Fig. 4: Multiplexed epigenetic editing at age-hyper- and age-hypomethylated genomic regions.

a, Scheme of multiplexed epigenetic editing at five genomic regions that gain DNAm with aging. b, Scatter-plot of DNAm changes across the three replicas. CpGs corresponding to the targeted genes are highlighted in the corresponding color. None of the bystander modifications reached statistical significance. c, Cumulative distribution function comparing the entirety of CpGs from the BeadChip with age-associated CpGs (4,389 age-hypo and 5,328 age-hypermethylated CpGs). d, Correlation of differential gene expression with differential methylation. CpGs and transcripts were matched by gene IDs. Pairs related to one of the five target genes are highlighted in the corresponding color. e, Scheme of multiplexed epigenetic editing at five genomic regions that lose DNAm with aging. f,g, Three days after transfection, DNAm changes were analyzed by a volcano plot (f) and a scatter-plot (g). The volcano plot shows DNAm changes and the limma P value (Benjamini–Hochberg adjusted). CpGs corresponding to the five target genes are highlighted by the corresponding colors (n = 3). h, At 15 days after transfection, the gains in DNAm in the target regions were hardly observed anymore. i, At day 3 after transfection, Gaussian kernel density estimate at age-associated CpGs (4,389 age-hypo and 5,328 age-hypermethylated CpGs) showed that bystander modifications were enriched at genomic regions that gain methylation with age. j, Cumulative distribution of DNAm at age-associated CpGs at day 3 after transfection. Notably, bystander effects are underrepresented at age-hypo and overrepresented at age-hypermethylated CpGs (both P < 10⁻¹⁵, two-sided, two-sample Kolmogorov–Smirnov test). k,l, At day 15 after transfection kernel density estimate and cumulative distribution did not reveal bystander effects anymore.