A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.
A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.
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DOI：
10.1016/j.stem.2016.01.021
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发表时间：
2016-04-07
期刊：
影响因子：
23.9
通讯作者：
Pyle AD
中科院分区：
文献类型：
--
作者：
Young CS;Hicks MR;Ermolova NV;Nakano H;Jan M;Younesi S;Karumbayaram S;Kumagai-Cresse C;Wang D;Zack JA;Kohn DB;Nakano A;Nelson SF;Miceli MC;Spencer MJ;Pyle AD
DMD中的突变破坏阅读框架，阻止肌营养不良蛋白翻译，并引起杜氏肌营养不良症（DMD）。在这里，我们描述了一个适用于60% DMD患者突变的CRISPR/Cas9平台。我们将该平台应用于DMD衍生的hiPSC，其中高达725 kb的成功缺失和非同源末端连接重新构建了DMD基因。这是迄今为止在DMD中显示的最大的CRISPR/Cas9介导的缺失。使用hiPSC允许评估疾病相关细胞类型中的肌营养不良蛋白。来自重构的hiPSC克隆系的心肌细胞和骨骼肌肌管具有恢复的肌营养不良蛋白。内部缺失的肌营养不良蛋白是功能性的，如通过体外和体内肌营养不良蛋白糖蛋白复合物的改善的膜完整性和恢复所证明的。此外，miR 31在重构后减少，与贝克尔肌营养不良症中的观察结果相似。这项工作证明了使用单个CRISPR对纠正大多数DMD患者的阅读框架的可行性。
Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed for evaluation of dystrophin in disease relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.