• Raymond Wong

New preprint: Using CRISPR to study genes contributing to age-related macular degeneration

Updated: Aug 25

Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells

Tu Nguyen, Daniel Urrutia-Cabrera, Luozixian Wang, Jarmon G. Lees, Jiang-Hui Wang, Sandy S.C. Hung, Alex W. Hewitt, Thomas L Edwards, Shiang Y. Lim, Chi Luu, Robyn Guymer, Raymond C.B. Wong



Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that they retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 causes a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.

Read the paper here.