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Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides


Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development.

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Fig. 1: Design of ADAR-directing ASOs and characterization in engineered ADAR-expressing cell lines (293 Flp-In T-REx).
Fig. 2: Applying RESTORE to edit endogenous transcripts (GAPDH and ACTB, each with a targeted 5′ UAG triplet in the 3′ UTR) in various cell lines by transfection with ASOs, performed in presence or absence of IFN-α, as indicated.
Fig. 3: Applying RESTORE for ORF editing with ASO v25, off-target analysis, and editing of disease-relevant sites.

Data availability

This manuscript provides Supplementary Information on primary data and further controls (Supplementary Figs. 115), and it contains a table of ASOs (Supplementary Table 1), a list of target sequences (Supplementary Note 1), and spreadsheets with significantly differently edited sites (Supplementary Datasets 13). The original next-generation sequencing data have been deposited in the NCBI GEO database under accession code GSE121573. Code is available at


  1. Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83–96 (2016).

    Article  CAS  Google Scholar 

  2. Vogel, P. & Stafforst, T. Site-directed RNA editing with antagomir deaminases-a tool to study protein and RNA function. ChemMedChem. 9, 2021–2025 (2014).

    Article  CAS  Google Scholar 

  3. Gagnidze, K., Rayon-Estrada, V., Harroch, S., Bulloch, K. & Papavasiliou, F. N. A new chapter in genetic medicine: RNA editing and its role in disease pathogenesis. Trends Mol. Med. 24, 294–303 (2018).

    Article  CAS  Google Scholar 

  4. Rossi, A. et al. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524, 230–233 (2015).

    Article  CAS  Google Scholar 

  5. Stafforst, T. & Schneider, M. F. An RNA-deaminase conjugate selectively repairs point mutations. Angew. Chem. Int. Ed. 51, 11166–11169 (2012).

    Article  CAS  Google Scholar 

  6. Montiel-Gonzalez, M. F., Vallecillo-Viejo, I., Yudowski, G. A. & Rosenthal, J. J. C. Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc. Natl Acad. Sci. USA 110, 18285–18290 (2013).

    Article  CAS  Google Scholar 

  7. Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

    Article  CAS  Google Scholar 

  8. Woolf, T. M., Chase, J. M. & Stinchcomb, D. T. Toward the therapeutic editing of mutated RNA sequences. Proc. Natl Acad. Sci. USA 92, 8298–8302 (1995).

    Article  CAS  Google Scholar 

  9. Wettengel, J., Reautschnig, P., Geisler, S., Kahle, P. J. & Stafforst, T. Harnessing human ADAR2 for RNA repair—recoding a PINK1 mutation rescues mitophagy. Nucleic Acids Res. 45, 2797–2808 (2017).

    CAS  PubMed  Google Scholar 

  10. Picardi, E. et al. Profiling RNA editing in human tissues: towards the inosinome atlas. Sci. Rep. 5, 14941 (2015).

    Article  CAS  Google Scholar 

  11. Bennett, C. F., Baker, B. F., Pham, N., Swayze, E. & Geary, R. S. Pharmacology of Antisense Drugs. Annu. Rev. Pharmacol. Toxicol. 57, 81–105 (2017).

    Article  CAS  Google Scholar 

  12. Vogel, P., Schneider, M. F., Wettengel, J. & Stafforst, T. Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the gRNA. Angew. Chem. Int. Ed. Engl. 53, 6267–6271 (2014).

    Article  CAS  Google Scholar 

  13. Heep, M., Mach, P., Reautschnig, P., Wettengel, J. & Stafforst, T. Applying human ADAR1p110 and ADAR1p150 for site-directed RNA editing-G/C substitution stabilizes gRNAs against editing. Genes (Basel) 8, 34 (2017).

    Article  Google Scholar 

  14. Patterson, J. B., Thomis, D. C., Hans, S. L. & Samuel, C. E. Mechanism of interferon action: double-stranded RNA-specific adenosine deaminase from human cells is inducible by alpha and gamma interferons. Virology 210, 508–511 (1995).

