Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Australia.
Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Australia.
CRISPR J. 2024 Apr;7(2):100-110. doi: 10.1089/crispr.2023.0065. Epub 2024 Apr 5.
Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.
遗传性视网膜疾病（IRDs）是一组由视网膜表达基因中的致病变异引起的致盲遗传疾病。在这项研究中，我们试图通过使用 CRISPR 激活（CRISPRa）在患者来源的成纤维细胞中诱导视网膜基因的表达，来开发一种快速评估 IRD 基因变异致病性的方法。我们证明了来自色素性视网膜炎患者的成纤维细胞中 的表达被 CRISPRa 诱导，从而能够研究与特定变异相关的致病机制。我们显示 c.4005 + 1G>A 变异导致 CRISPR 激活的成纤维细胞和来自同一 RP12 患者的视网膜类器官（RO）中第 11 外显子跳跃。c.652 + 5G>C 变异被证明增强了 CRISPR 激活的成纤维细胞中外显子 2 的跳跃，并在成纤维细胞和 RO 中差异影响 异构体的表达。我们的研究证明了一种在患者来源的成纤维细胞中进行 IRD 基因变异转录筛选的易于接近的平台，该平台可潜在地应用于任何基因变异的快速致病性评估。
