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

• Published in: Molecular therapy 2017-01, Vol.25 (1), p.24-43
• Main Authors: van Agtmaal, Ellen L., André, Laurène M., Willemse, Marieke, Cumming, Sarah A., van Kessel, Ingeborg D.G., van den Broek, Walther J.A.A., Gourdon, Geneviève, Furling, Denis, Mouly, Vincent, Monckton, Darren G., Wansink, Derick G., Wieringa, Bé
• Format: Article
• Language: English
• Subjects: (CTG⋅CAG)n repeat; Animals; Bacterial Proteins - genetics; Base Sequence; Cell lines; Clonal deletion; Clustered Regularly Interspaced Short Palindromic Repeats; Codon; CRISPR; CRISPR-Associated Protein 9; CRISPR-Cas Systems; CRISPR/Cas9; Deoxyribonucleic acid; Disease Models, Animal; DM1 myoblasts; DMPK protein; DNA; DNA repeat expansion; Double-strand break repair; dsDNA break repair; Dystrophy; Endonucleases - genetics; Epigenetics; Experiments; Fibroblasts - metabolism; Gene Editing; Gene Expression; Gene Order; Genes; Genetic Loci; Genetics; Genome editing; Genomes; Genomic Instability; Humans; Kinases; Life Sciences; Mice; Myoblasts; Myotonic dystrophy; Myotonic Dystrophy - genetics; Myotonin-Protein Kinase - genetics; NHEJ; Original; Polyglutamine; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Deletion; Software; somatic cell therapy; Target recognition; therapeutic genome editing; trinucleotide instability; Trinucleotide Repeat Expansion; Trinucleotide Repeats
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2016.10.014

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. van Agtmaal et al. demonstrate that excision of the expanded (CTG⋅CAG)n repeat from the DMPK gene in myotonic dystrophy patients can be reliably achieved by dual CRISPR/Cas9 cleavage at either side of the repeat and that this offers a strategy to adjust anomalous effects of expanded DMPK transcripts in myoblasts.