    Article  CAS  Google Scholar 

  15. Kim, D.-H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).

    Article  CAS  Google Scholar 

  16. Xu, X., Wang, Y. & Liang, H. The role of A-to-I RNA editing in cancer development. Curr. Opin. Genet. Dev. 48, 51–56 (2018).

    Article  CAS  Google Scholar 

  17. Valente, E. M. et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).

    Article  CAS  Google Scholar 

  18. Vogel, P., Hanswillemenke, A. & Stafforst, T. Switching protein localization by site-directed RNA editing under control of light. ACS Synth. Biol. 6, 1642–1649 (2017).

    Article  CAS  Google Scholar 

  19. Vogel, P. et al. Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs. Nat. Methods 15, 535–538 (2018).

    Article  CAS  Google Scholar 

  20. Singh, S. K., Koshkin, A. A., Wengel, J. & Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun. (Camb). 455–456 (1998).

  21. Obika, S. et al. Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering. Tetrahedr. Lett. 38, 8735–8738 (1997).

    Article  CAS  Google Scholar 

  22. O’Shea, J. J. et al. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu. Rev. Med. 66, 311–328 (2015).

    Article  Google Scholar 

  23. Lomas, D. A. & Mahadeva, R. α1-antitrypsin polymerization and the serpinopathies: pathobiology and prospects for therapy. J. Clin. Invest. 110, 1585–1590 (2002).

    Article  CAS  Google Scholar 

  24. Woodard, L. E. & Wilson, M. H. piggyBac-ing models and new therapeutic strategies. Trends Biotechnol. 33, 525–533 (2015).

    Article  CAS  Google Scholar 

  25. Vallecillo-Viejo, I. C. et al. Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme. RNA Biol. 15, 104–114 (2018).

    Article  Google Scholar 

  26. Vogel, P. & Stafforst, T. Critical review on engineering deaminases for site-directed RNA editing. Curr. Opin. Biotechnol. 55, 74–80 (2018).

    Article  Google Scholar 

  27. Eggington, J. M., Greene, T. & Bass, B. L. Predicting sites of ADAR editing in double-stranded RNA. Nat. Commun. 2, 319 (2011).

    Article  Google Scholar 

  28. Prakash, T. P. et al. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796–8807 (2014).

    Article  CAS  Google Scholar 

  29. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    Article  CAS  Google Scholar 

  30. Fukuda, M. et al. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing. Sci. Rep. 7, 41478 (2017).

    Article  CAS  Google Scholar 

  31. Antonelli, G., Scagnolari, C., Moschella, F. & Proietti, E. Twenty-five years of type I interferon-based treatment: a critical analysis of its therapeutic use. Cytokine Growth Factor Rev. 26, 121–131 (2015).

    Article  CAS  Google Scholar 

  32. Malecki, M. J. et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol. Cell. Biol. 26, 4642–4651 (2006).

    Article  CAS  Google Scholar 

  33. Nüssler, A. K. et al. Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver. In Vitro Cell. Dev. Biol. Anim. 35, 190–197 (1999).

    Article  Google Scholar 

  34. Ramaswami, G. et al. Accurate identification of human Alu and non-Alu RNA editing sites. Nat. Methods 9, 579–581 (2012).

    Article  CAS  Google Scholar 

  35. Ramaswami, G. et al. Identifying RNA editing sites using RNA sequencing data alone. Nat. Methods 10, 128–132 (2013).

    Article  CAS  Google Scholar 

  36. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  37. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    Article  CAS  Google Scholar 

  38. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  39. Kent, W. J. BLAT--the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).

    Article  CAS  Google Scholar 

  40. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

    Article  Google Scholar 

  41. Ramaswami, G. & Li, J. B. RADAR: a rigorously annotated database of A-to-I RNA editing. Nucleic Acids Res. 42, D109–D113 (2014).

    Article  CAS  Google Scholar 

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We gratefully acknowledge the donation of primary fibroblasts from E. M. Valente (Università degli Studi di Salerno, Fisciano, Italy), of the hepatocytes cell line AKN-1 from A. Nüssler (BG Klinik, Tübingen, Germany), and of the U2OS Flp-In cell line from E. Schiebel (Universität Heidelberg, Germany). We gratefully acknowledge support from the Deutsche Forschungsgemeinschaft to T.S. (STA 1053/3-2; STA 1053/7-1). This work is supported by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63) with an intramural innovation grant for J.W. This work is supported by National Institutes of Health grants R01GM102484 and R01GM124215 to J.B.L.

