Abstract
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.
Highlights
Introduction of thetdTomato sequence into the 3′-terminus of the Myl[2] gene did not significantly injure cardiomyocytes in vitro 6 days after transduction of AAV69, or heart tissues in vivo 28 days after direct injection of AAV6
Transduction of AAV6 into cardiomyocytes isolated from murine adult heart tissue by the Langendorff perfusion method failed to induce HDR9, probably because of the low transduction efficiency or the low viability of isolated adult cardiomyocytes after viral transduction
Intron-spanning PCR using cDNA samples obtained from injected myocardium and Sanger sequence analysis confirmed that the tdTomato sequence was precisely introduced into the 3′-terminus of the Myl[2] gene (Fig. 1D and Supplementary Fig. 1D)
Summary
TdTomato sequence into the 3′-terminus of the Myl[2] gene did not significantly injure cardiomyocytes in vitro 6 days after transduction of AAV69, or heart tissues in vivo 28 days after direct injection of AAV6. These data suggest that Mlc2v-tdTomato fusion protein produced by the recombined genetic locus did not significantly affect sarcomere function, and the Myl[2] gene might be an appropriate target for real-time imaging of sarcomere structure even in the in vivo heart. Our method rapidly introduced the tdTomato sequence into the 3′-terminus of the MYL2 gene in differentiated contracting cardiomyocytes, and this technique can potentially be used in various disease-specific hiPSC-CMs
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