Development of a CRISPRi Human Retinal Pigmented Epithelium Model for Functional Study of Age-Related Macular Degeneration Genes

Abstract

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected <i>TMEM97</i> as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of <i>TMEM97</i> in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of <i>TMEM97</i> in RPE and supports a potential role of <i>TMEM97</i> in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes.