Comprehensive CRISPR Editing of iPSCs for Dynamic Disease Modeling in Early-Onset Alzheimer's Research

Early-onset Alzheimer's disease (EOAD) is a devastating neurodegenerative disorder characterized by cognitive decline beginning before age 65, primarily caused by mutations in PSEN1, PSEN2, or APP genes, and leading to death within 10-15 years of symptom onset. The PSEN1-E280A mutation represents one of the most extensively studied EOAD variants and causes predictable pathological changes decades before clinical symptoms emerge. Currently, no disease-modifying treatments exist for EOAD, and research progress is severely hampered by the lack of comprehensive human cellular models capable of monitoring multiple disease pathways simultaneously in live cells. While the genetic basis of EOAD is known, the complex interplay between amyloid processing, tau pathology, autophagy dysfunction, and mitochondrial impairment in human neurons remains poorly understood. We propose to develop comprehensive human cellular models of EOAD using our innovative ONE-HDR system, a CRISPR-based technology that enables both mutation insertions and multiplex endogenous protein tagging in induced pluripotent stem cells (iPSCs). We will generate isogenic iPSC lines carrying the PSEN1- E280A mutation while simultaneously tagging with fluorescent proteins up to four key endogenous proteins for real-time monitoring of tau dynamics, autophagy processes, mitochondrial integrity, and cellular morphology in live cells without using staining or immunofluorescence. These cells will be differentiated into neurons to create a physiologically relevant platform for studying phenotypic alterations in models with the PSEN1-E280A mutation and screening a small set of compounds through dual intervention strategies targeting both early differentiation defects and mature neuronal pathology. The successful completion of this project will provide: 1) validated human cellular models of PSEN1-E280A EOAD with multiplex pathway monitoring in live cells, 2) optimized protocols for high-content imaging screening in iPSC-derived neurons without staining or immunofluorescence, and 3) identification of tool compounds effective at different disease stages that can potentially normalize EOAD-related cellular dysfunction. The completion of this project will accelerate both our understanding of PSEN1 function in neuronal physiology and the development of potential therapeutics for EOAD patients, while establishing a transformative platform for studying other mutations implicated in EOAD or other neurodegenerative diseases.