biostudies-literaturePahlevan Kakhki MEuropean Research Council52https://www.ebi.ac.uk/biostudies/studies/S-EPMC12924462biostudies-literatureUnknown27(1)<h4>Background</h4>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.<h4>Results</h4>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 witGenome biologyComprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.PMC12924462818170van den Hoeven GNeedhamsen MRangani FVenkata S Badam TKenny LAndreou IKular LRao Prakash CEwing EKalomoiri MJagodic MZheleznyakova GPahlevan Kakhki MCovacu RStarvaggi Cucuzza CRaghavan AfalseComprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.<h4>Background</h4>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.<h4>Results</h4>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 wit2026-01-01T00:00:00Z2026 Jan2026-07-16T15:14:37.477Z2026-07-10T03:09:40.001ZS-EPMC129244624162060810.1186/s13059-026-03967-6