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Authors and Affiliations



T.M., S.M., A.B., P.R., J.W., P.V. and T.S. conceived, performed and analyzed the experiments. Q.L. and J.B.L. analyzed and all authors interpreted next-generation sequencing data. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Thorsten Stafforst.

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Competing interests

T.S., J.W. and P.V. hold a patent on site-directed RNA editing (PCT/DE2016/000309). T.S., J.W., P.R. and T.M. are inventors of a filed patent based on the work published here.

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Integrated supplementary information

Supplementary Figure 1 Screening to improve the ADAR-recruiting domain.

A plasmid borne screening assay was applied to screen for improved ADAR-recruiting domains. For this, plasmids expressing the respective ASO as a chemically unmodified guideRNA from a U6 promotor were prepared. The guide RNA plasmids were co-overexpressed together with a reporter contruct (firefly luciferase) in 293 Flp-In T-REx cells expressing a specific ADAR isoform (A1p110 = ADAR1p110; A1p150 = ADAR1p150). Editing yields were determined by Sanger sequencing. Data are shown as the mean±SD, N=2 independent experiments

Supplementary Figure 2 Sequencing traces for editing of a 5′ UAG site in the 3′ UTR of GAPDH in 293 Flp-In T-REx ADAR cells.

Exemplary editing traces for the editings shown in Figure 1C in the manuscript, but including additional controls (“No RNA” = empty transfection; “18nt ASO no R/G” = ASO lacking the ADAR-recruiting domain; “unmod” means chemically unmodified, in-vitro transcribed ASOs of the indicated design v1, v4 or v9.4. Red asterisks indicate the editing sites.

Supplementary Figure 3 Editing yields for targeting a 5′ UAG codon in the 3′ UTR of GAPDH with chemically unmodified, in vitro transcribed ASOs in 293 Flp-In T-REx ADAR cells.

Unmodified in-vitro transcribed ASOs v1, v4 and v9.4 (5 pmol / 96well) were transfected into the respective ADAR-expressing Flp-In cell line. Data are shown as the mean±SD, N=3 independent experiments. A1p110 = ADAR1p110; A1p150 = ADAR1p150

Supplementary Figure 4 Western blot analysis of ADAR knockdown.

The western blot shown in Figure 2D in the manuscript was merged from images generated with two different exposure times. The part showing the ADAR bands comes from a 30 second exposure. The part showing β-actin from a 3 second exposure. The pictures were captured by the FusionCapt Advance SL4 (16.09b) software installed on the Fusion SL Vilber Lourmat (Vilber) western blot analyzer. No further image processing with respect to contrast or brightness was done. The western blot was done in technical duplicate.

Supplementary Figure 5 Determination of the effective dose (ED50) of the respective ASO for editing GAPDH in the respective 293 Flp-In T-REx ADAR cells.

Shown is an experiment completely analog to that shown in the manuscript in Figure 2E, but in the indicated ADAR-expressing 293 Flp-In T-REx cell. Data are shown as the mean±SD, N=3 independent experiments.

Supplementary Figure 6 Effect of cotransfection of a nontargeting ASO v9.5 or the chemically stabilized ADAR-recruiting domain v9.5 alone on the GAPDH 3′-UTR editing with ASO v9.5 in ADAR1p150-expressing 293 Flp-In T-REx cells.

