Fig. 6.

Epigenetic editing with gene reexpression, promoter demethylation and reactivation of tumor-suppressive function is achieved by VP160 and TET1 effectors on CLDN10B. For epigenetic editing of CLDN10B, HEK cells were transfected with according CRISPR-dCas vectors (either VP160 or TET1), RNA isolated, reversely transcribed and expression analyzed by RT-PCR. DNA was isolated, BS treated and CLDN10B CGI pyrosequenced. a dCas9-TET1 increases the endogenous background level (non-guided) of CLDN10B expression, compared to untreated HEK cells. b CLDN10B was demethylated by VP160 and TET1, that reactivated CLDN10B expression (c) and its tumor-suppressive function (d). d Wound healing assay in 2D cell culture reveals reduced cell migration after EpiEdit treatment. Scratch was placed 72 h post transfection, wound closure was examined by measuring the cell-free area at 0 h and 48 h (120 h post transfection) (Suppl. Figure 18 a)). e–g d Cas9-VP160 treatment of HEK cells in 2D and dynamic 3D cell culture. 3 Mio. cells were transferred 72 h post transfection (2D) into dynamic 3D cell culture (analyzed 120 h post transfection) (Suppl. Figure 18 b)). Demethylation of CLDN10B by dCas9-VP160 (e) leads to reexpression f and consequently to a reduced microscopic spheroid size (g + h)