title: CRISPR/Cas9-Induced (CTG⋅CAG)nRepeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.
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.
authors.affiliation: Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
authors.forename: Ellen L
authors.initials: EL
authors.lastname: van Agtmaal
authors.affiliation: Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
authors.forename: Laurène M
authors.initials: LM
authors.lastname: André
authors.affiliation: Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: Willemse
authors.affiliation: Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Cumming
authors.affiliation: Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
authors.forename: Ingeborg D G
authors.initials: IDG
authors.lastname: van Kessel
authors.affiliation: Radboud Institute for Molecular Life Sciences, Department of Cell Biology, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA, Nijmegen, the Netherlands.
authors.forename: Walther J A A
authors.initials: WJAA
authors.lastname: van den Broek
authors.affiliation: Inserm UMR 1163, 75015 Paris, France; Imagine Institute, Paris Descartes-Sorbonne Paris Cité University, 75270 Paris, France.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Gourdon
authors.affiliation: UPMC Université Paris 06, Inserm UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, 75252 Paris, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Furling
authors.affiliation: UPMC Université Paris 06, Inserm UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, 75252 Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Mouly
authors.affiliation: Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Darren G
authors.initials: DG
authors.lastname: Monckton
authors.affiliation: 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.
authors.forename: Derick G
authors.initials: DG
authors.lastname: Wansink
authors.affiliation: 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.
authors.forename: Bé
authors.initials: B
authors.lastname: Wieringa
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003062:Codon; D004195:Disease Models, Animal; D004720:Endonucleases / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing; D015870:Gene Expression; D023061:Gene Order; D056426:Genetic Loci; D042822:Genomic Instability; D006801:Humans; D051379:Mice; D009223:Myotonic Dystrophy / Q000235:genetics*; D065636:Myotonin-Protein Kinase / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019680:Trinucleotide Repeat Expansion; D018911:Trinucleotide Repeats
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D065636:Myotonin-Protein Kinase; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: (CTG⋅CAG)n repeat; CRISPR/Cas9; DM1 myoblasts; NHEJ; dsDNA break repair; myotonic dystrophy; somatic cell therapy; therapeutic genome editing; trinucleotide instability