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

Int J Mol Sci. 2023 Feb 8;24(4):3417. doi: 10.3390/ijms24043417.


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.

Keywords: CRISPR interference; TMEM97; age-related macular degeneration; retinal degeneration; retinal pigmented epithelium.

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Epithelium / metabolism
  • Genome-Wide Association Study*
  • Humans
  • Macular Degeneration* / metabolism
  • Oxidative Stress
  • Retinal Pigment Epithelium / metabolism