Volume 2 - Issue 3, May - June 2026

CRISPR-Cas9 genome editing has revolutionized biomedical research and holds transformative therapeutic promise; however, off-target (OT) cleavage events represent a cardinal safety concern whose epigenetic consequences remain incompletely characterized. Here we report a systematic investigation of OT-induced alterations in DNA methylation across six therapeutically relevant genomic loci in human HEK293T, HCT116, and primary CD34⁺ haematopoietic progenitor cells. Using a combined GUIDEseq, CUT&RUN-bisulfite sequencing, and long-read Oxford Nanopore methylation profiling pipeline, we detected reproducible CpG methylation perturbations at 83% of confirmed OT cleavage sites, with changes ranging from −27% (hypomethylation) to +31% (hypermethylation) relative to unedited controls. Critically, methylation alterations at the VEGFA site 1 and DNMT3A loci persisted through twelve cell divisions, indicating heritable epigenetic remodelling independent of primary sequence mutation. Mechanistically, we demonstrate that OT-associated double-strand breaks recruit the de novo methyltransferase DNMT3A and impair TET2-mediated demethylation through competitive chromatin occupancy. These epigenetic lesions are functionally consequential: aberrant promoter hypermethylation at the DNMT3A OT site correlated with a 3.4-fold increase in DNMT3A transcript abundance and a downstream shift in global methylation homeostasis. We further show that highfidelity Cas9 variants (eSpCas9, HiFi Cas9) substantially reduce but do not eliminate OT methylation perturbation, and that temporal delivery control via ribonucleoprotein (RNP) electroporation minimises epigenetic collateral damage. These findings establish OT methylation dysregulation as a clinically significant and quantifiable safety parameter for therapeutic genome editing, and we propose an integrated epigenomic safety assessment framework for pre-clinical and regulatory review.

Enuma Chukwunweike Henry, Ikhenemue O. Oseni, Braimah Nusrat,"CRISPR-Cas9 Off-Target Effects on Epigenetic Methylation Patterns: Implications for Genome Editing Safety and Therapeutic Applications" International Journal of Advanced Multidisciplinary Research and Educational Development, V2(3): Page(1012-1018) May-June 2026. ISSN: 3107-6513. www.ijamred.com. Published by Scientific and Academic Research Publishing.