Journal
SCIENTIFIC REPORTS
Volume 7, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-017-09716-x
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Funding
- JSPS KAKENHI [16K09500, 26461129]
- Japanese Ministry of Health, Labor, and Welfare
- Japan agency for medical research and development
- Banyu Foundation
- Novartis Pharma
- Actelion Pharmaceuticals Japan
- Center for Medical Research and Education, Graduate School of Medicine, Osaka University
- Grants-in-Aid for Scientific Research [16K09500, 17K16004, 17K08540, 15H04820, 26461129] Funding Source: KAKEN
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Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in nondividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2. Imaging-based sequential evaluation of endogenously tagged protein revealed that HDR occurs in cardiomyocytes, independently of DNA synthesis. We sought to repair a pathological mutation in Tnnt2 in cardiomyocytes of cardiomyopathy model mice. An sgRNA that avoided the mutated exon minimized deleterious effects on Tnnt2 expression, and AAV-mediated HDR achieved precise genome correction at a frequency of similar to 12.5%. Thus, targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.
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