Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects

TL;DR

This study compares different CRISPR/dCas9 tools for DNA methylation editing and finds that while some are more effective, they also cause unintended changes in gene activity.

Contribution

The study reveals that multimerization of DNA methyltransferase 3A increases editing potency but introduces widespread methylation and transcriptional changes.

Findings

Multimerization of DNA methyltransferase 3A enhances editing potency but causes early methylation at promoter regions.

Non-targeting gRNAs induce long-lasting methylation-independent transcriptional changes in RNA and energy metabolism genes.

CRISPRoff shows fewer and less stable off-target effects but still causes transcriptome alterations.

Abstract

CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.

We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.

Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

The online version contains supplementary material available at 10.1186/s13059-026-03967-6.

Figures