FIG. 2.

Traffic light reporter assessment of DNA repair fates. (A) Schematic of the TLR reporter. The FANCC CRISPR/Cas9 target sequence is contained within the dashed lines and was inserted into the GFP portion of the construct, resulting in an out-of-frame GFP. The +3 picornaviral 2A sequence allows the downstream nonfunctional +3 mCherry to escape degradation of the nonfunctional GFP.³⁷ Following target site cleavage in the presence of an exogenous GFP donor (box labeled “dsGFP donor”) the GFP gene is repaired by HDR and expresses GFP (expression indicated by green box; inactive mCherry shown as gray box). DNA repair by NHEJ can result in a frameshift that restores the mCherry ORF, resulting in red fluorescence (indicated by red-filled mCherry box; gray indicates inactive GFP). (B–E) Representative FACs plot of 293T TLR cell line treatment with CRISPR/Cas9 nuclease and nickase. A stable 293T cell line with an integrated copy of the FANCC TLR construct was generated that, at its basal level, was GFP negative and expressed <0.5% mCherry (B). This low level of expression is because of integration errors.³⁷ (C) Donor-only-treated cells showing no endogenous HDR. (D and E) FANCC-TLR-293T cell line transfected with the target gRNA, GFP donor, and the Cas9 nuclease (D) or nickase (E) with GFP (x-axis) and mCherry (y-axis) measured at 72 hr posttransfection. (F–H) Quantification and graphical representation of four different experiments and three different donor concentrations observed from FACs plots similar to those shown in (B–E). GFP data are plotted as green bars, and mCherry is plotted as red. The three different GFP donor concentrations (250, 500, and 1000 ng) are detailed. The basal level of mCherry from 2B was subtracted from all treatment groups. For the nuclease, p<0.05 for NHEJ (mCherry) vs. GFP (HDR). For the nickase, p<0.05 for GFP (HDR) vs. mCherry (NHEJ). (I) HDR ratio for nuclease and nickase. To determine the HDR ratio, the percentage of cells expressing GFP was divided by those expressing mCherry at each donor concentration. Mean±SD are graphed, and for nickase vs. nuclease at 250 ng of donor, p= 0.7, at 500 ng of donor p= 0.04, and at 1000 ng of donor p<0.001. TLR, traffic light reporter.