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Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Abstract

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.

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Fig. 1: Engineering cadRNAs.
Fig. 2: Transcriptome-wide and target transcript-level specificity profiles of cadRNAs.
Fig. 3: In vitro activity of cadRNAs.
Fig. 4: In vivo activity of cadRNAs.

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Data availability

RNA-seq data for Fig. 2a and Extended Data Figs. 4 and 5c are accessible at the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE164956. Any other data can be obtained from the corresponding author upon reasonable request. Publicly available datasets used in this study are as follows: GRCh38, release 32, https://www.gencodegenes.org/human/release_32.html GRCm39, release M27, https://www.gencodegenes.org/mouse/release_M27.htmlSource data are provided with this paper.

Code availability

Code is available from the corresponding author upon reasonable request.

References

  1. Melcher, T. et al. A mammalian RNA editing enzyme. Nature 379, 460–464 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. Bass, B. L. & Weintraub, H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 55, 1089–1098 (1988).

    Article  CAS  PubMed  Google Scholar 

  3. Bass, B. L. & Weintraub, H. A developmentally regulated activity that unwinds RNA duplexes. Cell 48, 607–613 (1987).

    Article  CAS  PubMed  Google Scholar 

  4. Mannion, N. M. et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 9, 1482–1494 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tomaselli, S. et al. Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma. Genome Biol. 16, 5 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Schoft, V. K., Schopoff, S. & Jantsch, M. F. Regulation of glutamate receptor B pre-mRNA splicing by RNA editing. Nucleic Acids Res. 35, 3723–3732 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wagner, R. W., Smith, J. E., Cooperman, B. S. & Nishikura, K. A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs. Proc. Natl Acad. Sci. USA 86, 2647–2651 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. Peng, Z. et al. Comprehensive analysis of RNA-seq data reveals extensive RNA editing in a human transcriptome. Nat. Biotechnol. 30, 253–260 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Tan, M. H. et al. Dynamic landscape and regulation of RNA editing in mammals. Nature 550, 249–254 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Levanon, E. Y. et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat. Biotechnol. 22, 1001–1005 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Woolf, T. M., Chase, J. M. & Stinchcomb, D. T. Toward the therapeutic editing of mutated RNA sequences. Biochemistry 92, 8298–8302 (1995).

    CAS  Google Scholar 

  14. 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 

  15. 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  PubMed  PubMed Central  Google Scholar 

  16. 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 

  17. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. Katrekar, D. et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16, 239–242 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Qu, L. et al. Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol. 37, 1059–1069 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Monteleone, L. R. et al. A bump-hole approach for directed RNA editing. Cell Chem. Biol. 26, 269–277 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Sinnamon, J. R. et al. In vivo repair of a protein underlying a neurological disorder by programmable RNA editing. Cell Rep. 32, 107878 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vallecillo-Viejo, I. C., Liscovitch-Brauer, N., Montiel-Gonzalez, M. F., Eisenberg, E. & Rosenthal, J. J. C. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Zamecnik, P. C. & Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl Acad. Sci. USA 75, 280–284 (1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dias, N. & Stein, C. A. Antisense oligonucleotides: basic concepts and mechanisms. Mol. Cancer Ther. 1, 347–355 (2002).

    CAS  PubMed  Google Scholar 

  29. Chung, H. et al. Human ADAR1 prevents endogenous RNA from triggering translational shutdown. Cell 172, 811–824 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Paul, C. P., Good, P. D., Winer, I. & Engelke, D. R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Litke, J. L. & Jaffrey, S. R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat. Biotechnol. 37, 667–675 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed Central  CAS  Google Scholar 

  33. Lehmann, K. A. & Bass, B. L. The importance of internal loops within RNA substrates of ADAR1. J. Mol. Biol. 291, 1–13 (1999).

