Long-read sequencing for the assessment of therapeutic CRISPR/Cas9 strategy for the removal of aberrant DM1-causing CTG repeats in DMPK gene locus
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ABSTRACT:
PROVIDER: PRJNA1170263 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30910700_1.fastq.gz | Fastqsanger.gz | |||
| SRR30910696_1.fastq.gz | Fastqsanger.gz | |||
| SRR30910697_1.fastq.gz | Fastqsanger.gz | |||
| SRR30910698_1.fastq.gz | Fastqsanger.gz | |||
| SRR30910699_1.fastq.gz | Fastqsanger.gz |
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Project description:The genetic cause of Myotonic Dystrophy Type 1 (DM1) is the expansion of unstable CTG-repeats in the 3´ untranslated region of the DMPK gene. Therapeutic gene editing for treatment of monogenic diseases could in principle remove definitively the disease-causing genetic defect. Here we describe the application of the CRISPR/Cas9 strategy to remove the CTG-expansion in the DMPK gene in myogenic cells derived from a DM1 patient that is heterozygous for the expansion. The efficacy of on-target genomic events was assessed by performing long-read DNA sequencing (Oxford Nanopore Technologies) after applying Cas9-mediated target enrichment of the Regions Of Interest (ROI), enabling the simultaneous analysis (a) of cuts by both sgRNAs resulting in the effective excision of the CTG expansion, (b) of cuts induced only by a single sgRNA, (c) of the potential occurrence of aberrant events such as inversions, and (d) the existence of non-edited sequences, within the same sample.
2025-02-26 | GSE279054 | GEO
Project description:Bisulfite sequencing was performed on GM04604 (UN-B), GM06077 (DM1-A), and GM04648 (DM1-B) patient cells to determine the levels of CpG methylation present at two CTCF sites closely flanking the CTG repeats of DMPK.
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Project description:RNA-seq on proliferating myoblasts of the DM11 line carrying 13 and 2600 CTG repeats in the DMPK gene, compared to their genome-edited counterparts without both the 13 and 2600 CTG repeat or just lacking the 2600 CTG repeat.
2019-04-11 | GSE127296 | GEO
Project description:Myotonic dystrophy type 1 (DM1), the most frequent inherited muscular dystrophy in adults, is caused by the CTG repeat expansion in the 3’UTR of the DMPK gene. Mutant DMPK RNA accumulates in nuclear foci altering diverse cellular functions including alternative splicing regulation. DM1 is a multisystemic condition, with debilitating central nervous system alterations. Although a defective neuroglia communication has been described as a contributor of the brain pathology in DM1, the specific cellular and molecular events potentially affected in glia cells have not been totally recognized. Thus, to study the effects of DM1 mutation on glial physiology, in this work we have established an inducible DM1 model derived from the MIO-M1 cell line expressing 648 CUG repeats. This new model recreated the molecular hallmarks of DM1 elicited by a toxic RNA gain-of-function mechanism: accumulation of RNA foci colocalized with MBNL proteins and dysregulation of alternative splicing. By applying a microarray whole-transcriptome approach, we identified several gene changes associated with DM1 mutation in MIO-M1 cells, including the immune mediators CXCL10, CCL5, CXCL8, TNFAIP3, and TNFRSF9, as well as the microRNAs miR-222, miR-448, among others, as potential regulators. A gene ontology enrichment analyses revealed that inflammation and immune response emerged as major cellular deregulated processes in the MIO-M1 DM1 cells. Our findings indicate the involvement of an altered immune response in glia cells, opening new windows for the study of glia as potential contributor of the CNS symptoms in DM1. In this dataset, we include the expression data obtained from MIO-M1-CTG(648) and MIO-M1-CTG(0) with and witouth Doxycycline treatment (1µg/ml) by 8 days . These data are used to obtain genes that are differentially expressed in response to mutant (CUG expanded) DMPK RNA expression.
2023-10-31 | GSE164057 | GEO
Project description:Expanded CAG/CTG repeats underlie thirteen neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD). Upon expansion, CAG/CTG repeat loci acquire heterochromatic characteristics. This observation raises the hypothesis that repeat expansion provokes changes to higher-order chromatin conformation and thereby affects both gene expression in cis and the genetic instability of the repeat tract. Here we tested this hypothesis directly by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD patient-derived cells. Surprisingly, chromatin contacts remain unchanged upon repeat expansion at both loci. This was true for expanded alleles with different DNA methylation levels and CTCF binding. Repeat tract sizes ranging from 15 to 1,700 repeats displayed strikingly similar chromatin interaction profiles. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the three-dimensional chromatin conformation of the surrounding genomic region. Our findings argue that extensive changes in heterochromatic properties are not enough to alter chromatin conformation at expanded CAG/CTG repeat loci. We conclude that 3D chromatin conformation is unlikely to drive repeat expansions or changes in gene expression in expanded CAG/CTG repeat disorders.
