High Throughput Combinatorial CRISPRa Screening to Identify Regulators of Neuronal Maturation

Abstract

Neurons derived from induced pluripotent stem cells (iPSCs) are powerful tools for modeling neurological diseases, but current methods often produce cells with an immature, fetal-like phenotype, limiting their relevance. Here, we developed a high-throughput CRISPR activation (CRISPRa) screening platform to systematically identify transcription factor (TF) combinations that synergistically enhance neuronal maturation. Using a SYN1 promoter-driven fluorescent reporter to enrich for mature neurons, we screened a library of 1,920 TFs and epigenetic modifiers paired with the neurogenic factor NEUROG3 (NGN3). The screen identified multiple novel TF combinations that significantly improved neuronal differentiation. We focused on a top hit, the epigenetic modifier KMT2B, and demonstrated that co-activation of KMT2B and NGN3 generates neurons with significantly enhanced transcriptional, morphological, and functional maturity compared to NGN3 alone. KMT2B+NGN3-derived neurons exhibited gene expression profiles more closely correlated with adult human brain tissue and showed enrichment for GWAS risk genes associated with major psychiatric disorders. These neurons also displayed longer neurites, higher synaptic puncta density, and more robust, coordinated network activity on multi-electrode arrays. Mechanistically, ATAC-seq revealed that KMT2B+NGN3 co-activation remodels the chromatin landscape to increase accessibility at loci critical for axonal guidance and synaptic plasticity while suppressing pathways associated with pluripotency. This work establishes a scalable platform for dissecting the combinatorial logic of neuronal maturation and provides an optimized protocol for rapidly generating more physiologically relevant human neurons for in vitro disease modeling.