A Massively Parallel CRISPR-Based Screening Platform for Modifiers of Neuronal Activity

Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens for neuronal activity, using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2. Using this screening method, we evaluated 1343 genes for their effect on excitability in human iPSC-derived neurons, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimer's disease not previously described to affect neuronal excitability. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal activity in health and disease. Human iPSCs were transduced with pooled sgRNA library lentivirus at a low MOI (10% transduction efficiency) to minimize multiple infections in which a cell receives more than one sgRNA. Then, iPSCs were selected with puromycin, differentiated into neurons via induction of NGN2 expression, and grown 21 days. Neurons were then dissociated with papain and 20k cells were loaded into a single well of a 10X Chromium GEM-X 3’ chip for GEM preparation. Single cell library preparation was conducted according to the protocol provided for Chromium GEM-X Single Cell 3’ Reagent Kit v4. Enrichment of sgRNA sequences was performed with three semi-nested PCR reactions.