Fig. 3: Efficacy and safety profile of improved REPRESS (iREPRESS).

A A₂₉ conversion rate as measured by deep sequencing. B Relative mature miR-21−5p level measured by TaqMan assay. ASCs were Mock-transduced (Mock group), co-transduced with Bac-REPRESS/Bac-cr21 (REPRESS group) or co-transduced with Bac-REPRESS/Bac-cr21/Bac-Cre (iREPRESS group). Bac-REPRESS expressed the entire REPRESS cassette flanked by two loxP sequences. Bac-cr21 expressed the crRNA targeting pri-miR-21 at d = −5 near the 5′ basal junction of the pre-miR-21 hairpin. Bac-Cre expresses Cre recombinase to excise and circularize the loxP-flanked REPRESS for sustained expression (see Supplementary Fig. 11 for schematic illustration). C, D Transcriptome-wide analysis of ASCs co-transduced with iREPRESS using cr21 (C) or non-targeting (NT) crRNA (D), with the Mock group as the reference. RNA-seq data are presented as log2(fold change) vs. log(mean expression). E, F Global miRNA analysis of ASCs co-transduced with iREPRESS using cr21 (E) or NT crRNA (F). Statistical significance was determined by two-sided Wald test followed by correction using Storey’s method to generate q values. Change with FALSE q value was cut off and volcano plot is presented in ‒log10(p value) vs. log2(fold change). Red and blue dots (C–F) represent significantly upregulated and downregulated genes in sequencing data. G, H Transcriptome-wide A-to-I off-target analysis of ASCs co-transduced with iREPRESS using cr21 (G) or NT crRNA (H). I, J miRNAome A-to-I off-target analysis of ASCs co-transduced with iREPRESS using cr21 (I) or NT crRNA (J). Orange dot represents on-target editing. Total RNA or miRNA was harvested after 3 days for RNA or small-RNA seq experiments. Statistical analyses were carried out with two-way ANOVA (A, B) followed by Tukey multiple comparison test. Data represent means±SD of three independent culture experiments. Source data are provided as a Source data file.