年龄相关性黄斑变性（AMD)是常见的与年龄相关不可逆致盲性眼病。全基因组关联分析（GWAS）发现ARMS2/HTRA1是AMD高危基因，作用机制尚不明确，而近期AMD药物临床研究对于疗效是否会因高危基因的差异而不同争议不断。我们前期研究发现这两个高危基因的iPS-RPE细胞经老化后SOD2表达、抗氧化能力等与野生型有差异，但因这两个基因紧密连接，常规基因编辑方法难以区别研究，而且野生型与高危型细胞其他基因背景信息不一致，干扰进一步通路分析。本项目我们将利用靶向基因修饰新技术CRISPR/case9，向野生型iPS细胞分别引入ARMS2以及HTRA1高危序列SNP，达到区别研究、排除背景信息干扰的目的，再与原细胞对比研究，全蛋白组学技术寻找高危基因特异差异蛋白，探索AMD致病机理；同时将基因编辑成功的iPS-RPE细胞进行视网膜下移植，验证CRISPR/case9技术的基因修饰安全性。

Age-related macular degeneration (AMD) is a major cause of retinal damage. Genome-wide association studies (GWASs) and linkage analyses have provided strong risk factors for AMD. The two SNP are tightly linked and lie in the neighboring HTRA1 and ARMS2 genes. How these mutations might cause sight to deteriorate is unclear, the underlying molecular mechanisms of AMD are unknown. Recently, our unbiased proteome analysis suggested the antioxidant mechanisms of super oxide dismutase (SOD) are perturbed in affected cells and that, over time, this introduces reactive oxidative species (ROS) mediated cellular insults that eventually manifest as AMD. If ROS metabolism is indeed disrupted in AMD, then we might finally begin to address the causes of the disease. We developed a protocol that recapitulates aging in these patient-stem-cell-derived retinal cells by treated with A2E and blue light. We can manipulate the stem cell by using genome Gene-editing technology-CRISPR/Cas9, targeting disease-associated SNPs, to determine the individual contributions of each. Use the CRISPR/Cas9 system to convert HTRA1 and ARMS2 alleles from low-risk to high risk in patient-derived stem cells. Determine the individual contribution of human HTRA1 and ARMS2 alleles to AMD pathogenesis. Test whether CRISPR conversion from low to high-risk AMD alleles affects ROS levels in cells. Test the function of gene-edited patient-stem-cell-derived RPE by transplantation into mice. By applying these powerful methodologies, we believe we can finally identify the root causes of AMD and so begin to develop new therapies.