Abstract
Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.
Highlights
Duchenne muscular dystrophy (DMD) is one of the most severe muscle diseases affecting ~1 in 5000 boys and causing premature death
For proof-of-concept, we chose to develop an assay to screen for sgRNAs restoring dystrophin expression for a DMD exon-52 deletion
This specific deletion was selected because a humanized mouse model, del52hDMD/mdx, carrying copies of human DMD with a deletion of exon 52 in an mdx background is available [22]
Summary
Duchenne muscular dystrophy (DMD) is one of the most severe muscle diseases affecting ~1 in 5000 boys and causing premature death. It is caused by mutations in the DMD gene encoding the protein dystrophin, which has over 3600 amino acid residues. Dystrophin links the cytoskeleton actin to the transmembrane dystroglycan complex to maintain the integrity of the membrane of the contracting muscle fibers [1]. Dystrophin deficiency causes membrane fragility and muscle degeneration. Cardiomyocytes and diaphragm muscles are all affected, patients progressively lose muscle tissue and function, and patients mostly die from respiratory failure, cardiomyopathy, and heart failure in the 2nd-4th decade of life.
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