4.7 Article

Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes

Journal

SCIENTIFIC REPORTS
Volume 7, Issue -, Pages -

Publisher

NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-017-09716-x

Keywords

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Funding

  1. JSPS KAKENHI [16K09500, 26461129]
  2. Japanese Ministry of Health, Labor, and Welfare
  3. Japan agency for medical research and development
  4. Banyu Foundation
  5. Novartis Pharma
  6. Actelion Pharmaceuticals Japan
  7. Center for Medical Research and Education, Graduate School of Medicine, Osaka University
  8. 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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