Wang Jiang-Hui, Urrutia-Cabrera Daniel, Lees Jarmon G, Mesa Mora Santiago, Nguyen Tu, Hung Sandy S C, Hewitt Alex W, Lim Shiang Y, Edwards Thomas L, Wong Raymond C B
Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, VIC 3002, Australia.
Ophthalmology, Department of Surgery, University of Melbourne, Melbourne, VIC 3010, Australia.
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 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of 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 in RPE and supports a potential role of 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.
年龄相关性黄斑变性（AMD）是一种致盲性疾病，其特征在于视网膜色素上皮（RPE）功能障碍，最终导致神经感觉视网膜的破坏或丧失。全基因组关联研究已经确定了超过60个AMD的遗传风险因素；然而，这些基因中的许多基因在人类RPE中的表达谱和功能作用仍然不清楚。为了促进对AMD相关基因的功能研究，我们通过生成表达dCas9-KRAB的稳定ARPE19细胞系，开发了一种具有整合CRISPR干扰（CRISPRi）用于基因抑制的人类RPE模型。我们对人类视网膜进行了转录组分析，以对AMD相关基因进行优先级排序，并选择 作为敲低研究的候选基因。使用特异性sgRNA，我们表明在ARPE19中敲低 可降低活性氧（ROS）水平，并对氧化应激诱导的细胞死亡发挥保护作用。这项工作首次对RPE中的 进行了功能研究，并支持了 在AMD病理生物学中的潜在作用。我们的研究突出了使用CRISPRi研究AMD遗传学的潜力，并且这里生成的CRISPRi RPE平台为AMD相关基因的功能研究提供了一种有用的体外工具。
