RESUMEN
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.
Asunto(s)
Exones/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Sistemas de Lectura/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Distrofina/genética , Distrofina/metabolismo , Genes Reporteros/genética , Humanos , Mutación INDEL/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) affects besides muscle also the brain, resulting in memory and behavioral problems. The consequences of dystrophinopathy on gross macroscopic alterations are unclear. To elucidate the effect of full-length dystrophin expression on brain morphology, we used high-resolution post-mortem MRI in mouse models that either express 0% (mdx), 100% (BL10) or a low amount of full-length dystrophin (mdx-XistΔhs). While absence or low amounts of full-length dystrophin did not significantly affect whole brain volume and skull morphology, we found differences in volume of individual brain structures. The results are in line with observations in humans, where whole brain volume was found to be reduced only in patients lacking both full-length dystrophin and the shorter isoform Dp140.