Figure 7.

Gene-Editing Endpoints at Euchromatin versus Heterochromatin after ODN Donor Delivery in HEK.EGFP^TetO.KRAB Cells

(A) Schematics of ODN design and target site before and after RGN engagement. The RGN complex Cas9:gEGFP is presumed to generate a 3′-ended DNA flap complementary and non-complementary to ODN.as and ODN.s, respectively. HDR-based gene editing with ODN.s and ODN.as donors should result in EGFP-to-EBFP conversion via the knocking in of exogenous DNA encoding the EBFP flurochrome (cyan nucleotides) flanked by sequences complementary to the target site, i.e., 36- and 80-nt-long arms. Open arrowheads, position of the DSB induced by Cas9:gEGFP; orange triplet, PAM. (B) Probing HDR-based gene editing with sense and antisense ODNs. HEK.EGFP^Tet.KRAB cells were transfected with ODN.s or with ODN.as, each mixed with expression plasmids coding for either non-cutting Cas9:gNT or cutting Cas9:gEGFP complexes. HDR and NHEJ quantification in HEK.EGFP^Tet.KRAB cells was assessed by EBFP- and EGFP-directed flow cytometry, respectively. (C) ODN-based gene editing. Dual-color flow cytometric quantification of HDR and NHEJ frequencies in HEK.EGFP^TetO.KRAB cells is shown. HER.TLR^TetO.KRAB cells, incubated (+) or not incubated (−) with Dox, were exposed to the indicated experimental conditions. Bars correspond to mean ± SD of the indicated number (n) of independent experiments (biological replicates done on different days). (D) Relative participation of HDR and NHEJ pathways during ODN-mediated repair of DSBs taking place at heterochromatin versus euchromatin. Panel D displays the results shown in (C) as the ratios between the frequencies of NHEJ and HDR in HEK.EGFP^TetO.KRAB cells exposed or not exposed to Dox.