A/Prof Raymond Wong's team has developed a CRISPRi system to knockdown genes in human retinal pigmented epithelial cells. Using this system, A/Prof Wong found the gene TMEM97 play a role in regulating oxidative stress – a part of ageing in the macula. The findings provide a deeper understanding of the underlying causes of AMD and help prioritise new gene targets for treatments.
Development of a CRISPRi Human Retinal Pigmented Epithelium Model for Functional Study of Age-Related Macular Degeneration Genes
Jiang-Hui Wang, Daniel Urrutia-Cabrera, Jarmon G Lees, Santiago Mesa Mora, Tu Nguyen, Sandy S C Hung, Alex W Hewitt, Shiang Y Lim, Thomas L Edwards, Raymond C B Wong
International Journal of Molecular Sciences, 2023 Feb 8;24(4):3417.
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 TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 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 TMEM97 in RPE and supports a potential role of TMEM97 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.
Read the publication here.