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. 2021 Apr 3;22(7):3741. doi: 10.3390/ijms22073741

Figure 3.

Figure 3

Co-editing of ts gene and ABL1 locus improves recovery of ABL-edited cells. (A) Schematic of the construct targeting ABL1 to insert a 1.95 kb CMV-CloverGFP-pA cassette into exon 2. The guide sequence is shown above in bold, with the PAM sequence in pink (the guide is on the minus strand). The donor plasmid has the cassette flanked by 1 kb homology arms. (B–F) Co-editing of TAFts and CMV-CloverGFP cassette in ABL1. HEK293 TAF1 ts (clone 1) cells were transfected in biological duplicates with CMV-Clover in ABL donor plasmid, ssODN for the rescue of TAF1ts, and plasmids for expression of Flag-Cas9 constructs and sgRNAs targeting TAF1ts and ABL1, or TAF1ts and a non-targeting sgRNA for the control. Two days post-transfection, cells from each well were replated into two dishes, with one maintained at 37 °C and passaged every 4–5 days over the course of the experiment (one month), with samples taken for SDS-PAGE and immunoblot at the first passage (shown in panel B). The other dish was transferred to 39.5 °C. The medium was changed as needed, but these cells were not passaged. (C) GFP expression in heat-selected colonies was assessed by photographing the cells using the Cy3 filter (GFP) and brightfield. The numbers of colonies are indicated at the lower righthand corners of the images. (D) Summary of GFP-expression in heat-selected colonies, n = 2, error bars represent SEM, * p < 0.05. (E) Pools of the colonies that had grown at 39.5 °C (“selected”) and samples of the cells continuously grown at 37 °C (“unselected”) were analyzed by SDS-PAGE and immunoblot. (F) Analysis of genomic DNA of heat-selected cells. Genomic DNA from the pools of selected cells was PCR-amplified using the control or recombination-specific primer pairs indicated. The recombination-specific primer set generates a 1.4 kb band, and the control set a 1.1 kb band. (G) qPCR was performed on genomic DNA from the pools of selected and non-selected cells using primers specific for the left homology arm and the CMV sequence within the donor plasmid. Primers specific to genomic DNA were used for normalization. N = 2, error bars represent SEM.