Extended Data Fig. 5: Pyrosequencing validation of targeted epigenetic modification.

a) Genomic tracks of differential DNAm at day 7 in a 10k base window centered at the corresponding target site (dashed vertical line) in the five targeted genes. b) Pyrosequencing measurements of demonstrating stable DNAm levels at PDE4C and FHL2 (triplicates). HEK293T cells were either transfected guides for PDE4C, FHL2, ELOVL2, KLF14, and TEAD1 together with dCas9-DNMT3A, or scramble guide RNA and dCas9-DNMT3A. The epigenetic modifications both regions were stable for at least 17 days. DNAm was significantly higher in experimental conditions across all timepoints (P value < 0.05, unpaired t-test). c) Gaussian kernel density estimate comparing the entirety of CpGs from the BeadChip with age-associated CpGs (4389 age-hypo and 5328 age-hypermethylated CpGs). In these experiments hardly any reproducible bystander effects were observed. d) Transcriptomic changes upon multiplexed epigenetic editing at the age-hypermethylated CpGs. 10 genes were significantly up- and 11 genes significantly down-regulated (adjusted P < 0.05; >2 fold change).