An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing

Fig. 1. Adding a U7 or U1 promoter and SmOPT U7 hairpin to the antisense gRNA improves RNA editing. a 100 nt antisense guide RNAs targeting adenosines in the RAB7A 3’UTR, FANCC 3’UTR, SMAD4 coding sequence, or SOD1 start codon (TIS) were expressed using the hU6, hU7, or mU7 promoters, either alone or possessing an additional SmOPT sequence and human or mouse U7 hairpin on the 3’end. Plasmids also contained the CMV promoter to overexpress ADAR2 (gray) or GFP (endogenous ADAR, yellow). RNA was measured from HEK-293T cells 2 days post transfection. Mean values ± SD are shown; n = 3 biological replicates. P values were calculated by Mixed-effects analysis using Dunnett’s multiple comparisons test (two-sided). b Sequences containing two or three copies of the hnRNP A1 binding motif (UAGGGW, underlined) were added onto the 5′ end of antisense guide RNAs expressed using a human U1 promoter and possessing a SmOPT mU7 hairpin. RNA was measured from HEK-293T cells with endogenous ADAR levels 2 days after plasmid transfection. Mean values ± SD are shown; n = 5 biological replicates. P values were calculated by Mixed-effects analysis using Dunnett’s multiple comparisons test (two-sided). c Landing…

Fig. 2. Pooled screen of SmOPT and U7 hairpin variants using split-pool single cell barcoding. a Mutagenesis screen workflow. 1) Three antisense guide RNAs targeting the RAB7A 3’UTR, SNCA 3’UTR, or GAPDH CDS were each cloned onto 252 mutagenesis variants along the Sm binding or U7 hairpin sequences, in duplicate pools. 2) Plasmid libraries were sequenced to match the guide RNA with a 14 N random GuideID barcode located in the GFP 5’UTR. 3) Plasmid libraries were integrated into the Landing Pad 293T cell line. 10 M cells, each containing a single copy of construct, were pooled, fixed, and permeabilized. 4) Four custom primers reverse transcribed GFP, RAB7A, SNCA, or GAPDH transcripts within each fixed cell. Each RT primer possessed an additional 10 N umi and an anchor sequence, which was annealed to a Splint scaffold. 5) Cells were split equally across 60 different wells, each containing a different 7mer subcode that was ligated onto the reverse transcribed cDNA. Cells were then washed, pooled, and split again across 60 new subcode wells. For 60 subcodes, 3 rounds of split-pool ligation yields 216,000 possible combinations. Finally, labeled cells were split into separate wells of 10,000…

Fig. 3. Engineered SmOPT U7 variants can improve editing across multiple endogenous targets. The original SmOPT mU7 hairpin sequence (white) or three combination variants (SmOPT-11A + mU7-3GG, SmOPT-11A + mU7-12C, and SmOPT-11A + mU7-3GG + mU7-12C) were cloned onto six antisense guide RNAs, with or without a 5′ double hnRNP A1 binding motif, and individually transfected into Landing Pad 293T cells. Following puromycin selection for successfully integrated single-copy guide RNA constructs, RNA was measured 13 days post transfection. Negative controls consist of samples where the transfected plasmid targeted a different gene. Mean values are shown for 2 biological replicates. Results across all antisense guide RNAs (n = 12; 6 gene targets +/− hnRNP) were analyzed in aggregate by two-way ANOVA; P values comparing each combination variant to the original SmOPT mU7 hairpin were calculated using Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.

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…

Fig. 5. Comparison and combination with alternative gRNA design approaches. Antisense guide RNAs targeting the RAB7A 3’UTR (100.50, 80.40, or 60.30) with a 5′ double hnRNP A1 binding motif were compared to three previously published guide RNAs: CLUSTER 3xRS 19-13-11-20p8; cadRNA 200.100.loops.interspersed.v3; and LEAPER circ-arRNA151. Published RAB7A RNA editing values for transient transfection of HEK-293 cells are marked. Guide RNAs were expressed using either the hU6 promoter (open bars) or an improved synthetic mU7 promoter (striped bars). Antisense guide RNAs possessed no SmOPT U7 hairpin (gray), or the triple-variant SmOPT U7 hairpin (purple). In some cases, the guide RNA sequences were flanked by circularizing ribozymes to form circular guide RNAs (as noted). Plasmids were individually transfected into Landing Pad 293T cells. For transient transfection, RNA editing was measured 2 days later. For single-copy genomic integration, RNA editing was measured 13 days post transfection, following puromycin selection for cells containing the guide RNA construct. Mean values ± SD are shown; n = 3 biological replicates. Source data are provided as a Source Data file.

Fig. 6. Engineered SmOPT U7 hairpin variants also improve published ASOs for DMD exon skipping. a Antisense Oligo RNA expression constructs alongside a constitutive GFP and puromycin resistance marker were flanked by insulator sequences (ins) and terminal repeats (TR) for the piggybac transposase. b Placement of the ASO binding sites on DMD exon 2 or 51, which were expressed with or without a 5′ double hnRNP A1 binding motif. c ASOs were expressed with the original SmOPT U7 hairpin (white) or the triple combination variant (SmOPT-11A + mU7-3GG + mU7-12C, purple), using either the natural hU1 promoter and mU7 terminator (open bars) or an improved synthetic mU7 cassette (striped bars). Constructs were randomly integrated into the genome of RD rhabdomyosarcoma cells using piggyBac transposase, followed by 7 days of puromycin selection for successful integrants. GFP+ myoblasts were then differentiated for 10 days to induce expression of the full-length DMD Dp427m transcript. Exon skipping was measured using droplet digital PCR. Top: Representative ddPCR plots with probes specific for exon retained or exon skipped mRNA isoforms. Bottom: Percent exon skipping. Empty vector control is a…

Fig. 7. Improved SmOPT U7 gRNAs achieve high levels of RNA editing in Hurler syndrome mice. a AAV vector used for guide RNA delivery. A 90.65 antisense Hurler guide RNA with −6 + 30 loops targeting the mouse Idua W392X pre-mRNA or a 100 nt antisense Scramble negative control were combined with a 5′ hnRNP domain and the triple variant SmOPT U7 hairpin. 2E12 scAAV PHP.eB were retroorbitally injected into mIDUA-W392X mutant mice. In addition, mIDUA-W392X mice injected with saline only or wild-type C57BL/6 mice were included as controls. Brain tissue (anterior cerebrum) was harvested 4 weeks later. b In vivo cerebral cortex samples were analyzed for: RNA editing of the target Idua-W392X mutation; IDUA enzyme activity; Glycosaminoglycan accumulation; Guide RNA expression level relative to the native U1 snRNA; and Idua transcript levels relative to Gapdh mRNA. Mean values ± SD are shown; n = 3 mice. Results were analyzed by one-way ANOVA with multiple pairwise comparisons. c Bystander A > G editing along the region of Idua exon 9 targeted by the AAV PHP.eB Hurler guide. The W392X mutation is highlighted in red. Editing of the subsequent adenosine results in a synonymous GCA > GCG mutation…