Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.
Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.
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DOI：
10.1038/s41598-020-72216-y
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发表时间：
2020-09-18
影响因子：
4.6
通讯作者：
Sakata Y
中科院分区：
文献类型：
--
作者：
Kohama Y;Higo S;Masumura Y;Shiba M;Kondo T;Ishizu T;Higo T;Nakamura S;Kameda S;Tabata T;Inoue H;Motooka D;Okuzaki D;Takashima S;Miyagawa S;Sawa Y;Hikoso S;Sakata Y
有丝分裂后的心肌细胞被认为不允许精确的靶向整合，包括CRISPR/Cas9基因组编辑后的同源定向修复（HDR）。在这里，我们证明了通过心室内注射腺相关病毒（AAV）直接递送大量编码指导RNA（gRNA）和修复模板DNA的转基因促进表达Cas9的成年鼠心肌细胞中的精确靶向基因组替换。无论是全身注射AAV还是直接注射腺病毒都不能促进靶向整合，这表明心肌细胞中需要高拷贝数的单链转基因。值得注意的是，AAV介导的体外和体内心肌细胞中的靶向整合依赖于范可尼贫血途径，这是单链模板修复机制的关键组成部分。在从诱导的多能干细胞分化的人心肌细胞中，AAV介导的靶向整合在分化后荧光标记Mlc 2 v蛋白，独立于DNA合成，并且能够实时检测单层搏动心肌细胞中的肌节收缩。我们的研究结果为非分裂心肌细胞的靶向基因组置换提供了广泛的应用。
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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通过非分裂心肌细胞中的同源指导修复靶向基因组替代。
影响因子：
4.6
作者：
Ishizu T;Higo S;Masumura Y;Kohama Y;Shiba M;Higo T;Shibamoto M;Nakagawa A;Morimoto S;Takashima S;Hikoso S;Sakata Y
通讯作者：
Sakata Y
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
48
作者：
Burridge, Paul W.;Matsa, Elena;Shukla, Praveen;Lin, Ziliang C.;Churko, Jared M.;Ebert, Antje D.;Lan, Feng;Diecke, Sebastian;Huber, Bruno;Mordwinkin, Nicholas M.;Plews, Jordan R.;Abilez, Oscar J.;Cui, Bianxiao;Gold, Joseph D.;Wu, Joseph C.
通讯作者：
Wu, Joseph C.
影响因子：
64.8
作者：
Laflamme, Michael A.;Murry, Charles E.
通讯作者：
Murry, Charles E.
影响因子：
--
作者：
Guan, Xuan;Wang, Zejing;Childers, Martin K.
通讯作者：
Childers, Martin K.