A plant-derived epigenetic editing platform that introduces non-CG methylation into human cells. PpDNMT3 creates methylation features also found in neurons and modulates gene expression, expanding the epigenome space available for engineering and disease research.

UNMET NEED

• DNA methylation is a central mechanism controlling gene expression and cell identity.
• In most human cells, methylation occurs mainly at CG sites, limiting the genomic regions accessible to methylation-based regulation.
• Non-CG methylation is prominent in neurons and embryonic stem cells and contributes to neuronal gene regulation.
• New tools are needed to introduce non-CG methylation in additional cell types to study, manipulate, and potentially repair abnormal gene-expression programs.

OUR SOLUTION

Using the moss DNA methyltransferase PpDNMT3 to induce non-CG methylation and regulate gene expression in human cells.

Published supporting data:

• PpDNMT3 induces robust, genome-wide non-CG methylation in HEK293/HEK293T human cells.
• Non-CG methylation reaches levels comparable to human neurons and embryonic stem cells, with a similar preference for CAC sites.
• PpDNMT3 targets genes, transposons, and genomic regions with few or no CG sites, expanding the addressable epigenome.
• PpDNMT3 alters gene expression; combined CG and non-CG hypermethylation is associated with stronger transcriptional repression.
• The platform can potentially be combined with programmable DNA-targeting systems such as dCas9 for locus-specific epigenome editing.

POTENTIAL FUTURE APPLICATIONS- Manipulation/repair if gene expression for therapeutic purposes

1. Targeted epigenome editing: Combine PpDNMT3 with programmable DNA-targeting systems such as dCas9 to regulate selected genes, including at CG-poor regions.
2. Neurological disease research: Test neuron-associated non-CG methylation in neuronal models to investigate neurodevelopmental and neurodegenerative disease mechanisms.
3. Disease models and cell engineering: Create cellular models with defined epigenetic states and evaluate effects on cell identity and differentiation.
4. Functional epigenomics: Test causal effects of non-CG methylation at promoters, gene bodies, enhancers, and repetitive elements.
5. DNA mapping and labeling: Explore PpDNMT3-dependent non-CG methylation as a distinctive genomic label for mapping applications.

INTELLECTUAL PROPERTY
Patent application filed: US patent application No. 62/813,805.

REFERENCES

1. Yaari R. et al. RdDM-independent de novo and heterochromatin DNA methylation by plant CMT and DNMT3 orthologs. Nature Communications 10, 1613 (2019).
2. Domb K. et al. DNA methylation mutants in Physcomitrella patens elucidate individual roles of CG and non-CG methylation in genome regulation. PNAS 117, 33700-33710 (2020).
3. Domb R. et al. A plant DNMT3 induces non-CG methylation and affects gene expression in human cells. iScience 29, 117542 (2026). DOI: 10.1016/j.isci.2026.117542.