Correction of the exon 2 duplication in DMD myoblasts by a single CRISPR/Cas9 system. Homo sapiens

Exonic duplications account for 10-15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a novel CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one gRNA directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications independently from the duplication extension. Overall design: CGH analysis of the Dystrophin gene in eight DMD patients with exon 2 duplications

外显子重复占杜氏肌营养不良（Duchenne muscular dystrophy, DMD）——一种严重的遗传性神经肌肉疾病——全部致病突变的10%~15%。本研究报道了一种基于成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR）/Cas9的新型策略，通过靶向缺失技术校正DMD基因中最常见的2号外显子重复，并在患者来源的肌源性细胞中验证了该方法的编辑效能。研究证实，在无需筛选的前提下，经基因组编辑的成肌细胞分化得到的肌管中，野生型肌营养不良蛋白（dystrophin）的转录与蛋白表达均得以恢复。仅需一条靶向内含子重复区域的向导RNA（gRNA）即可完成重复外显子的移除，此举不仅提升了编辑效率，同时降低了脱靶效应的发生风险。本研究为携带致病重复突变的患者开辟了全新的治疗视角，且该策略不受重复序列延伸范围的限制。整体实验设计：对8例携带2号外显子重复的DMD患者的肌营养不良蛋白基因开展比较基因组杂交（Comparative Genomic Hybridization, CGH）分析。