期刊
SCIENTIFIC REPORTS
卷 10, 期 1, 页码 -出版社
NATURE RESEARCH
DOI: 10.1038/s41598-020-72216-y
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资金
- JSPS KAKENHI [18K08069, 18K19543, 19K08489, 19K16518]
- Japanese Ministry of Health, Labor, and Welfare
- Japan agency for medical research and development [19bm0804008h0003]
- Novartis Pharma
- Takeda Pharmaceutical Company
- Lilly
- MSD
- Daiichi Sankyo
- Mitsubishi Tanabe Pharma
- Cell Science Research Foundation
- Grant for Basic Research of the Japanese Circulation Society (2018)
- Actelion Pharmaceuticals Japan
- Center of Medical Innovation and Translational Research
- Center for Medical Research and Education in Graduate School of Medicine, Osaka University
- Grants-in-Aid for Scientific Research [18K19543, 19K08489, 19K16518, 18K08069] Funding Source: KAKEN
Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.
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