Abstract
Purpose :
CRX variants that are associated with Leber congenital amaurosis (LCA7) have been shown to disrupt photoreceptor maturation and function. CRX plays a vast role in photoreceptor gene regulation and chromatin remodeling. Therefore, to better understand mutant CRX-mediated changes in photoreceptor cells, we performed a single cell multiome experiment utilizing an in vitro human LCA7 retinal organoid model.
Methods :
Retinal organoids were grown from four hiPSC lines: CRX+/+ (control), CRX+/T155ins4, CRX+/K88Q, CRX+/- (CRISPR-edited line). After 240 days in culture (D240), retinal organoids were collected and flash frozen (n=4 organoids per line). 8,000 nuclei per sample were isolated and used for joint single cell RNA sequencing and ATAC sequencing assays. Sequencing data were processed using Cell Ranger ARC and analyzed using Seurat and Signac packages. ChromVAR was used to score transcription factor binding motif enrichment in single cells.
Results :
Analysis of cell clusters between the control (CRX+/+) and both LCA7 lines (CRX+/T155ins4, CRX+/K88Q) reveals differences in the ratio of cell types, including mature and immature photoreceptor cells, as well as glia. In particular, the CRX+/T155ins4 line exhibits a drastic increase in the proportion of glial cells, with a large reduction in the proportion of mature rods and cones compared to control. The CRX+/- line, in which the entire K88Q allele has been excised, shows cell proportions most similar to those of the control line. Examination of expression profiles within photoreceptor clusters shows a decrease for key CRX target genes for both LCA7 lines, including NRL, NR2E3, RCVRN, ARR3, and RHO. These changes are partially ameliorated in the gene-edited hemizygous line (CRX+/-). Transcription factor (TF) binding motif enrichment analyses reveal a decrease in various motifs for the LCA7 lines, including CRX and OTX2 motifs. In contrast, the LCA7 lines exhibit enrichment of binding motifs for the STAT (cell growth and differentiation) and RFX (ciliogenesis) TF families.
Conclusions :
These data advance our understanding of how domain-specific CRX mutations in patients affect photoreceptor cells, which could help provide us with new therapeutic targets for LCA7.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.