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. 2016 Oct 7;45(5):2797–2808. doi: 10.1093/nar/gkw911

Figure 2.

Figure 2.

Editing under transient expression in 293T cells. (A) Scheme of the mRNA/guideRNA duplex of the editing experiment shown in B. (B) Fluorescence imaging of the GFP channel (50 ms exposure time for editing and positive control, 150 ms for negative controls) and RNA sequencing results for cellular editing, 48 h post co-transfection of R/G-guideRNA, ADAR2 and the W58X GFP reporter. E396A ADAR2 is a catalytically inactive mutant. For the editing experiment a duplicate is shown. (C–E) Optimization of cellular editing. (C) Inclusion of a 3΄-terminal hairpin and variation of the length of the mRNA binding platform from 18 to 29 nt, black arrows indicate the different lengths used. For additional traces and fluorescence imaging see Supplementary Figure S10. Red arrows indicate the targeted base, red* indicate off-target editing in the sequencing traces. (D) Editing dependency when varying the number of nucleotides (2–9) intervening the targeted adenosine and the R/G-motif. (E) Editing versus off-target editing using the optimal guideRNA architecture (7 intervening bases, 16 nt mRNA template, 3΄-hairpin) depending on the amount of transfected ADAR2. If not indicated differently, plasmid amounts were 300 ng ADAR2, 300 ng W58X GFP, 1300–1600 ng guideRNA per well in 24-well-plates. For details see Supplementary Figures S8–S15.