A high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons is established and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.
Abstract
While advances in omics profiling rapidly expand the catalog of genes associated with brain activity in health and disease, functional annotation lags far behind. Here, we establish a high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons. By converting cumulative neuronal activity into a stable, flow cytometry-readable signal, this approach enables systematic interrogation through pooled screening. Using a focused library of memory-associated genes, we recover known regulators and identify TMEM50A, a previously uncharacterized protein, as an essential regulator of neuronal activity. TMEM50A forms a complex with LEPROTL1 and associates with ESCRT-III machinery on multivesicular bodies (MVBs). TMEM50A loss impairs MVBs function, remodels the neuronal surface proteome, reduces synapse density, and alters behavior in mice. This platform enables systematic discovery of neuronal activity regulators and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.
Current brain atlases are largely descriptive, cataloging correlative molecular snapshots such as gene expression signatures yet offering limited functional insight. Here, we develop a scalable, cell-type-resolved in vivo CRISPR interference (CRISPRi) platform enabling systematic gene function profiling in the mouse brain. Through genome-wide screens across four neuronal populations at three time points spanning youth to aging, we identify neuronal essential genes missed in vitro and define a consensus set of 269 neuronal core essential genes. The data reveal cell-type-specific genetic vulnerabilities, including divergent dependencies validated for exosome component 9 (Exosc9) and osteopetrosis-associated transmembrane protein 1 (Ostm1) between excitatory and inhibitory neurons. We uncover aging-specific dependencies enriched in mitochondrial and translational pathways, aligning with transcriptional changes in the aging human brain. Finally, we establish the CRISPRinvivo data portal as a community resource for in vivo screening. Altogether, this work provides a broadly applicable platform for in vivo functional genomics and a framework for building comprehensive gene-function brain atlases.
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