Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes
| Autor: | Seiji Takashima, Daisuke Okuzaki, Satoki Nakamura, Yasushi Sakata, Takamaru Ishizu, Satoshi Kameda, Shungo Hikoso, Tomoaki Higo, Yasuaki Kohama, Takumi Kondo, Tomoka Tabata, Shigeru Miyagawa, Mikio Shiba, Yuki Masumura, Hiroyuki Inoue, Yoshiki Sawa, Shuichiro Higo, Daisuke Motooka |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Male
0301 basic medicine Myosin Light Chains DNA recombination Transgene Induced Pluripotent Stem Cells lcsh:Medicine Mice Transgenic 030204 cardiovascular system & hematology Gene delivery Biology medicine.disease_cause Article S Phase 03 medical and health sciences 0302 clinical medicine Genome editing medicine Animals Humans CRISPR Myocytes Cardiac Transgenes lcsh:Science Induced pluripotent stem cell Adeno-associated virus Cells Cultured BRCA2 Protein Multidisciplinary Fanconi Anemia Complementation Group A Protein DNA synthesis Cas9 lcsh:R Gene Transfer Techniques Cell Differentiation Dependovirus Cardiovascular biology Cell biology Fanconi Anemia HEK293 Cells 030104 developmental biology Genetic engineering lcsh:Q Cardiac Myosins RNA Guide Kinetoplastida |
| Zdroj: | Scientific Reports Scientific Reports, Vol 10, Iss 1, Pp 1-13 (2020) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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