Characterizing on-target and off-target mCG deposition efficiency by dC9-D3A direct fusion system. (A) Schematics of the dCas9 (dC9) and TALE (T) constructs used, indicating positioning of nuclear localization sequences (NLS), protein tags (human influenza hemagglutinin [3xHA, 3xTy1]), promoter choice (glycerol kinase promoter [hPGK], human elongation factor-1 alpha promoter [hPEF1a]), solubility tag (protein G B1 domain [GB1]), selectable marker (puromycin resistance [puro]), single-chain Fv antibody against GCN4 domain (αGCN4), and human DNTM3A catalytic domain (D3A). (B) Timeline and outline for experimental design for measuring DNA methylation and TF occupancy (CTCF and NRF1). (C) Targeted DNA methylation deposition to the UNC5C promoter in HeLa cells, measured by bsPCR-seq. sgRNA placement is shown with yellow arrows; dotted lines indicate interval for CGs included in quantitation. (D) Western blot of relative dC9-D3A protein abundance (anti-Ty1) per 50 µg of total cell lysate: (lane 1) untransfected HeLa cells, (lanes 2,4) 48 h post transfection (hpt), (lanes 3,5) 48 hpt and 48 h puromycin (puro) selection, loading control anti-Tubulin. Arrow indicates dC9-D3A.
