Figure 3.

Comparing Gene-Editing Outcomes at Euchromatin versus Heterochromatin after Plasmid Vector Delivery of Donor DNA

(A) Plasmid^d-based gene editing. Dual-color flow cytometric quantification of HDR and NHEJ frequencies in HER.TLR^TetO.KRAB cells are shown. HER.TLR^TetO.KRAB cells incubated (+) or not incubated (−) with Dox were mock-transfected or were transfected with Plasmid^d mixed with constructs encoding the indicated RGN complexes. Two different transfection protocols (A and B) were used to deliver the DNA constructs into target cells. Bars represent mean ± SD of the indicated number (n) of independent experiments (biological replicates done on different days). (B) Representative dot plots corresponding to HER.TLR^TetO.KRAB cells transfected with Plasmid^d mixed with expression constructs coding for Cas9:gNT or Cas9:TLR.1 complexes initially treated or not treated with Dox. (C) Relative engagement of HDR and NHEJ pathways during plasmid-mediated repair of DSBs created at heterochromatin versus euchromatin. Panel C displays the data shown in (A) as the ratios between the frequencies of NHEJ and HDR in HER.TLR^TetO.KRAB cells exposed or not exposed to Dox.