Target-stabilized base editors enable robust high-fidelity RNA editing

target-stabilized-base-editors-enable-robust-high-fidelity-rna-editing
Target-stabilized base editors enable robust high-fidelity RNA editing

References

  1. Crick, F. Central dogma of molecular biology. Nature 227, 561–563 (1970).

    Google Scholar 

  2. Manning, K. S. & Cooper, T. A. The roles of RNA processing in translating genotype to phenotype. Nat. Rev. Mol. Cell Biol. 18, 102–114 (2017).

    Google Scholar 

  3. Moore, M. J. & Proudfoot, N. J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009).

    Google Scholar 

  4. Wright, C. J., Smith, C. W. J. & Jiggins, C. D. Alternative splicing as a source of phenotypic diversity. Nat. Rev. Genet. 23, 697–710 (2022).

    Google Scholar 

  5. Tian, B. & Manley, J. L. Alternative polyadenylation of mRNA precursors. Nat. Rev. Mol. Cell Biol. 18, 18–30 (2017).

    Google Scholar 

  6. Nishikura, K. Functions and regulation of RNA editing by ADAR deaminases. Annu. Rev. Biochem. 79, 321–349 (2010).

    Google Scholar 

  7. Eisenberg, E. & Levanon, E. Y. A-to-I RNA editing – immune protector and transcriptome diversifier. Nat. Rev. Genet. 19, 473–490 (2018).

    Google Scholar 

  8. Liddicoat, B. J. et al. RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science 349, 1115–1120 (2015).

    Google Scholar 

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

    Google Scholar 

  10. Pestal, K. et al. Isoforms of RNA-editing enzyme ADAR1 independently control nucleic acid sensor MDA5-driven autoimmunity and multi-organ development. Immunity 43, 933–944 (2015).

    Google Scholar 

  11. Garrett, S. & Rosenthal, J. J. C. RNA editing underlies temperature adaptation in K+ channels from Polar Octopuses. Science 335, 848–851 (2012).

    Google Scholar 

  12. Yablonovitch, A.L. et al. Regulation of gene expression and RNA editing in adapting to divergent microclimates. Nat. Commun. 8, 1570 (2017).

  13. Pullirsch, D. & Jantsch, M. F. Proteome diversification by adenosine to inosine RNA-editing. RNA Biol. 7, 205–212 (2010).

    Google Scholar 

  14. Dadush, A. et al. DNA and RNA base editors can correct the majority of pathogenic single nucleotide variants. Npj Genom. Med. 9, https://doi.org/10.1038/s41525-024-00397-w (2024).

  15. Booth, B. J. et al. RNA editing: Expanding the potential of RNA therapeutics. Mol. Ther. 31, 1533–1549 (2023).

    Google Scholar 

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

    Google Scholar 

  17. Katrekar, D. 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).

    Google Scholar 

  18. Yi, Z. Y. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat. Biotechnol. 40, 946–955 (2022).

    Google Scholar 

  19. 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).

    Google Scholar 

  20. Reautschnig, P. et al. CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo. Nat. Biotechnol. 40, 759–768 (2022).

    Google Scholar 

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

    Google Scholar 

  22. Reautschnig, P. et al. Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA. Nat. Biotechnol.43, 545–557 (2024).

  23. Monian, P. et al. Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides. Nat. Biotechnol. 40, 1093–1102 (2022).

    Google Scholar 

  24. Yi, Z. Y. et al. Utilizing AAV-mediated LEAPER 2.0 for programmable RNA editing in non-human primates and nonsense mutation correction in humanized Hurler syndrome mice. Genome Biol. 24, 243 (2023).

    Google Scholar 

  25. Wang, Y. R., Park, S. & Beal, P. A. Selective recognition of RNA substrates by ADAR deaminase domains. Biochemistry 57, 1640–1651 (2018).

    Google Scholar 

  26. Zambrano-Mila, M.S. et al. Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2. Nat. Commun. 14, 8212 (2023).

  27. Uhlen, M. et al. Tissue-based map of the human proteome. Science 347, 1260419 (2015).

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

    Google Scholar 

  29. Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019).