    Article  CAS  PubMed  Google Scholar 

  34. Chen, Y. G. et al. Sensing self and foreign circular RNAs by intron identity. Mol. Cell 67, 228–238 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang, D. et al. Characterization of an MPS I-H knock-in mouse that carries a nonsense mutation analogous to the human IDUA-W402X mutation. Mol. Genet. Metab. 99, 62–71 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Garcia-Rivera, M. F. et al. Characterization of an immunodeficient mouse model of mucopolysaccharidosis type I suitable for preclinical testing of human stem cell and gene therapy. Brain Res. Bull. 74, 429–438 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Frankish, A. et al. GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 47, D766–D773 (2019).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Zhu, A., Ibrahim, J. G. & Love, M. I. Heavy-tailed prior distributions for sequence count data: removing the noise and preserving large differences. Bioinformatics 35, 2084–2092 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300 (1995).

    Google Scholar 

  44. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).

  45. Liu, Y., Wilson, T. J., McPhee, S. A. & Lilley, D. M. J. Crystal structure and mechanistic investigation of the twister ribozyme. Nat. Chem. Biol. 10, 739–744 (2014).

    Article  CAS  PubMed  Google Scholar 

  46. Felletti, M., Stifel, J., Wurmthaler, L. A., Geiger, S. & Hartig, J. S. Twister ribozymes as highly versatile expression platforms for artificial riboswitches. Nat. Commun. 7, 12834 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Miao, H. et al. A long noncoding RNA distributed in both nucleus and cytoplasm operates in the PYCARD-regulated apoptosis by coordinating the epigenetic and translational regulation. PLoS Genet. 15, e1008144 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank G. Chen, L. Hodge, U. Parekh, R. Perales and other members of the Mali laboratory for discussions, advice and help with experiments. This work was generously supported by University of California, San Diego Institutional Funds and National Institutes of Health (NIH) grants (R01HG009285 (P.M.), R01CA222826 (P.M.), R01GM123313 (P.M.) and 1K01DK119687 (D.M.)). This publication includes data generated at the University of California, San Diego IGM Genomics Center using an Illumina NovaSeq 6000 that was purchased with funding from an NIH SIG grant (S10 OD026929). Schematics were created using BioRender.

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Authors

Contributions

D.K. and P.M. conceived the study and wrote the paper. D.K., P.M., J.Y., Y.X., A.S. and Y.S. performed experiments. D.M. quantified RNA editing activity from RNA-seq data.

Corresponding author

Correspondence to Prashant Mali.

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Competing interests

D.K., J.Y. and P.M. have filed patents based on this work. P.M. is a scientific co-founder of Shape Therapeutics, Boundless Biosciences, Navega Therapeutics and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. Y.S. is an employee of Shape Therapeutics. D.K. is now an employee of Shape Therapeutics. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Michael Jantsch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of genetically encoded cadRNAs.

(a) RNA editing efficiencies achieved 48 hours and 96 hours post transfection of circular.200.100 and ribozyme.mutant.200.100 plasmids. Ribozyme.mutant.200.100 was created by substituting two key residues in both twister ribozymes (P3 ribozyme: residue 15 G to U and residue 16 U to G; P1 ribozyme: residue 22 A to G and residue 26 C to U) of the construct circular.200.10045,46. Values represent mean ± SEM (n = 3; p = 0.0021, p = 0.0112; unpaired t-test, two-tailed). (b) Schematic representation of various products detected by inward and outward binding primers used for quantification. The outward binding primers selectively amplify the cadRNA. The inward binding primers amplify uncleaved and cleaved-unligated fractions in addition to cadRNA. Values represent mean ± SEM (n = 3). (c) Cells transfected with circular.200.100 and ribozyme.mutant.200.100 plasmids were treated with actinomycin D for 1, 6 and 16 hours starting at 24 hours post transfections. qPCRs were carried out using inward binding primers from panel (b) and expression levels were normalized to untreated samples. (d) Levels of circular.100.50 and linear.100.50 adRNA were measured in the nucleus and cytoplasm. GFP transfected cells were included as controls. U1 snRNA and GAPDH were used to normalize for the nuclear and cytoplasmic compartments respectively. Relative U1 snRNA and GAPDH levels seen in the nuclear vs cytoplasmic fractions were consistent with other published work47. Values represent mean ± SEM (n = 3). All experiments were carried out in HEK293FT cells.

Source data

Extended Data Fig. 2 Curbing bystander editing of the RAB7A transcript.

