Fig. 4: An improved synthetic U7 promoter further increases RNA editing and guide RNA expression.

a Arrangement of the synthetic guide RNA expression cassette; locations of the modified DSE, PSE, and 3’box elements within the mouse U7 promoter and terminator are highlighted. b Antisense guide RNAs targeting the RAB7A 3’UTR (100.50) or SNCA 3’UTR (80.40), with or without a 5′ double hnRNP A1 binding motif, were cloned onto the original SmOPT U7 hairpin sequence (white) or the triple variant (SmOPT-11A + mU7-3GG + mU7-12C, purple). Guide RNAs were expressed using either the natural hU1 promoter and mU7 terminator (open bars) or an improved synthetic mU7 cassette (striped bars). Plasmids were individually transfected into Landing Pad 293T cells. RNA editing was measured 13 days post transfection, following puromycin selection for cells containing single-copy integrations of the guide RNA construct. Expression levels of guide RNA and target mRNA were measured using ddPCR and normalized to U1 snRNA or HPRT1 mRNA housekeeping genes, respectively (ratio of copies per µl). Mean values ± SD are shown; n = 3 biological replicates. Results were analyzed in aggregate by three-way ANOVA: for RNA editing, P < 0.0001 for all three main effect variables (promoter, SmOPT U7 hairpin, and guide RNA); for guide RNA quantification, P < 0.0001 for the promoter and guide RNA main effect variables, but not significant for the SmOPT U7 hairpin. For each antisense guide RNA, P values are indicated for pairwise comparisons using one-way ANOVA with Tukey’s test (ns, not significant). Source data are provided as a Source Data file.