Graduate Student University of Washington, United States
Abstract: Retinitis Pigmentosa (RP) and Macular Telangiectasia (MacTel) are inherited retinal disorders (IRDs) that cause vision loss. While these diseases have established genetic risks, >30% of cases cannot be explained by protein-coding variants alone. Cis-regulatory elements (CREs) are noncoding DNA sequences that regulate gene expression and mutations in CREs have been linked to several IRDs. However, regulatory variants are difficult to characterize because CREs can be cell-type specific, act at a distance, and are often not conserved across model systems. To address these challenges, human retinal organoids (ROs) derived from induced pluripotent stem cells (iPSCs) have emerged as a powerful model for studying CREs and retinal diseases. Here, we present two complementary single-cell approaches for multiplexed testing of CREs in ROs to assess changes in target gene expression and cell state. The first method pools CRE knockout ROs derived from CRISPR-edited iPSCs, each labeled with a unique probe barcode, and profiled by single-cell RNA sequencing (scRNA-seq). This approach enables characterization of different CREs in a parallelized and multiplexed manner, facilitating the analysis of up to 16 samples simultaneously. The second method involves electroporating CRISPR/Cas9-expressing plasmids into developing human ROs to target candidate CREs, followed by scRNA-seq of the pooled, electroporated organoid cells. The individual gRNAs serve as barcodes for de-multiplexing, allowing the identification of essential CREs linked to IRDs. Using these high-throughput techniques, we identified CREs essential for expression of the RP-associated gene NRL and the MacTel-associated gene TTC39B. Disrupting an enhancer of NRL significantly decreased the expression of rod-specific transcription factors and shifted cell fate from rod to cone, while disrupting an enhancer of TTC39B demonstrated a cell-type specific decrease in gene expression. These findings uncover novel CRE-mediated disease mechanisms in previously unexplored genomic regions. Together, these methods represent powerful tools for the functional interrogation of CREs in ROs, advancing our understanding of epigenetic regulation in retinal development and disease.
