October 23, 2019  |  

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

Authors: Dastidar, Sumitava and Ardui, Simon and Singh, Kshitiz and Majumdar, Debanjana and Nair, Nisha and Fu, Yanfang and Reyon, Deepak and Samara, Ermira and Gerli, Mattia F M and Klein, Arnaud F and De Schrijver, Wito and Tipanee, Jaitip and Seneca, Sara and Tulalamba, Warut and Wang, Hui and Chai, Yoke Chin and In't Veld, Peter and Furling, Denis and Tedesco, Francesco Saverio and Vermeesch, Joris R and Joung, J Keith and Chuah, Marinee K and VandenDriessche, Thierry

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
DOI: 10.1093/nar/gky548
Year: 2018

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