Histograms of percent A-to-G editing within a 200 bp window around the target adenosine in the RAB7A transcript as quantified by Sanger sequencing. The target adenosine is located at position 0. The dsRNA stretch formed between the antisense and the target are shown below each histogram. Design 1 (cadRNA): Unmodified circular.200.100 antisense, in addition to the A-C mismatch at position 0, two mismatches are seen at positions +66 and +91 that were created to avoid a stretch of poly Us to allow for transcription from a U6 promoter. Design 2 (cadRNA.loops.interspersed.v1): Loops of size 8 bp created at position −5 and +30 relative to the target adenosine and additional 8 bp loops added at 15 bp intervals along the antisense strand. Design 3 (cadRNA.loops.interspersed.v2): As compared to v1, a G-mismatch was positioned opposite a highly edited A (at position +9), an additional 8 bp loop was added at position −81 and the loop at position +49 was changed to a 12 bp loop. Design 4 (cadRNA.loops.interspersed.v3): As compared to v1, the 8 bp loop at +30 was changed to a 12 bp loop starting at position +27, one additional 8 bp loop was added at position −81 and the loop at position +49 was changed to a 12 bp loop. Values represent mean % editing (n = 2). All experiments were carried out in HEK293FT cells.

Source data

Extended Data Fig. 3 Characterization of IVT synthesized cadRNAs.

qPCRs were carried out on cDNA synthesized from IVT-circular.200.100 adRNA and IVT-ribozyme.mutant.200.100 adRNA using primers binding to the ligation stem and ribozyme sequence. n.d.: not detected. Values represent mean ± SEM (n = 3).

Source data

Extended Data Fig. 4 In vivo specificity of cadRNAs.

2D histograms comparing the transcriptome-wide A-to-G editing yields observed with an AAV delivered construct (y-axis) to the yields observed with the control AAV construct (x-axis). Each histogram represents the same set of reference sites, where read coverage was at least 10 and at least one putative editing event was detected in at least one sample. Nsig is the number of sites with significant changes in editing yield. Points corresponding to such sites are shown with red crosses. The on-target editing efficiency values obtained in the RNA seq are highly inflated due to a large number of reads coming from the cadRNAs mapping onto the target and thus have been omitted from the 2D histograms. The on-target editing values obtained via Sanger sequencing for the four samples analyzed by RNA seq were mCherry-M1: 0%, mCherry-M2: 0%, 2x.circular.200.100-M1: 42.94% and 2x.circular.200.100-M2: 41.32% respectively. M1 and M2 refer to injected mouse 1 and 2.

Source data

Extended Data Fig. 5 Transcriptomic changes associated with in vivo cadRNA expression.

(a) qPCRs were carried out on IFN-inducible genes involved in sensing of dsRNA 2 weeks and 8 weeks post AAV injections. Values represent mean ± SEM (n = 3; p-values for 2 week long experiment, 2x.circular.200.100 vs mCherry, for genes from left to right p = 0.0721, p = 0.0353, p = 0.8082, p = 0.0748, p = 0.0303; p-values for 8 week long experiment, 2x.circular.200.100 vs mCherry, for genes from left to right p = 0.7276, p = 0.6020, p = 0.3838, p = 0.3491, p = 0.2746; unpaired t-test, two-tailed). (b) qPCRs were carried out on ADAR variants 2 weeks and 8 weeks post AAV injections. Values represent mean ± SEM (n = 3; p-values for 2-week long experiment, 2x.circular.200.100 vs. mCherry, for ADAR variants from left to right p = 0.3165, p = 0.1885, p = 0.2815; p-values for 8 week long experiment, 2x.circular.200.100 vs. mCherry, for genes from left to right p = 0.8150, p = 0.1440, p = 0.9532; unpaired t-test, two-tailed). (c) Transcriptome-wide differentially expressed genes in the two groups: 2x.circular.200.100 vs. mCherry are highlighted in red.

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Sanger sequencing trace files.

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Katrekar, D., Yen, J., Xiang, Y. et al. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs. Nat Biotechnol 40, 938–945 (2022). https://doi.org/10.1038/s41587-021-01171-4

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