Fig. 5

Schematic illustration of hTERT promoter region for targeted THOR demethylation. A Schematic representation of the CRISPR-dCas9 system to specifically demethylate THOR. A catalytically inactive mutant Cas9 (dCas9) is fused with TET1 demethylase enzyme for erasing DNA methylation marks in the specific region of THOR. B Eight sgRNAs targeting THOR region of the human TERT gene were selected. The sgRNAs recognizing their respective target sites are shown in blue, with the arrows pointing toward the protospacer adjacent motif (PAM) sequence. The sequenced region is 351 bp long and located upstream to the transcription start site (TSS). CpG sites are represented as dots and were sequenced with a specific panel of primers. Nine CpG sites proximal to the TSS are highlighted in light green (Fragment A2); fifteen CpGs located in mid THOR are highlighted in light blue (Fragment A3) and six CpGs correspondent to an upstream region are highlighted in light orange (Fragment A4). The fragment A1 corresponds to the core promoter, where hTERT promoter mutations (C228T and C250T) occur. The negative strand (5’ to 3’) and gRNAs targeting the positive strand are represented from 3’ to 5’