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Raymond Wong, Jiang Hui Wang, Daniel Urrutia-Cabrera, Jarmon Lees, Santiago Mesa Mora, Tu Nguyen, Sandy Shen-Chi Hung, Alex W Hewitt, Thomas Edwards; Generation of a CRISPRi RPE model to study age-related macular degeneration genetics. Invest. Ophthalmol. Vis. Sci. 2022;63(7):4607 – F0399.
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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. Development of a simple-to-use in vitro RPE model would help us study the complex genetics and pathogenesis of AMD.
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. To demonstrate the potential of this CRISPRi RPE system, we performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for further knockdown study. Following CRISPRi-mediated knockdown of TMEM97, we analysed cell viability and oxidative stress levels to determine its functional role in ARPE19.
Characterisation of CRISPRi ARPE19 show that the cell line retains stable expression of dCas9-KRAB for at least 2 months. Moreover, the CRISPRi ARPE19 cell line expresses RPE marker ZO-1 and retains the ability to form a polarised monolayer with hexagonal RPE morphology following nicotinamide treatment, supporting the quality of the derived cell line. Our results show that CRISPRi enables significant knockdown of TMEM97 in ARPE19 using specific sgRNAs. In particular, knockdown of TMEM97 in ARPE19 reduces reactive oxygen species (ROS) levels and exerts 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 demonstrates the potential for using CRISPRi to study AMD-associated genes in RPE, and the CRISPRi RPE platform generated here provide a useful in vitro tool for functional studies of AMD-associated genes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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