Figure 5.

ObLiGaRe is mediated via NHEJ. (A) Illustration of the reporter DGF cassette in the AAVS1 locus. ZFN sites are the same as in Figure 3. A functional GFP is generated after HR using the HA donor, while a functional mCherry is inserted upon ObLiGaRe using the ObLiGaRe donor. The two donors are about the same size. (B) Determination of fluorescent cell number after cotransfection of Nras ZFN, ObLiGaRe, and HA donors by FACS. Detection of GFP and mCherry cells were gated using parental cells (WT, left), which did not show any fluorescent cells upon cotransfection. When the reporter cell line was cotransfected with the three constructs, GFP and mCherry cells were detected in their corresponding gates (DGF, right). (C) Representation of the percentage of GFP- and mCherry-positive cells out of total live cells in WT HCT116 cells, of reporter HCT116 cells without treatment (DGF), or after treatment with 10 μM Nu7026 or 4 mM caffeine. Error bars, SD; n = 3.