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Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells

Journal Contribution - Journal Article

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.
Journal: Nucleic Acids Research
ISSN: 0305-1048
Issue: 16
Volume: 46
Pages: 8275-8298
Number of pages: 24
Publication year:2018
Keywords:myotonic dystrophy, myogenic cells, myotonic dystrophy patient-derived iPS
  • DOI: https://doi.org/10.1093/nar/gky548
  • Scopus Id: 85061296032
  • WoS Id: 000450950500025
  • ORCID: /0000-0003-1901-6750/work/61973314
  • ORCID: /0000-0001-5596-7129/work/62819879
  • ORCID: /0000-0002-0853-9890/work/78128336