Abstract 1952: Novel mouse models enabling locus-specific manipulation of DNA methylation.

Abstract Introduction: DNA methylation is a key epigenetic mechanism that regulates development and disease, including cancer. However, current tools for precise, locus-specific epigenetic gene editing remain limited. Because phenotypic outcomes often result from interactions among multiple genes, there is a critical need for systems that can modulate methylation across several genomic loci simultaneously. Equally important, tight spatial and temporal control of such epigenetic modulation is essential to accurately dissect causal relationships between DNA methylation and gene function. To address these challenges, we developed mouse models that enable controlled, cell type-specific, and time-dependent epigenetic modulation in vivo. Methods: We applied the CRISPR-dCas9-SunTag system for targeted DNA methylation editing, using TET1 for demethylation and DNMT3A/3L for methylation. To generate mouse models, we employed homologous recombination to introduce knock-in constructs at genomic safe harbors. Specifically, the dCas9-SunTag-TET1-GFP system was inserted into the Rosa26 locus and the dCas9-SunTag-DNMT3A/3L-mCherry system was integrated into the Hipp11 (H11) locus. Expression of the dCas9-based epigenetic editors was regulated through recombinase systems: Cre or Flp recombination activated expression, while Dre recombination removed the entire construct, thereby terminating expression. Specific guide RNA (gRNAs) directed targeted demethylation or methylation at single or multiple genomic sites. Results: For both mouse lines, correct knock-in was confirmed by Southern blot and DNA sequencing. Germline transmission was successful, and offspring developed normally with the expected Mendelian ratios. Mouse embryonic fibroblasts (MEFs) derived from these lines were used to validate inducible expression mediated by Cre or Flp recombination. Inducibility was confirmed by Cas9 Western blot, RT-PCR analysis of TET1 or DNMT3A/3L transcripts, and fluorescent reporter expressions. Using gRNA targeting multiple promoters, including tumor suppressor genes p16 and Hic1 and oncogenes Tfap2a and VEGF, we observed robust and site-specific DNA methylation editing. Furthermore, targeted methylation of the p16 promoter in MEFs resulted in transcriptional silencing and bypass of the senescence checkpoint. Conclusion: We successfully established mouse models that enable precise, locus-specific manipulation of DNA methylation through inducible CRISPR-dCas9-SunTag-based epigenetic editing. These models provide a versatile platform for dissecting the causal relationships between DNA methylation and gene function in vivo. Given their flexibility and inducible control, these systems will be broadly applicable to researchers investigating functional epigenomics, developmental regulation, and the epigenetic mechanisms underlying human diseases. Citation Format: Julia M. Salamat, Li Yang, Xiaomin Chen, Eduardo Lopez, Lanlan Shen. Novel mouse models enabling locus-specific manipulation of DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1952.