Human CRX Regulates Photoreceptor Cells Development via Bidirectional Transcriptional Control in Retinal Organoids

CRX (cone-rod homeobox) is a key regulator of retinal photoreceptor development, yet its human-specific functions remain poorly understood due to scarce human retinal tissues and significant species differences. Here, we established a human CRX-mCherry fluorescent reporter retinal organoid (RO) model to dissect CRX-mediated gene regulation. Using FACS, RNA-seq, and CUT&Tag-seq, we identified CRX target genes and revealed its dual regulatory role: it activates photoreceptor-specific genes (e.g., RP1L1, linked to inherited retinal degeneration) in a dose-dependent manner, while suppressing non-photoreceptor genes (e.g., PCDH8 and PROX1). Notably, we first generated the human CRX CUT&Tag dataset, providing direct insights into CRX's genome-wide regulatory landscape in photoreceptor cell development. These findings demonstrate that CRX functions as both a transcriptional activator and repressor, ensuring photoreceptor-specific gene expression and preventing aberrant cell fate transitions. Our study provides critical insights into the role of human CRX in retinal development and implications for retinal degenerative diseases. Overall design: We established a CRX-mCherry reporter fluorescent hiPSC line, which was subsequently differentiated into retinal organoids (ROs). On day 90 (D90), ROs were dissociated into single cells through enzymatic digestion. The cell suspension was then subjected to fluorescence-activated cell sorting (FACS) to isolate mCherry-positive and mCherry-negative cell populations. These two distinct populations were collected separately for RNA-seq analysis (designated as mN and mP). Furthermore, we performed ChIP-seq on D90 CRX-mCherry ROs to investigate CRX binding sites across the genome. Two experimental conditions were established: (1) a negative control group without CRX antibody (designated as -Ab), and (2) an experimental group with CRX antibody (designated as +Ab) to specifically capture CRX-associated chromatin regions.