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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83658 | GEO | 2017/06/23

SECONDARY ACCESSION(S): PRJNA326658

REPOSITORIES: GEO

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Correction of Exon 2, Exon 2-9 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

Project description:Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2 to 9, and exons 8 to 9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the corrected myotubes indicated rescue of dystrophin related molecular pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. This resulted in the restoration of dystrophin expression and reversal of transcriptome pathological signatures in the corrected patient-derived cells. Our study contributes valuable insights into the safety and efficacy about the application of the single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

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Fine mapping of a de novo interstitial 10q22-q23 duplication in a patient with congenital heart disease and microcephaly

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Mouse Segmental Duplication and Copy-Number Variation

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Correction of Exon 2, Exon 2-9 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

Project description:Correction of Exon 2, Exon 2-9 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

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Mouse Segmental Duplication and Copy-Number Variation

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Transcriptome analysis of the TA muscles from WT and Dmd Exon 51 Knockout mice

Project description:Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other associated cell types. Here, we describe the creation of a new mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with the nuclei of myofibers, as well as other mononucleated cell types that contribute to DMD disease pathogenesis, we performed single nucleus transcriptomics of skeletal muscle of mice with exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide new insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

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Correction of the Exon 2, Exon 2-8 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

Project description:Correction of the Exon 2, Exon 2-8 and Exons 8-9 duplications in DMD patient myogenic cells by a Single CRISPR/Cas9 system

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Epigenetic analysis of the DMD locus

Project description:The Dystrophin gene (DMD) is the largest gene in the human genome, mapping on Xp21, spanning 2.2Mb and accounting for approximately 1% of the entire human genome. Mutations in this gene cause Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 hours, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA pol II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. More importantly, epigenetic marks highlighted the existence of four novel cis-DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

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