Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

Duchenne肌营养不良症（DMD）是由肌营养不良蛋白基因突变引起的一种严重的进行性肌肉疾病。大多数DMD突变是过早终止肌营养不良蛋白的缺失。肌营养不良蛋白基因第50外显子的缺失是引起DMD的最常见的单个外显子缺失。可以通过跳过外显子51来纠正此类突变，从而恢复抗肌萎缩蛋白阅读框。使用聚簇的规则间隔的短回文重复序列/ CRISPR关联的9（CRISPR / Cas9），我们通过删除外显子50生成了DMD小鼠模型。这些ΔEx50小鼠表现出严重的肌肉功能障碍，可通过全身递送编码CRISPR的腺相关病毒来纠正/ Cas9基因组编辑组件。我们优化了肌营养不良蛋白阅读框校正的方法，该方法使用了单个引导RNA，可产生重构突变并允许外显子51跳过。结合Cas9的肌肉特异性表达，这种方法最多可恢复整个骨骼肌和肌肉中肌营养不良蛋白蛋白表达的90％。 ΔEx50小鼠的心脏。使用基因组DNA的单次切割永久绕过DMD突变的这种方法代表了迈向临床校正DMD突变以及其他潜在神经肌肉疾病突变的一步。