Epigenetic editing at individual age-associated CpGs affects the genome-wide epigenetic aging landscape [MSC_hyperL_D11]

Aging is reflected by genome-wide DNA methylation changes, but it is largely unclear how these epigenetic modifications are regulated. In this study, we explored the possibility to interfere with epigenetic clocks by epigenetic editing at individual CpG sites. CRISPR-guided approaches (dCas9-DNMT3A and CRISPRoff) facilitated targeted methylation at age-associated genomic regions that remained stable for up to three months. Furthermore, epigenetic editing evoked many genome-wide off-target effects, which were highly reproducible and enriched at other age-associated CpGs – thus, they are not random off-target effects but seem to resemble co-regulated epigenetic bystander modifications. 4C chromatin conformation analysis at age-associated sites revealed increased interactions with bystander modifications and other age-associated CpG sites. Subsequently, we multiplexed epigenetic modifications in HEK293T, mesenchymal stromal cells, and T cells at five genomic regions that become either hypermethylated or hypomethylated upon aging. While epigenetic editing at age-hypomethylated CpGs appeared less stable, it also resulted preferentially in bystander modifications at CpGs with highest correlation with chronological age. Notably, modifications were simultaneously observed at CpGs that gain and lose methylation with age. These results demonstrate that targeted epigenetic editing can modulate the epigenetic aging network in its entirety and thereby interfere with epigenetic clocks. We analyzed genome-wide DNA-methylation changes with targeted epigenetic editing. Human mesenchymal stromal cells were electroporated with gRNA and dCAS9-DNMT3A/3L. Controls were transfected with plasmides expressing scramble guide RNAs and dCAS9-DNMT3A/3L. Timepoint of isolation is denoted in "time point".