Altering DNA Methylation Levels at Multiple Age-linked CpGs

May 15, 2025 By Stuart P. Atkinson

Histograms of methylated-CpG counts per read for two editing systems. — Use of dCas9–DNMT3A and CRISPRoff for targeted methylation CpG editing. From Liesenfelder et al.

Altering DNA Methylation Levels at Multiple Age-linked CpGs

Part 1 of this series from the Epigenome Technologies blog reported on the first half of an exciting recent Nature Aging (Liesenfelder et al.) from researchers led by Wolfgang Wagner (RWTH Aachen University Medical School). The initial stages of this paper described how CRISPR-guided editing of DNA methylation (DNAm) levels at individual

The Epigenome Technologies blog now brings you Part 2 of this series, which reports on the impact of multiplexed HEK293T cells and then in primary human T cells and mesenchymal stromal cells, given that targeting multiple age-associated CpGs might increase any interference observed within the proposed epigenetic aging network. The range of products and services that Epigenome Technologies provides can empower your research aims with flexible, high-resolution technologies that turn hidden regulatory layers into actionable discoveries ready to transform our understanding of health and disease.

sgRNA screen DNAm scatter, volcano, and expression plots. — CRISPR–DNMT3A sgRNA library screens in HEK293T cells. From Liesenfelder et al.

Multiplexing Epigenetic Editing at CpGs Undergoing Age-related Increase in DNA Methylation

The authors targeted regions associated with genes (PDE4C, FHL2, ELOVL2,KLF14, and TEAD1) that undergo aging-associated gains in DNAm, given the high correlation of DNAm with chronological age (Han et al. and Hannum et al.), their low methylation in HEK293T/blood cells, and the relative ease of designing guide (g)RNAs

EPIC Illumina BeadChip analysis demonstrated that increased DNAm levels displayed long-term stability
Unfortunately, they also detected:
a less pronounced increase in DNAm than previously observed
a lack of significant bystander effects or enrichment in age-associated regions
few alterations at the transcriptomic level
and no clear correlation between gene expression and DNAm changes
The PDE4C gene did undergo a nearly 50-fold increase in expression after multiplexed epigenome editing

Density and cumulative DNAm-change plots at days 3 and 15. — Distribution of DNA methylation changes at various timepoints all CpGs (blue), hypo-CpGs (gold) and hyper-CpGs (red), shown as density and cumulative curves. From Liesenfelder et al.

Multiplexing Epigenetic Editing at CpGs Undergoing an Age-associated Loss of DNA Methylation

Given these unexpected findings, the study next explored the restoration of DNAm at five genes (COL1A1, AKAP8L, CSNK1D, MEIS1-AS3, and IGSF11) losing DNAm during aging

While many CpGs gained DNAm, the results of epigenetic editing disappeared after 15 days, with results suggesting that DNAm gains at sites losing DNAm during aging display a lower level of stability than at sites that gain DNAm during aging

Multiplexing epigenetic editing at CpGs losing DNA methylation during aging induced bystander effects (19,254 hypermethylated and 88 hypomethylated sites) that did not relate to predicted gRNA-related off-target regions at day 3 (which disappeared by day 15)

Bystander effects displayed a significant enrichment for CpGs that gain DNAm during aging and a lesser enrichment for CpGs losing DNAm during aging
This surprising finding also demonstrates coherent age-associated DNAm alterations

Bar charts of epigenetic age differences across clocks for donors. — Effects of editing age-associated CpGs on epigenetic clock predictions. From Liesenfelder et al.

Understanding How Multiplexed Epigenetic Editing Impacts the T Cell Aging Network

Multiplexed epigenetic aging at sites that undergo age-related increases in DNAm (PDE4C, FHL2, ELOVL2, KLF14, and TEAD1) in primary T cells induced site-specific DNAm after 21 days

Epigenetic editing in these primary cells induced hypo-/hyper-methylated bystander effects enriched at CpGs that gain or lose DNAm with aging in blood

Multiplexed epigenetic aging at sites losing DNAm during aging (COL1A1, AKAP8L, CSNK1D, MEIS1-AS3, and IGSF11) did not support evident DNAm alterations after 10 days and only induced moderate bystander effects

These findings again suggest the greater stability of editing at CpGs that gain DNAm during aging
Overall, this moderate effect prompted a lesser impact on the genome-wide aging network

Analysis of how these alterations would impact epigenetic clocks revealed an acceleration in epigenetic aging of up to 10 years after epigenetic editing at CpGs that gain DNAm during aging

Transient effects at CpGs losing DNAm during aging induced a less coherent impact on epigenetic clocks

DNAm control vs edited scatter for hyper-CpGs in MSCs. — Editing age-hyper CpGs in primary mesenchymal stromal cells: (a) experiment design schematic; (b) day 11 scatter of DNAm(control) versus DNAm(experiment) for five target loci. From Liesenfelder et al.

Understanding How Multiplexed Epigenetic Editing Impacts the Mesenchymal Stromal Cell Aging Network

Analysis of bystander effects at age-associated CpGs also employed another primary cell type - mesenchymal stromal cells

Targeting epigenetic editing of PDE4C, FHL2, ELOVL2, KLF14, and TEAD1 prompted a moderate increase in DNAm, with no clear association between bystander effects and transcriptomic changes
Bystander effects in mesenchymal stromal cells increased at CpGs that gain or lose DNAm with long-term culture (which induced senescence) and displayed enrichment in age-associated CpGs

Network of chromatin-interacting CpG nodes colored by hyper/hypomethylation. — Network schematic of bystander CpG methylation changes via chromatin interactions: red (age-hypermethylated) and orange (age-hypomethylated) nodes sized by correlation, with arrows indicating directional influence. From Liesenfelder et al.

Conclusions: Epigenetic Editing at Multiple Age-related CpGs Can Impact the Epigenetic Clock

Overall, the second half of this fascinating paper further suggests that epigenetic editing interferes with age-associated DNAm patterns and impacts an epigenetic aging network; indeed, epigenetic editing at CpGs that gain DNAm during aging accelerated epigenetic age in T cells (even if the clocks involved include some of the target regions involved). While the current data demonstrating the simultaneous modulation of CpGs that gain and lose DNAm during aging by epigenetic editing suggest the unfeasibility of directing epigenetic aging, they will help to guide future research that will more fully define the aging network and define new means of affecting aging at the epigenetic level.

A deeper understanding of the impact of editing at distinct epigenetic levels or even at the single-cell level may offer the means to push this groundbreaking research forward ; can Epigenome Technologies help in this endeavor? The profiling of multiple histone modifications combined with simultaneous RNA sequencing at the single-cell level may provide an understanding of the complementary role of another level of epigenetic regulation. Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. As such, applying Paired-Tag technology may enable giant leaps forward in understanding gene regulation and, in this case, the impacts of epigenetic editing on aging.

For more on the consequences of altering DNA methylation at multiple age-linked CpGs, see Nature Aging, March 2025.