Generating Isogenic Myoblast Cell Models by CRISPR/Cas9-mediated Editing, Regulating and Targeting Genes in the Myotonic Dystrophy Type 1 (DM1) Locus - Repeat Expansions & Myotonic Dystrophy (REDs) Accéder directement au contenu
Communication Dans Un Congrès Année : 2017

Generating Isogenic Myoblast Cell Models by CRISPR/Cas9-mediated Editing, Regulating and Targeting Genes in the Myotonic Dystrophy Type 1 (DM1) Locus

Résumé

Introduction: Recently, we have reported promising first steps towards use of CRISPR/Cas9-therapy for DM1, aimed at removal of the (CTG•CAG)n-repeat. Still, further fundamental and therapeutic studies are needed to better understand the complex etiology of disease in tissues of DM1 patients. Methods: By use of genome editing and promoter targeting in myoblasts from DM1 patients, unaffected individuals or transgenic DM1 mice we have generated different new DM1 cell models. A summary of requirements for - and effects of - genome targeting with different Cas9-guide NA combinations will be given. Results: Complete precise excision of the repeat tract from normal and mutant DMPK alleles, with sizes between 0.1-8 kbp, could be achieved at high frequency by dual-simultaneous CRISPR/Cas9-cleavage at either side of the (CTG*CAG)n sequence. Introduction of only one dsDNA break in either the 5'- or 3' unique flanks of the expanded trinucleotide repeat is undesirable, as it promotes uncontrollable instability with deletion of large segments from the expanded region, rather than clean DNA repair. Importantly, aspects of impaired myogenic differentiation were normalized upon loss of large repeats. We have also used the CRISPR/Cas9 system as a tool to generale isogenic myoblast models with (i) a deleted G-quadruplex motif/CTCF site in the 3'UTR region of the DMPK gene, and (iii) altered expression of genes from the DM1 locus. Discussion: Cast/CRISPR-based genome editing and targeting is a versatile approach for studly of how repeats expansion and structural elements in the 3'UTR of DMPK mRNA affect myogenic potential an RNA-processing or -nucleocytoplasmic partitioning in cell models for DM1
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hal-04019231 , version 1 (08-03-2023)

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  • HAL Id : hal-04019231 , version 1

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Ellen L van Agtmaal, Laurane M Andre, Mariekel Willemse, Remco van Cruchten, Ingeborg DGT van Kessel, et al.. Generating Isogenic Myoblast Cell Models by CRISPR/Cas9-mediated Editing, Regulating and Targeting Genes in the Myotonic Dystrophy Type 1 (DM1) Locus. International Myotonic Dystrophy Consortium Meeting IDMC-11, International Myotonic Dystrophy Consortium, Sep 2017, San Francisco, United States. ⟨hal-04019231⟩
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