Functional study of the AMD-associated gene TMEM97 in retinal pigmented epithelium using CRISPR interference

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 >65 genetic risk factors for AMD, including the locus. encodes the Sigma-2 receptor which is involved in apoptosis and cytotoxicity across a range of neurodegenerative diseases. However, the expression pattern of in the human retina and its functional role in retinal cells has remained elusive. Here we utilised CRISPR interference (CRISPRi) to investigate the functional role of in the retina. Transcriptome analysis of all major cell types within the human retina showed that is expressed in the RPE, retinal ganglion cells (RGCs) and amacrine cells. Using CRISPRi, we performed loss-of-function study of in the human RPE cell line, ARPE19. We generated a stable ARPE19 cell line expressing dCas9-KRAB which facilitated knockdown of using specific sgRNAs. Our results show that knockdown of in ARPE19 exerts a protective effect against oxidative stress-induced cell death. This work provides the first functional study of in RPE and supports the role of in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi cell line generated here provided an useful tool for functional studies of other AMD-associated genes.