This is an additional control experiment. The on-target is the 5´-UAG codon in the 3´-UTR of GAPDH. A surveyed potential off-target is the 5´-UAG site in the 3´-UTR of ACTB. SERPINA ASO v9.5 acts as a non-targeting control, as the target (SERPINA1) is not expressed in this cell line. Another control is the ADAR-recruiting domain v9.5. This is the isolated, chemically stabilized ADAR-recruiting domain lacking any specificity domain. An ASO v9.5 against the on-target was co-transfected with either the non-targeting control or the control lacking a specificity domain. On-target editing requires the presences of the matching ASO. The surveyed potential off-target (ACTB) was not edited to detectable level under any condition. The on-target yield was not perturbed by the presence of the non-targeting ASO or the ADAR-recruiting domain alone, suggesting that only the combination of matching specificity and ADAR-recruiting domain enables site-directed RNA editing. It further suggests that the natural editing capacity is not limiting the editing reaction. (5 pmol ASO/96 well have been used)

Supplementary Figure 7 Effect of cotransfection of a nontargeting ASO v9.5 or the chemically stabilized ADAR-recruiting domain v9.5 alone on the GAPDH 3′-UTR editing with ASO v9.5, but for the recruitment of endogenous ADAR in HeLa cells without IFN-α.

This control experiment is the exact copy of the expriment shown in the preceding Supplementary Figure but was carried out in HeLa cells, recruiting endogenous ADAR. Exactly the same results have been observed and the same conclusions can be drawn.

Supplementary Figure 8 Editing of 5′ UAG codons in the ORF of GAPDH versus 3′ UTR in ADAR-expressing 293 Flp-In T-REx cells.

Editing of two different 5´-UAG codons in the ORF of GAPDH in 293 Flp-In T-REx ADAR cells (ORF #1 and #2). A) ORF site #2; here the comparison was made to the editing of the 5´-UAG codon in the 3´-UTR; and all three ADAR-expressing 293 Flp-In T-REx cell lines are included. B) The editing of the ORF site #1 was only tested in ADAR1-expressing Flp-In T-REx cell lines. The results are very similar. Further editing experiments, as shown in Figure 3B, target ORF site #1. Data in A) and B) are shown as the mean±SD, N=3 independent experiments. A1p110 = ADAR1p110; A1p150 = ADAR1p150.

Supplementary Figure 9 Sequencing traces for editing of a 5′ UAG site (ORF site 2) in the ORF of GAPDH in 293 Flp-In T-REx ADAR cells.

Exemplary editing traces for the editings shown in Supplementary Figure 8A, but including additional controls (“No RNA” = empty transfection; “18nt ASO no R/G” = ASO lacking the ADAR-recruiting domain. Red asterisks indicate the editing sites. A reverse primer was used for sequencing.

Supplementary Figure 10 Editing yields for the editing of a 5′ UAG codon in the ORF of GAPDH in HeLa cells with ASO v25 containing a chemically unmodified versus modified ADAR-recruiting domain.

Here, an ASO v25 with a chemically unmodified ADAR-recruiting domain (unmod R/G), was compared to an ASO of the same sequence with addititional chemical modifiaction (all pyrimidine nucleotides in the ADAR-recruiting domain are backbone 2’-O-methylated). ASOs were transfected in HeLa cells. Data are shown as the mean±SD, N=3 independent experiments.

Supplementary Figure 11 Analysis of off-target editing sites with increased editing yield upon ASO treatment.

a) Besides the targeted site in GAPDH, 9 off-target editing sites were identified in ASO + IFN-α-treated cells compared to the control (no ASO + IFN-α). Six of them (CHARC1, SOD2 #1-#5) were known editing sites and found to be already edited in the control, N=2 independent experiments. b) and c) The regions around the off-target sites were aligned to the ASO-interacting region (40 nt) of the GAPDH transcript using MUSCLE ( The red A indicates the edited site and nucleotides matching to the target sequence of the ASO in GAPDH are highlighted in turquois. The sequence alignment suggests that the editing at the three novel editing sites (PRR11, GPR64, EFHD2) is clearly induced by misguiding through the ASO. Notably, the strongest off-target (PRR11) might be controllable by further chemical modification of the specificity domain of the ASO. Four of the nine off-target sites (SOD2 #2-5) lack any strong homology to target region, also the edited codon is different from 5´-UAG. This makes it very unlikely that the off-target editing at such sites was induced by the ASO via direct binding to the off-target site, also because those sites all reside in secondary RNA structure (Alu elements). However, we found a potential ASO binding site in the 3´-UTR of SOD2 (panel d) around nt 2100ff. (refering to NM_000636.3) that resides around 300 nt 5´ to the first Alu element (nt 2380-2670) and around 1300 nt 5´ to the second Alu element (nt 3400-3525). Since all SOD2 off-target sites reside in the two Alu elements one could imagine an ASO-dependent induction of the editing by either increase of the local ADAR concentration or by assisting the formation of an editable RNA secondary structure in the Alu element.