    Google Scholar 

  30. Huang, X.X. et al. Programmable C-to-U RNA editing using the human APOBEC3A deaminase. Embo J. 39, https://doi.org/10.15252/embj.2020104741 (2020).

  31. Buchumenski, I. et al. Global quantification exposes abundant low-level off-target activity by base editors. Genome Res. 31, 2354–2361 (2021).

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  34. Marina, R.J., Brannan, K.W., Dong, K.D., Yee, B.A. & Yeo, G.W. Evaluation of engineered CRISPR-Cas-mediated systems for site-specific RNA editing. Cell Rep. 33, 108350 (2020).

  35. Xu, C. L. et al. Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes. Nat. Methods 18, 499–506 (2021).

    Google Scholar 

  36. Liu, Y.J. et al. REPAIRx, a specific yet highly efficient programmable A > I RNA base editor. Embo J. 39, https://doi.org/10.15252/embj.2020104748 (2020).

  37. Yan, H. & Tang, W.X. Programmed RNA editing with an evolved bacterial adenosine deaminase. Nat. Chem. Biol. 20, 1361–1370 (2024).

  38. Wu, J. H. et al. Live imaging of mRNA using RNA-stabilized fluorogenic proteins. Nat. Methods 16, 862–865 (2019).

    Google Scholar 

  39. Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    Google Scholar 

  40. Gadella, T. W. J. et al. mScarlet3: a brilliant and fast-maturing red fluorescent protein. Nat. Methods 20, 541–545 (2023).

    Google Scholar 

  41. Guharoy, M., Bhowmick, P., Sallam, M. & Tompa, P. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system. Nat. Commun. 7, 10239 (2016).

  42. Crowe, C. et al. Mechanism of degrader-targeted protein ubiquitinability. Sci. Adv. 10, eado6492 (2024).

    Google Scholar 

  43. Fu, H. L., Yang, Y. X., Wang, X. B., Wang, H. & Xu, Y. DeepUbi: a deep learning framework for prediction of ubiquitination sites in proteins. Bmc Bioinform. 20, 86 (2019).

    Google Scholar 

  44. Yeh, C. W. et al. The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes. Embo J. 40, e105846 (2021).

    Google Scholar 

  45. Koren, I. et al. The eukaryotic proteome is shaped by E3 ubiquitin ligases targeting C-terminal degrons. Cell 173, 1622–1635 (2018).

    Google Scholar 

  46. Smith, C. A., Calabro, V. & Frankel, A. D. An RNA-Binding chameleon. Mol. Cell 6, 1067–1076 (2000).

    Google Scholar 

  47. Smith, C. A., Crotty, S., Harada, Y. & Frankel, A. D. Altering the context of an RNA bulge switches the binding specificities of two viral Tat proteins. Biochemistry 37, 10808–10814 (1998).

    Google Scholar 

  48. Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).

  49. Calabro, V., Daugherty, M. D. & Frankel, A. D. A single intermolecular contact mediates intramolecular stabilization of both RNA and protein. Proc. Natl. Acad. Sci. USA 102, 6849–6854 (2005).

    Google Scholar 

  50. Rogers, S., Wells, R. & Rechsteiner, M. Amino-acid-sequences common to rapidly degraded proteins – the pest hypothesis. Science 234, 364–368 (1986).

    Google Scholar 

  51. Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010).

    Google Scholar 

  52. Lee, D. H. & Goldberg, A. L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 8, 397–403 (1998).

    Google Scholar 

  53. Wang, Y. R., Havel, J. & Beal, P. A. A phenotypic screen for functional mutants of human adenosine deaminase acting on RNA. Acs Chem. Biol. 10, 2512–2519 (2015).

