Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

• Published in: PloS one 2020-01, Vol.15 (1), p.e0228072-e0228072
• Main Authors: Mata López, Sara, Balog-Alvarez, Cynthia, Vitha, Stanislav, Bettis, Amanda K, Canessa, Emily H, Kornegay, Joe N, Nghiem, Peter P
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Biology and Life Sciences; Codons; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA binding proteins; DNA sequencing; Dogs; Duchenne muscular dystrophy; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Dystrophy; EDTA; Engineering and technology; Fluorescent antibody technique; Force measurement; Gene Editing - methods; Gene expression; Gene mutation; Gene sequencing; Genes; Genetic modification; Genetic research; Genetic Therapy - methods; Genome editing; Homology; Immunofluorescence; Injection; Medicine and Health Sciences; Messenger RNA; Microscopy; Muscular dystrophy; Muscular Dystrophy, Duchenne - genetics; Muscular Dystrophy, Duchenne - therapy; Mutation; Myoblasts; Myoblasts - cytology; Myoblasts - metabolism; Myotubes; Neomycin; Nuclease; Nucleases; Plasmids; Proteins; Repair; Research and Analysis Methods; Retirement benefits; RNA; Stop codon; Transcription; Transcription activator-like effector nucleases; Transcription Activator-Like Effector Nucleases - genetics; Utrophin; Veterinary colleges
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0228072

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.