ENGINEERING EPIGENOME EDITING PLATFORMS FOR REVERSIBLE GENE EXPRESSION REGULATION IN HUMAN CELLS

Abstract

Background: Epigenome editing technologies provide a reversible control of gene expression by targeting chromatin remodeling in human cells, representing a promising alternative to permanent genome modification.
Objective: Here, we sought to design and test programmable epigenome editing platforms that can reversibly activate or repress disease-associated genes with high specificity and minimal genomic perturbation.
Methods: CRISPR/dCas9 based activator and repressor systems fused with epigenetic modifiers such as VP64 and KRAB domains were constructed and delivered in HEK293 and induced pluripotent stem cells by lentiviral transduction. qPCR, RNA sequencing, and chromatin immunoprecipitation assays were used to analyze gene expression and epigenetic alterations.

Findings: The engineered platforms demonstrated up to 8.5-fold activation of OCT4 expression and 85% repression efficiency of SOX2 within 72 hours. Reversibility analysis demonstrated ~90% restoration of baseline expression following withdrawal of epigenome editing constructs, and off-target effects were <5%.

Conclusion: The results show that engineered epigenome editing systems can efficiently and reversibly regulate gene expression in human cells, and point to their potential applications in precision medicine, regenerative therapy, and functional genomics.