CRISPR epigenome editors show complex on- and off-target effects
Advanced CRISPR/dCas9-based epigenome editors are designed to precisely alter DNA methylation at specific genomic regions without introducing double-strand breaks, offering promising strategies for research and future therapies. A new study from researchers at Center for Molecular Medicine (CMM) and Karolinska Institutet, published in Genome Biology, shows that the relationship between intended (on-target) and unintended (off-target) effects is more complex than previously appreciated. While these systems are widely used to study gene regulation and disease mechanisms, their broader impact on the epigenome has not been comprehensively profiled.
The researchers benchmarked multiple CRISPR/dCas9 epigenome editing platforms. Using complementary genome-wide high-resolution approaches, they demonstrate that off-target DNA methylation changes can occur even when targeting is highly specific at the DNA sequence level. Importantly, the extent and pattern of these off-target effects varied depending on the effector domain and experimental context.
“Our results show that epigenome editing is not simply a local event at the intended locus. There can be broader epigenetic consequences, and these need to be carefully evaluated, especially if these tools are to be translated into clinical applications,” says the study’s first author, Majid Pahlevan Kakhki, research specialist at the Department of Clinical Neuroscience at Karolinska Institutet.
Beyond methodological benchmarking, the study provides conceptual insight: on-target efficiency and off-target activity are not independent phenomena. Instead, they appear to be linked through complex chromatin and regulatory network dynamics. These findings highlight the need for standardized validation pipelines when applying epigenome editing in functional genomics or therapeutic development.
“Our work highlights that careful and rigorous evaluation of specificity is essential to ensure accurate interpretation of functional studies and further optimizations can hopefully bring this exciting frontier to safe clinical translations in the future,” says Professor Maja Jagodic, who led the study with Dr. Lara Kular.
The research was conducted at CMM and Karolinska Institutet and has been funded by, among others, Swedish Research Council, EUbOPEN consortium, Swedish Brain Foundation, Knut and Alice Wallenberg Foundation and European Research Council (ERC).
Publication (CMMers are highlighted in bold):
Majid Pahlevan Kakhki, Fatemeh Rangani, Ewoud Ewing, Chiara Starvaggi Cucuzza, Galina Zheleznyakova, Maria Kalomoiri, Lea Kenny, Anika Raghavan, Chandana Rao Prakash, Gabe van den Hoeven, Tejaswi Venkata S. Badam, Ruxandra Covacu, Ioanna Andreou, Maria Needhamsen, Lara Kular* & Maja Jagodic*. Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on- and off-target effects. Genome Biology, 27, 52 (2026). https://doi.org/10.1186/s13059-026-03967-6
*These authors contributed equally to the study.