Figure 5.

Specificity profile of DEMs. (A) ATAC-seq results. Circos plot showing whole genome accessibility in genomic DNA extracted from primary lymphocytes nucleofected either with DEM #6 (grey) or its inactive counterpart dDMT #6 (black). The line indicates the DEM #6 target site on chromosome 3 (CCR5). (B) Accessibility at CCR5 (top) and B2M (bottom) in the indicated samples. Normalized read counts pooled from two independent experiments are shown. Dashed vertical line indicates DEM #6 binding site. (C) DEM off-target activity at predicted sites. The extent of CpG methylation at potential DEM #6 off-targets (OT) predicted in silico was analyzed via next generation bisulfite sequencing. For each region we analyzed the average CpG methylation in a single amplicon encompassing the predicted off-target site. Histogram shows the average CpG methylation measured (mean ± S.E.M., n = 3). Statistical significance calculated with a two-tailed, paired Student's t-test (*P < 0.05). n/a denotes failure in obtaining a PCR product. (D) Accessibility at the off-target OT5 measured via ATAC-seq. (E) Venn diagram showing the overlap between ATAC-seq, RNA-seq and in silico off-target sites predicted via PROGNOS. The number of overlapping sites is indicated. Overlap with computational prediction of off-target sites is restricted to hits within 10-kb distance from the annotated transcription start sites (TSS) of the 84 de-regulated genes identified via RNA-seq analysis or from the 325 regions of lower chromatin accessibility resulting from ATAC-seq analysis respectively.