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Comprehensive profiling of transcription factors for reprogramming human astrocytes to neuronal cells through endogenous CRISPR-based gene activation

Reisman, S. J.; Halabi, D.; Miller, S. E.; Song, L.; Geraghty, S.; Sangvai, N.; Rice, G.; Safi, A.; Crawford, G. E.; Gersbach, C. A.

2025-10-12 cell biology
10.1101/2025.10.11.681828 bioRxiv
Show abstract

Neuronal loss is a hallmark of neurodegeneration and brain injury. Direct reprogramming of astrocytes into neurons has emerged as a promising approach to restore lost neurons. Comprehensive mapping and characterization of candidate astrocyte-to-neuron reprogramming factors is an essential step to realizing the potential of this strategy. Here, we established a CRISPR activation (CRISPRa)-based approach for neuronal reprogramming of primary human astrocytes. We conducted high-throughput CRISPRa screens of all human genes encoding transcription factors (TFs) to identify novel and efficient reprogramming factors. scRNA-seq characterization of top hits revealed that single TFs reprogram primary human astrocytes into multiple neuronal subtypes with distinct cell type-specific gene signatures. We demonstrate that INSM1 reprograms astrocytes to a glutamatergic neuron-like state and has broad neurogenic activity across different cell types and across human and mouse contexts. Finally, we conduct paired CRISPRa screens to identify cofactors that cooperate with INSM1 to enhance neuronal reprogramming and subtype specification, and elucidate genomic mechanisms of interaction and downstream regulators.

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