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

Sumitava Dastidar (1) , Simon Ardui , Kshitiz Singh , Debanjana Majumdar , Nisha Nair , Yanfang Fu , Deepak Reyon , Ermira Samara , Mattia F M Gerli , Arnaud F Klein (2) , Wito de Schrijver , Jaitip Tipanee , Sara Seneca , Warut Tulalamba , Hui Wang , Yoke Chin Chai , Peter In'T Veld , Denis Furling (2) , Francesco Saverio Tedesco , Joris R Vermeesch , J Keith Joung , Marinee K Chuah , Thierry Vandendriessche

1 Departement of gene therapy & regenerative medecine, Vrijz unversiteit Brussel
2 Centre de recherche en Myologie – U974 SU-INSERM

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 dys-trophy 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 ribonucle-oprotein (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 .

Domaines: Biotechnologies

Sumitava Dastidar, Simon Ardui, Kshitiz Singh, Debanjana Majumdar, Nisha Nair, et al.. Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells. Nucleic Acids Research, 2018, 46, pp.8275 - 8298. ⟨10.1093/nar/gky548⟩. ⟨hal-02357315⟩