    Google Scholar 

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

    Google Scholar 

  55. Matthews, M. M. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426–433 (2016).

    Google Scholar 

  56. Montiel-González, M.F., Vallecillo-Viejo, I.C. & Rosenthal, J.J.C. An efficient system for selectively altering genetic information within mRNAs. Nucleic Acids Res. 44, https://doi.org/10.1093/nar/gkw738 (2016).

  57. Wang, X. et al. Develop a compact RNA base editor by fusing ADAR with engineered EcCas6e. Adv. Sci. 10, 2206813 (2023).

  58. Lehmann, K. A. & Bass, B. L. Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities. Biochemistry 39, 12875–12884 (2000).

    Google Scholar 

  59. Tyagi, S. & Kramer, F. R. Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

    Google Scholar 

  60. Zhou, W. J. et al. Genetically encoded sensor enables endogenous RNA imaging with conformation-switching induced fluorogenic proteins. J. Am. Chem. Soc. 143, 14394–14401 (2021).

    Google Scholar 

  61. Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003).

    Google Scholar 

  62. Thomas, J.M. & Beal, P.A. How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs. Bioessays 39, https://doi.org/10.1002/bies.201600187 (2017).

  63. Park, S. et al. High-throughput mutagenesis reveals unique structural features of human ADAR1. Nat. Commun. 11, 5130 (2020).

  64. Yan, Z. et al. Next-generation IEDB tools: a platform for epitope prediction and analysis. Nucleic Acids Res. 52, W526–W532 (2024).

    Google Scholar 

  65. Vance, C. et al. ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules. Hum. Mol. Genet. 22, 2676–2688 (2013).

    Google Scholar 

  66. Shang, Y. L. & Huang, E. J. Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis. Brain Res. 1647, 65–78 (2016).

    Google Scholar 

  67. López-Erauskin, J. et al. ALS/FTD-linked mutation in FUS suppresses infra-axonal protein synthesis and drives disease without nuclear loss-of-function of FUS. Neuron 100, 1–15 (2018).

    Google Scholar 

  68. Wu, Z. J., Yang, H. Y. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).

    Google Scholar 

  69. Braak, H. et al. Amyotrophic lateral sclerosis-a model of corticofugal axonal spread. Nat. Rev. Neurol. 9, 708–714 (2013).

    Google Scholar 

  70. Wroe, R., Butler, A. W. L., Andersen, P. M., Powell, J. F. & Al-Chalabi, A. ALSOD: The amyotrophic lateral sclerosis online database. Amyotroph Lateral Scler. 9, 249–250 (2008).

    Google Scholar 

  71. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016).

    Google Scholar 

  72. Ono, M. et al. Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3) -: ANGPTL3 is cleaved and activated. J. Biol. Chem. 278, 41804–41809 (2003).

    Google Scholar 

  73. Romeo, S. et al. Rare loss-of-function mutations in family members contribute to plasma triglyceride levels in humans. J. Clin. Invest. 119, 70–79 (2009).

    Google Scholar 

  74. Davis, J. R. et al. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors. Nat. Biomed. Eng. 6, 1272–1283 (2022).

    Google Scholar 

  75. Bergmark, B. A. et al. Effect of Vupanorsen on non-high-density lipoprotein cholesterol levels in Statin-treated patients with elevated cholesterol: TRANSLATE-TIMI 70. Circulation 145, 1377–1386 (2022).

    Google Scholar 

  76. Kozak, M. An analysis of 5’-noncoding sequences from 699 vertebrate messenger-rnas. Nucleic Acids Res. 15, 8125–8148 (1987).

    Google Scholar 

  77. Miller, T. M. et al. Trial of antisense oligonucleotide Tofersen for ALS. New Engl. J. Med. 387, 1099–1110 (2022).

    Google Scholar 

  78. Snyder, M. P. et al. The human body at cellular resolution: the NIH Human Biomolecular Atlas Program. Nature 574, 187–192 (2019).

    Google Scholar 

  79. Porebski, B. T. & Buckle, A. M. Consensus protein design. Protein Eng. Des. Sel. 29, 245–251 (2016).

    Google Scholar 

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