR-TO tools

RNA molecules are essential for neuronal function, playing a key role in coordinating gene expression across different cellular compartments. Various RNAs, including messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA), respond rapidly to environmental cues and synaptic activity, supporting critical processes such as axonal growth, synaptic plasticity, and long-term memory formation. The spatial organization of RNAs within neurons enables localized protein synthesis at synapses, spanning distances from millimeters to meters, and is vital for nerve repair. Moreover, dysregulation of RNA-binding proteins, such as TDP-43, has been linked to various neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s Disease. Since these proteins are involved in alternative splicing, polyadenylation, transcription activation, translation, and RNA spatial localization4, it suggests a strong pathological link between RNA regulation and disease progression. Despite progress made in understanding other functional aspects of RNA-binding protein dysfunction (e.g., cryptic splicing), however, the functional role of spatial RNA localization in disease development remains largely underexplored, due to the lack of efficient tools for manipulating and perturbing endogenous RNA spatial localization to infer its causal physiological or pathological function. To investigate how RNA spatial mechanisms govern neuronal functions, my colleagues and I have recently developed a novel technology termed CRISPR-mediated transcriptome organization (CRISPR-TO) to perturb endogenous RNA spatial localization in neurons. I propose to use this novel CRISPR-TO tool to enable programmable control of endogenous RNA localization both in vitro and in vivo to study and create future treatment for neurodegenerative diseases. CRISPR-TO will first be applied in vitro using human induced pluripotent stem cell (hiPSC)-derived neurons from healthy individuals and neurodegenerative disease patients to identify RNA localization targets (Aim 1-2). These findings will then be translated in vivo via AAV delivery of CRISPR-TO to test how RNA spatial regulation influences disease progression and functional outcomes in preclinical models (Aim 3). If successful, this study can transform how we investigate RNA mislocalization in neurodegenerative diseases, uncovering novel mechanisms that contribute to disease progression and neuron regeneration. By enabling scalable, programmable control of endogenous RNA localization, we could pave the way for innovative spatial RNA therapeutic strategies, with implications far beyond ALS, extending to other neurological and systemic diseases where RNA localization plays a critical role. Project Number: 1F31NS147860-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Maylin Fu | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F03C-F (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11318023