Repurposing Site-Specific Recombinases for Targeted Epigenetic Silencing with the RecOFF Approach

Background/Objectives: Epigenome editing offers a promising alternative to conventional genome editing by enabling targeted control of gene expression without introducing permanent DNA mutations. However, many existing technologies are limited by large construct size that can impose substantial constraints on delivery and in vivo application. In this study, we introduce a novel compact epigenome editing platform based on site-specific recombinases (SSRs) that we term RecOFF. Methods: We evaluated the RecOFF epigenome editing tools across multiple experimental configurations to assess their ability to mediate transcriptional repression in reporter systems containing an epigenetically tunable promoter. Results: As a proof-of-concept, we repurposed the catalytically inactive Cre recombinase as a DNA-binding scaffold for gene silencing by fusing it to epigenetic silencer domains (CreOFF). Notably, a single CreOFF in vitro-transcribed (IVT)-mRNA electroporation was sufficient to induce stable and robust long-term silencing across binding site configurations, reaching up to 95% silencing efficiency. Bisulfite sequencing confirmed that repression was mediated by sustained DNA methylation at CpG sites within the promoter throughout this period. Finally, we coupled the catalytically inactive form of Brec1, an SSR reprogrammed from Cre through directed evolution for the excision of HIV-1 proviral DNA, with epigenetic silencer domains (Brec1-OFF), and showed that it significantly silences the HIV-1 long terminal repeat (LTR) promoter. Conclusions: By demonstrating the RecOFF approach with two distinct and well-characterized SSRs, we show that RecOFF provides a framework that can be adapted to different genomic targets using alternative recombinases, making these compact and potent tools a potentially useful addition to the current epigenome editing tools.