Epigenome editing-mediated restoration of expression by demethylation of CpG island shore in porcine fibroblasts.

Fibrillin-1, an extracellular matrix protein encoded by the gene, is crucial for maintaining connective tissue integrity. Mutations in result in haploinsufficiency, leading to Marfan syndrome, in which the expression of functional is correlated with disease onset and severity. Recent studies suggest that expression is modulated by DNA methylation, particularly within the CpG island shores of its promoter region. In porcine models, mRNA levels have been found to correlate with the proportion of hypomethylated alleles in the CpG island shore region. In this study, we employed epigenome editing using the dCas9-TET1 system to induce targeted DNA demethylation within the CpG island shore, which became hypermethylated after a prolonged culture of porcine fetal fibroblast cells. This approach effectively reduced methylation in the targeted region, and cells expressing the dCas9-TET1 system maintained hypomethylation across multiple passages. Critically, DNA demethylation of the CpG island shore restored expression in heterozygous knockout fibroblasts, which developed stochastic hypermethylation after extended culture. These findings highlight the potential of DNA methylation manipulation to restore expression in cells with a haploinsufficient genetic background.