2020-04-07 | GSE148176 | GEO
Project description:Introduction: Myotonic dystrophy of type 1 (DM1), the most common dystrophy in adults, is an autosomal dominant inherited disease, affecting around 1 in 8000 person. Patients suffering from DM1 develop essentially muscle disorders such as myotonia, muscle weakness, muscle loss and atrophy. The disease is caused by the mutation of the DMPK "Dystrophia Myotonica Protein Kinase" gene. The mutation correspond to an abnormally large expansion of CTG tri-nucleotides repeats located in the 3'-untranslated region of this gene. Expanded CTG repeats are normally transcribed, but accumulates in RNA aggregates that sequester RNA-binding proteins such as the splicing regulator MBNL1. Consequently, due to MBNL1 sequestration, DM1 is characterized by aberrant splicing of a wide number of mRNA, which are themselves responsible for the symptoms observed in the disease. Purpose: To determine as much as possible novel splicing misregulations taking place in DM1 skeletal muscle, we performed a paired-end RNA sequencing (RNA-seq) using muscles samples of normal individuals (CTRL, n=3) versus muscles of DM1 patients (DM1, n=3). The data was analyzed by a bioinformatical software, called MISO, in order to map the alternative splicing changes between normal and DM1 muscle. The aim of this study was to get a broad and precise view of the splicing changes occurring in DM1 muscle.
2017-08-24 | GSE85984 | GEO
Project description:Expanded CAG/CTG repeats underlie thirteen neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD). Upon expansion, CAG/CTG repeat loci acquire heterochromatic characteristics. This observation raises the hypothesis that repeat expansion provokes changes to higher-order chromatin conformation and thereby affects both gene expression in cis and the genetic instability of the repeat tract. Here we tested this hypothesis directly by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD patient-derived cells. Surprisingly, chromatin contacts remain unchanged upon repeat expansion at both loci. This was true for expanded alleles with different DNA methylation levels and CTCF binding. Repeat tract sizes ranging from 15 to 1,700 repeats displayed strikingly similar chromatin interaction profiles. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the three-dimensional chromatin conformation of the surrounding genomic region. Our findings argue that extensive changes in heterochromatic properties are not enough to alter chromatin conformation at expanded CAG/CTG repeat loci. We conclude that 3D chromatin conformation is unlikely to drive repeat expansions or changes in gene expression in expanded CAG/CTG repeat disorders. This SuperSeries is composed of the SubSeries listed below.
2020-04-07 | GSE148185 | GEO
Project description:This study tested candidate therapeutic antimiRs in a comprehensive new collection of primary myoblasts derived from myotonic dystrophy type 1 (DM1) patients. DM1 is caused by genetically unstable CTG repeat expansions in the DMPK gene and has multiple clinical presentations, likely because of variable repeat sizes at the tissue and organ levels. Expanded DMPK transcripts deplete Muscleblind-Like Splicing Regulator protein MBNL1, which in turn causes DM1 symptoms. A promising therapeutic approach increases the expression of MBNL1 by blocking natural repressors miR-23b and miR-218 employing antimiRs with chemical modifications designed for a better pharmacological profile in humans. In untreated primary cells, we demonstrate that upregulated therapeutic targets miR-23b and miR-218 and reduced MBNL1 protein levels inversely correlate with CTG repeat size, supporting that active MBNL1 protein repression synergizes with the sequestration by CUG repeats in DM1. Upon treatment with novel antimiRs, multiple readouts of RNA toxicity associated with typical DM1 alterations improved. MBNL1-dependent exon inclusions, and myoblast fusion and myotube area defects, showed robust correction over a wide range of CTG expansions. Unexpectedly, antimiR treatment also reduced DMPK transcript levels and the number of ribonuclear aggregates. Based on these data, we selected a leading antimiR for further transcriptomics characterization and demonstrated that it reversed up to 68% of dysregulated genes. The study revealed that the candidate antimiRs could provide therapy to clinically relevant DM1 forms over a wide range of repeat sizes and genetic backgrounds by simultaneously reducing MBNL1 sequestration and derepressing protein synthesis.
2024-05-01 | GSE237411 | GEO
Project description:Myotonic dystrophy type 1 (DM1) is a neuro-muscular disorder caused by CTG triplet expansion in the 3-UTR of the DMPK gene. Mutated transcripts aggregate in muscle nuclei and sequester the MBNL1 splicing factor. To assess the involvement of Mbl sequestration on transcriptpion deregulation in DM1, we performed genome wide analyses of gene expression of DM1 Drosophila model in three different genetic contexts: Mef>mblRNAi, Mef>600CTG, Mef>960CTG vs. Mef>lacZ control line.
2013-04-01 | E-MTAB-1469 | biostudies-arrayexpress
Project description:DM1 (OMIM #160900) is a chronic, slowly progressing multisystemic disease, with symptoms that include loss of muscle strength, myotonia and excessive fatigue. DM1 is caused by a dynamic expansion of CTG repeats, ranging from 50 to several thousands, in the 3’ untranslated region of the DMPK gene. Characteristic molecular features of the disease have been associated with a toxic RNA gain of function of the CUG expansions. Expanded CUG repeats have been demonstrated to be toxic per se in several cell types and animal models disrupting gene transcription and pre-mRNA alternative splicing. Deep sequencing is here used to characterize the deregulation of the RNAs associated to the RISC complex in the skeletal muscle of DM1 patients.
2018-07-15 | GSE108591 | GEO