Fig. 1: Epigenetic editing is stable but not coherent within the target region.

a, Schematic presentation of two CRISPR-guided epigenetic editors used in this study. Deficient CAS9 protein is linked to DNMT3A/3L at the C terminus (dCAS9-DNMT3A/3L, coexpressed with eGFP for selection)¹³, and CRISPRoff comprising DNMT3A/3L at the N terminus, and tagBFP and a KRAB domain at the C terminus¹⁵. Single guide RNAs were designed 50–200 bp distant of the target CpG. b, Manhattan plot illustrating the DNAm changes upon targeting the age-associated hypermethylated region in PDE4C (EPIC Illumina BeadChip data of chromosome 19; mean of three replicas; 14 days after transfection). Significant DNAm changes are highlighted in red: delta mean DNAm > 0.1 and an adj. P < 0.05 (limma P value, Benjamini–Hochberg adjusted). The target CpG in PDE4C is highlighted in black. c, Bisulfite amplicon sequencing of 26 neighboring CpGs at the target region of PDE4C. The bar plot depicts the frequency of methylated CpGs on individual reads, indicating that even on modified DNA strands not all neighboring CpGs become coherently methylated. 61.7% and 42.9% of reads have higher methylation levels than observed in the wild type, for dCAS9-DNMT3A and CRISPRoff, respectively. d, Time-course experiment of DNAm at PDE4C measured by bisulfite barcoded amplicon sequencing. The lines resemble the 26 different CpGs in the amplicons. e, Frequency of DNAm patterns in bisulfite barcoded amplicon sequencing data of PDE4C. Reads are clustered by their DNAm pattern. The binding region of one gRNA is indicated. The second gRNA binds two base pairs next to the CpG #26 and might explain the low methylation gain at #25 and #26.