Research ArticleMuscular Dystrophy

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

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Science Translational Medicine  29 Nov 2017:
Vol. 9, Issue 418, eaan8081
DOI: 10.1126/scitranslmed.aan8081

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Making the cut

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a fatal childhood muscle disease. To optimize the correction of DMD mutations by CRISPR/Cas9 gene editing, Amoasii et al. first generated mice that had exon 50 deleted, a common human mutational “hotspot” region of the dystrophin gene. The authors then reported a method in which a single cut in genomic DNA encoding dystrophin with CRISPR/Cas9 in these engineered mice resulted in up to 90% restoration of dystrophin expression in mouse skeletal and heart muscles. This method of permanently bypassing DMD mutations using a single genomic cut suggests that this strategy may have potential for efficiently correcting DMD mutations.

Abstract

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.

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