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. 2020 Nov 25;19(3):415–417. doi: 10.1111/pbi.13497

Figure 1.

Figure 1

Prime editing for precise genome modification in tomato. (a) Schematic diagram of the prime editing constructs in this study. (b) Dual‐luciferase reporter system for assessments of prime editing efficiencies. Dual‐LucM contains an inactive NanoLuc designated as NanoLucM. pegRNA‐12 and pegRNA‐13 target the mutated site to restore the NanoLuc activity. fLuc, firefly luciferase. (c) Comparison of prime editing efficiencies of pCXPE01, pCXPE02 and pCXPE03 in tomato using the Dual‐luciferase reporter system delivered by bombardment. Editing frequencies were calculated by NanoLuc/fLuc, counting the normal reporter Dual‐Luc as 100%. Values (mean ± s.e.m.) were calculated from three independent experiments (n = 3). P values were obtained using the two‐tailed Student’s t‐test. (d and e) Regenerated tomato shoots (d, indicated by arrows) and a representative T0 seedling (e) on hygromycin‐containing medium. Bar, 10 mm. (f) Summary of prime editing results of pCXPE03 in regenerated tomato shoots and T0 plantlets, as determined by NGS and Sanger sequencing, respectively. (g and h) Sequence chromatograms of prime‐edited T0 plants. Edited bases were indicated by red arrows. (b, f, g and h) Targets and their PAMs in sequences were underlined in black and red, respectively. PBS and RT sequences are underlined with solid and dashed lines, respectively. Nucleotides for substitutions are marked in red.