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.

有丝分裂后心肌细胞被认为不允许进行精确的靶向整合，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，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，可促进表达Cas9的成年小鼠心肌细胞中精确的靶向基因组替换。全身注射 AAV 或直接注射腺病毒都不会促进靶向整合，这表明心肌细胞需要高拷贝数的单链转基因。值得注意的是，AAV 介导的心肌细胞靶向整合在体外和体内都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。在诱导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Mlc2v蛋白，独立于DNA合成，并能够实时检测单层跳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中的靶向基因组替换提供了广泛的应用。