TY - JOUR
T1 - Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells
AU - Dastidar, Sumitava
AU - Ardui, Simon
AU - Singh, Kshitiz
AU - Majumdar, Debanjana
AU - Shivsankaran Nair, Nisha Nair
AU - Fu, Yanfang
AU - Reyon, Deepak
AU - Samara, Ermira
AU - Gerbil, Mattia FM
AU - Klein, Arnaud F.
AU - De Schrijver, Wito
AU - Tipanee, Jaitip
AU - Seneca, Sara
AU - Tulalamba, Warut
AU - Wang, Hui
AU - Chai, Yoke Chin
AU - In 'T Veld, Pieter
AU - Furling, Denis
AU - Tedesco, Francesco Saverio
AU - Vermeesch, Joris R.
AU - Joung, J. Keith
AU - Chuah, Marinee
AU - VandenDriessche, Thierry
PY - 2018/9/19
Y1 - 2018/9/19
N2 - 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.
AB - 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.
KW - myotonic dystrophy
KW - myogenic cells
KW - myotonic dystrophy patient-derived iPS
UR - http://www.scopus.com/inward/record.url?scp=85061296032&partnerID=8YFLogxK
U2 - 10.1093/nar/gky548
DO - 10.1093/nar/gky548
M3 - Article
SN - 0305-1048
VL - 46
SP - 8275
EP - 8298
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 16
ER -