Adjuvant genetic treatments enhance homology-directed precise gene editing in postmitotic hearts

Precise gene editing after CRISPR/Cas9-induced double stranded DNA breaks can be obtained by homology directed repair (HDR) using an exogenously provided DNA template. However, HDR is inefficient in adult post-mitotic tissues, such as the heart. Here, we wanted to stimulate HDR in cardiomyocytes (CMs) by transiently administering small RNAs that enhance this process. We screened a library of 2024 human microRNAs (miRNAs) and identified two families of miRNAs that share the same cellular mRNA targets and act as powerful HDR enhancers. The two prototypes for these families (miR-520c-3p and miR-302d-3p) induce expression of cellular factors involved in homologous recombination (including Mre11, Nbs1, Rad50 and of the single stranded DNA binding protein Rad51) in the absence of DNA damage. Both miRNAs also directly downregulate CYP26B1, the cytochrome involved in retinoic acid (RA) degradation. Cardiomyocyte treatment with both miRNAs increased expression of RA-associated genes, among which the Rec8 meiotic cohesin protein, which maintains sister chromatid pairing during meiosis and ensures homologous recombination between paired chromosomes 1. Overexpression of Rec8 alone markedly stimulated HDR in both cultured CMs and in the heart of neonatal and adult mice. These results indicate that precise gene correction is possible in postmitotic hearts by transiently inducing a pro-recombinogenic environment, thus paving the way to a definitive cure of inherited cardiomyopathies.