Fig. 2
From: Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids
Generation and delivery validation of all-in-one EF-1α driven sncRNA PE plasmids. A, B Overview of PE systems used in this study. A Regularly used CMV-driven multi plasmid PE systems. B Overview of the two EF-1α driven pAIO plasmids generated for this study. Plasmids are abbreviated to pAIO-PE2-GFP and pAIO-PE4max from here on. Grey arrow: CMV promoter; grey rectangle: PE system; green rectangle: GFP; black arrow: U6 promoter; blue rectangle: pegRNA; brown arrow: hsyn promoter; red rectangle: mCherry; orange arrow: EF-1α promoter. C, D Comparison of pCMV-PE2-GFP delivery versus pAIO-PE2-GFP delivery in C HEK293T cells and D hiPSC, using FACS. C (Left) Quantitative representation of the fluorescent fractions when transfected with the full multi plasmid PE2 system, pCMV-PE2-GFP and U6-pegRNA-mCherry (total, double positive and GFP only), compared to pAIO-PE2-GFP in HEK293T. C, D (Middle) GFP expression profile of C the transfected HEK293T or D nucleofected hiPSC pools. C, D (Right) Zoom in on the GFP positive cells of a representative sample set. D (Right) Quantitative representation of the fluorescent fractions when transfected with pCMV-PE2-GFP compared to pAIO-PE2-GFP in hiPSC. Bar plots show the mean edit percentages ± SD; replicates are presented as individual data points (see individual data values in Additional file 2: Table S1)