Abstract Introduction Recent advancements in precision genome-editing technologies, including prime editing (PE), have opened new avenues for correcting pathogenic variants(1,2). PE offers improvements over CRISPR-Cas9 systems by reducing off-target effects, avoiding double-strand breaks, and enabling various types of gene editing(3). Congenital heart disease (CHD) is one of the most common birth defects, with genetic variants such as those in NKX2-5 implicated in its aetiology(4). This study reports the first successful application of PE for the murine CHD model with Nkx2-5 R52G variant. Methods All the animal experiments were approved by an institutional ethics committee and strictly adhered to the relevant guidelines for animal experiments. We established a knock-in mouse model harbouring the Nkx2-5 R52G variant. While the Nkx2-5 R52G homozygous variant is embryonically lethal, heterozygous variant exhibits the phenotype of the atrial septal defect (ASD), the most common CHD in humans(5). To differentiate between embryos in which the variant was rectified by PE and those that were originally wild-type, the pegRNA was designed not just to correct the variant to the wild-type CGT (Arg) sequence, but to introduce a silent variant, changing it to CGG (Arg). This allowed us to identify embryos that had undergone successful editing. We employed the PE3 system. The cocktail containing the mRNA of PEmax, nicking gRNA, and pegRNA was injected into the cytoplasm of embryos. The zygotes were generated by in vitro fertilisation of wild-type oocytes and sperm from Nkx2-5 R52G/WT male mice. To evaluate the efficiency of PE, we collected the embryos 96 hours after injection and performed amplicon sequencing. We also examined whether ASD phenotype was rescued in mice with successful PE of the Nkx2-5 variant and assessed for off-target effects. Results The successful editing efficiency of PE was 9.3% in collected embryos. Furthermore, a total of 93 fetal hearts at embryonic day 19.5 were collected, 41 of which were heterozygous for the variant. Of these 41 heterozygous hearts, PE successfully repaired the variant in three (7.3%) individuals. The top five predicted off-target sites for both the pegRNA and the nicking gRNA were analysed in the three successfully edited individuals, but no evidence of off-target editing was found. Notably, none of the three mice with successful PE exhibited the ASD phenotype, nor did they show the abnormal gene expression pattern observed in the heart harbouring the Nkx2-5 R52G variant. Conclusion This study represents the first successful application of PE to correct a CHD-associated variant in the embryo. While our finding highlights the potential of PE as a therapeutic tool for CHD, significant challenges remain in improving the efficiency and reliability of this technology in early embryonic tissues.Graphic Abstract
Nishijo et al. (Sat,) studied this question.