Supplementary Figure 12 Analysis of off-target editing sites with attenuated editing upon ASO treatment.

a) Five editing sites, all located in Alu sequences, were found to be significantly less edited in ASO-transfected, IFN-α-treated cells compared to the control lacking ASO transfection (but treated with IFN-α), N=2 independent experiments. b) and c) The regions around the off-target sites were aligned to the ASO-interacting region (40 nt) of the GAPDH transcript using MUSCLE ( The red A indicates the respective edited site and nucleotides matching with the ASO target sequence on GAPDH are highlighted in turquois. For the most strongly affected site (MAGT1), but also for the other four sites, the ASO seems to be able to bind tightly in proximity to the respective editing sites and therefore interrupt the dsRNA secondary structure of the Alu repeat, which is required for editing. This suggests that the attenuated editing found at those sites is caused by direct interaction of the ASO with the off-target transcript and is not due to a global sequestering of the ADAR enzyme by the ASO.

Supplementary Figure 13 Effect of IFN-α and ASO treatment on ADAR1 expression and the natural editing homeostasis.

a) FPKM values describing overall ADAR1 (p110+p150) expression following IFN-α treatment and ASO administration. IFN-α treatment induced ADAR1 expression in HeLa cells in a similar manner independent of ASO transfection. N=2 independent experiments. b) Analysis of significantly differently edited sites after IFN-α treatment in HeLa cells (no ASO transfection). Editing appears globally increased following IFN-α treatment. Significance of 20271 edited sites was tested using Fisher’s exact test (two-sided, p<0.01, N≥50); 116 sites were detected as significantly differently edited. The NGS expriment was done in independent duplicate.

Supplementary Figure 14 Sequencing traces of editing the PiZZ mutation in SERPINA1, showing on- and off-target editing in the A-rich 5′ CAA codon.

Exemplary sequencing traces of the 3 experimental conditions shown in Figure 3E of the manuscript. Red arrows indicate off-target, red asterisks indicate on-target editing sites, a reverse primer was used for sequencing. Shown are additional controls for empty transfection, and for transfecting an ASO lacking the ADAR-recruiting domain (no R/G). A) Editing in ADAR1p150-expressing 293 Flp-In T-REx cells; B) Editing of SERPINA1 PiZZ in HeLa cells expressing SERPINA1 PiZZ either genomically integrated (piggyBac) or transiently overexpressed (plasmid). In particular ASO v25 gave substantial off-target editing with the proximal adenosine in the targeted 5´-CAA codon.

Supplementary Figure 15 Improvement of editing specificity in the ASO:mRNA hybrid.

Editing of the 5´-CAA codon to restore the E342K mutation in SERPINA1 (a reverse primer was used here!) comes along with off target editing at the nearest neighboring adenosine (see also preceding Supplementary Figure, panel B). To reduce proximal off-target editing, the 3 nt gap in the modification pattern of the ASO was reduced to a 2 nt gap by putting an additional chemical modification (2´-O-methyl uridine) opposite the off-target nucleotide. Two representative sequencing traces were selected from three very similar replicates which show that the additional chemical modification strongly reduces the proximal off-target edit while only modestly influencing the on target editing. (editing was performed in HeLa cells with SERPINA1 PiZZ cDNA overexpressed from a plasmid, 5 pmol/96 well ASO was transfected)

Supplementary information

Supplementary Information

Supplementary Figures 1–15, Supplementary Tables 1 and 2, and Supplementary Note 1

Reporting Summary

Supplementary Dataset 1

Effect of IFN-α treatment in the absence of ASO

Supplementary Dataset 2

Effect of ASO treatment in the absence of IFN-α

Supplementary Dataset 3

Effect of ASO treatment in the presence of IFN-α

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Merkle, T., Merz, S., Reautschnig, P. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat Biotechnol 37, 133–138 (2019).

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