Bassuk Alexander G, Zheng Andrew, Li Yao, Tsang Stephen H, Mahajan Vinit B
Department of Pediatrics, University of Iowa, Iowa City, IA, USA.
The Bernard &Shirlee Brown Glaucoma Laboratory, Departments of Ophthalmology, Pathology &Cell Biology, Institute of Human Nutrition, College of Physicians &Surgeons, Columbia University, New York, NY, USA.
Sci Rep. 2016 Jan 27;6:19969. doi: 10.1038/srep19969.
Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient's c.3070G > T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene's repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease.
从患者成纤维细胞产生的诱导多能干细胞（iPSC）有可能用作视网膜疾病自体移植细胞的来源。然而，患者来源的iPSC仍会携带致病突变。为了产生健康的患者来源细胞，可以使用基于成簇规律间隔短回文重复序列（CRISPR）/Cas9细菌系统的新基因编辑技术来修复突变，从而产生无需患者免疫抑制的移植物。我们测试了CRISPR/Cas9是否可用于患者特异性iPSC，以精确修复导致X连锁视网膜色素变性（XLRP）的RPGR点突变。从一名XLRP患者的皮肤穿刺活检培养的成纤维细胞被转导以产生携带患者c.3070G>T突变的iPSC。用CRISPR引导RNA、Cas9核酸内切酶和供体同源模板转导iPSC。尽管该基因具有重复且富含GC的序列，但13%的RPGR基因拷贝显示突变得到校正并转化为野生型等位基因。这是首次使用CRISPR纠正来自患有光感受器变性患者的iPSC中的致病突变的报告。这一重要的概念验证发现支持了基于个性化iPSC的视网膜疾病移植疗法的发展。
