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Development and applications of CRISPR-based functional genomics platforms in human iPSC-derived neurons


CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells such as neurons. Neurons possess unique cell biological properties that enable them to exert highly specialized physiological functions. To enable systematic characterization of neuronal cell biology, we established CRISPR interference (CRISPRi)- and CRISPR activation (CRISPRa)-based platforms in human neurons derived from induced pluripotent stem cells (iPSCs) that allow robust repression or activation of endogenous genes and large-scale genetic screens in human neurons. Using this toolkit, we performed multiple genome-wide screens to identity genes controlling neuronal survival under unstressed and oxidative stress conditions and genes regulating neuronal redox homeostasis. These screens provide numerous novel biological insights. We also demonstrate that our platforms can be coupled with single-cell RNA sequencing and longitudinal high-content imaging to reveal consequences of genetic perturbations on gene expression and neuronal morphology respectively. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

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