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Mol Ther. 2017 Jan 4;25(1):24-43. doi: 10.1016/j.ymthe.2016.10.014. Epub 2017 Jan 4.

CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.

Author information

1
Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
2
Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
3
Inserm UMR 1163, 75015 Paris, France; Imagine Institute, Paris Descartes-Sorbonne Paris Cité University, 75270 Paris, France.
4
UPMC Université Paris 06, Inserm UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, 75252 Paris, France.
5
Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands. Electronic address: rick.wansink@radboudumc.nl.
6
Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands. Electronic address: be.wieringa@radboudumc.nl.

Abstract

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

KEYWORDS:

(CTG⋅CAG)n repeat; CRISPR/Cas9; DM1 myoblasts; NHEJ; dsDNA break repair; myotonic dystrophy; somatic cell therapy; therapeutic genome editing; trinucleotide instability

PMID:
28129118
PMCID:
PMC5363205
DOI:
10.1016/j.ymthe.2016.10.014
[Indexed for MEDLINE]
Free PMC Article

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