Programmable editing of primary MicroRNA switches stem cell differentiation and improves tissue regeneration

Abstract

Programmable RNA editing is harnessed for modifying mRNA. Besides mRNA, miRNA also regulates numerous biological activities, but current RNA editors have yet to be exploited for miRNA manipulation. To engineer primary miRNA (pri-miRNA), the miRNA precursor, we present a customizable editor REPRESS (RNA Editing of Pri-miRNA for Efficient Suppression of miRNA) and characterize critical parameters. The optimized REPRESS is distinct from other mRNA editing tools in design rationale, hence enabling editing of pri-miRNAs that are not editable by other RNA editing systems. We edit various pri-miRNAs in different cells including adipose-derived stem cells (ASCs), hence attenuating mature miRNA levels without disturbing host gene expression. We further develop an improved REPRESS (iREPRESS) that enhances and prolongs pri-miR-21 editing for at least 10 days, with minimal perturbation of transcriptome and miRNAome. iREPRESS reprograms ASCs differentiation, promotes in vitro cartilage formation and augments calvarial bone regeneration in rats, thus implicating its potentials for engineering miRNA and applications such as stem cell reprogramming and tissue regeneration.

Fig. 1. Development of REPRESS for pri-miRNA editing.

A Mode of action of REPRESS. crRNA targeting to the ssRNA motif of pri-miRNA brings the dCas13-ADAR2_DD to the vicinity of basal junction, thus catalyzing specific A- > I conversion within the pre-miRNA hairpin. B The REPRESS expression cassette. dCas13 (dPspCas13b or dRfxCas13d) was fused with ADAR2_DD E488Q via various linkers. The fusion protein was flanked by two bipartite nuclear localization signals (bpNLS) to facilitate nuclear import. crRNA expression cassette was driven by U6 promoter with an architecture of spacer-direct repeat (DR) for dPspCas13b (pB-crRNA) or DR-spacer-DR for dRfxCas13d (pD-crRNA). C Schematic illustration of a segment of pri-miR-10a and the secondary structure of pre-miR-10 hairpin. The numbering of bases was defined according to the sequences retrieved from miRBase database. Translucent red box indicates the intended edited adenosine. D REPRESS variants encoding dPspCas13b (B-REPRESS 1–6) or dRfxCas13d (D-REPRESS 1–6) and various linkers. Corresponding editing efficiencies are also shown. E, F Editing efficiency using different spacer lengths (L). G Illustration of relative distance (d) between targeting position and the 5′ (‒) or 3′ (+) basal junctions. H–P Editing efficiencies at different targeting positions for various pri-miRNAs. Plasmids encoding REPRESS and crRNA were co-delivered into cells with a ratio of 1:3 (w:w). Total RNA was harvested after 1 day for reverse transcription and PCR amplification. PCR amplicons (300–500 bp for different pri-miRNA, 444 bp for pri-miR10a; see Supplementary Fig. 1 for how PCR amplicons were synthesized) encompassing the pre-miRNA and flanking sequences were Sanger sequenced. A- > I editing efficiencies were calculated by EditR. Statistical analyses were carried out with one-way (F, H, J, L, N) or two-way (P) ANOVA followed by Tukey multiple comparison test. Data represent means ± SD of three independent culture experiments. Source data are provided as a Source data file.

Fig. 2. Editing of pri-miRNAs knocked down various mature miRNAs in different cells.

A dREPRESS expression cassette harboring the fusion of dRfxCas13d and deactivated ADAR2_DD with E488Q and E396A mutations. B–G Relative mature miRNAs levels. Plasmids encoding optimized REPRESS or dREPRESS were co-delivered with crRNA expressing plasmids into cells with a ratio of 1:3 (w:w). miRNAs were collected at 1 day for TaqMan assay using primers specific to mature miRNAs. Statistical analyses were carried out with one-way ANOVA followed by Tukey multiple comparison test. Data represent means±SD of three independent culture experiments. Source data are provided as a Source data file.

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.

Fig. 4. iREPRESS switched ASCs from adipogenic towards chondrogenic differentiation.

Experiments and experimental groups are identical to those shown in Fig. 3. A, B Relative expression of adipogenic marker genes C/ebpα (A) and Ppar-γ (B). C, D Relative expression of chondrogenic marker genes Acan (C) and Col2a1 (D). E–G Representative images of Safranin O, Alcian Blue and Alizarin Red staining (n = 3). H–J Spectrophotometric analysis for oil droplet formation (adipogenesis, H), GAG (for chondrogenesis, I) and mineralization (for osteogenesis, J). Scale bar = 100 μm. Statistical analyses were carried out with one-way (H–J) and two-way ANOVA (A–D) followed by Tukey multiple comparison test. Quantitative data represent means±SD of three independent culture experiments. Source data are provided as a Source data file.

Fig. 5. iREPRESS-mediated miR-21 knockdown enhanced cartilage formation.

A Gross appearance of engineered cartilages. Representative images of (B) H&E, (C) Alcian Blue and (D) Col II staining (n = 3). Black arrow heads represent the positive stains. E, F Semiquantitative analysis of GAG and Col II. ASCs were mock- (Mock group) or iREPRESS-transduced (iREPRESS group) in 15-cm dishes. At 1 dpt, the cells were seeded into porous gelatin scaffolds (diameter = 6 mm; thickness = 1 mm; 5 × 10⁶ cells/scaffold; n = 3 for each group). The ASCs/gelatin constructs continued to be cultured in chondroinductive medium and assayed at 7 or 14 dpt. Statistical analyses were carried out with two-way ANOVA followed by Tukey multiple comparison test. Quantitative data represent means±SD of three independent culture experiments. Source data are provided as a Source data file.

Fig. 6. iREPRESS-engineered ASCs/scaffold constructs promoted calvarial bone healing.

A–C Representative 3D and hot-and-cold projections of the transverse and sagittal views of the calvarial bone at 4 (A), 8 (B) and 12 (C) weeks post-implantation. Orange arrow heads represent new bone formation. D–F Regenerated bone area (mm²), volume (mm³) and density (HU, Hounsfield Unit) of Mock (n = 4 rats, 7 dpt; n = 5 rats, 14 dpt) and iREPRESS (n = 6 rats) implanted animals. ASCs from the Mock and iREPRESS groups (as in Fig. 5) were seeded into gelatin scaffolds, cultured and harvested at 7 or 14 dpt. The constructs were implanted into the critical-sized (diameter = 6 mm) calvarial defects of SD rats. Bone regeneration was evaluated by μCT imaging. Percentage values were calculated by normalization to the bone area (28 mm²), volume (28 mm³) and density (4600 HU) of the original defects. Statistical analyses were carried out with two-way ANOVA followed by Tukey multiple comparison test. Quantitative data are represented as means±SD. Source data are provided as a Source data file.