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Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses

Nature Biomedical Engineering volume 4, pages 97–110 (2020)Cite this article

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Abstract

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann–Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.

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Fig. 1: Development of split-intein CBEs and ABEs.
Fig. 2: Optimization of split-intein base-editor AAVs.
Fig. 3: Systemic injection of v5 AAV9 editors results in cytosine and adenine base editing in heart, muscle and liver.
Fig. 4: AAV-mediated cytosine and adenine base editing in the CNS by two delivery routes.
Fig. 5: AAV-mediated cytosine and adenine base editing in the retina following sub-retinal injections of 2-week-old Rho-Cre;Ai9 mice.
Fig. 6: Base editing of Npc1I1061T in the mouse CNS.

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Data availability

The data that support the results of this study are available within the paper and its Supplementary Information. All unmodified reads for sequencing-based data in the manuscript are available from the NCBI Sequence Read Archive under accession number PRJNA532891. AAV genome sequences are provided in the Supplementary Information. Key plasmids from this work will be available from Addgene (depositor: D.R.L.), and other plasmids and raw data are available from the corresponding author on request.

Code availability

The custom code used in this study is provided in the Supplementary Information.

References

  1. Landrum, M. J. et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 42, D980–D985 (2014).

    CAS  PubMed  Google Scholar 

  2. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    PubMed  PubMed Central  Google Scholar 

  6. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    PubMed  Google Scholar 

  8. Ryu, S. M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

    CAS  PubMed  Google Scholar 

  9. Yeh, W. H., Chiang, H., Rees, H. A., Edge, A. S. B. & Liu, D. R. In vivo base editing of post-mitotic sensory cells. Nat. Commun. 9, 2184 (2018).

    PubMed  PubMed Central  Google Scholar 

  10. Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscl. Thromb. Vas. 37, 1741–1747 (2017).

    CAS  Google Scholar 

  11. Russell, S. et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849–860 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Carvalho, L. S. et al. Evaluating efficiencies of dual AAV approaches for retinal targeting. Front. Neurosci. 11, 503 (2017).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  14. Liu, D. R., Levy, J, M. & Yeh, W. H. AAV delivery of nucleobase editors. US patent 15/784,033 (2017).

  15. Truong, D. J. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    CAS  PubMed  Google Scholar 

  17. Wright, A. V. et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Zettler, J., Schutz, V. & Mootz, H. D. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 583, 909–914 (2009).

    CAS  PubMed  Google Scholar 

  19. Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Stevens, A. J. et al. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138, 2162–2165 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Shah, N. H., Eryilmaz, E., Cowburn, D. & Muir, T. W. Extein residues play an intimate role in the rate-limiting step of protein trans-splicing. J. Am. Chem. Soc. 135, 5839–5847 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR–Cas9. Nat. Biotechnol. 33, 102–106 (2015).

    CAS  PubMed  Google Scholar 

  23. Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).

    CAS  PubMed  Google Scholar 

  25. Grieger, J. C. & Samulski, R. J. Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps. J. Virol. 79, 9933–9944 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Choi, J. H. et al. Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons. Mol. Brain 7, 17 (2014).

    PubMed  PubMed Central  Google Scholar 

  28. Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    CAS  PubMed  Google Scholar 

  29. Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Gray, S. J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum. Gene Ther. 22, 1143–1153 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Mendell, J. R. et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl. J. Med. 377, 1713–1722 (2017).

    CAS  PubMed  Google Scholar 

  33. Wu, Z., Asokan, A. & Samulski, R. J. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol. Ther. 14, 316–327 (2006).

    CAS  PubMed  Google Scholar 

  34. Duan, D. Systemic AAV micro-dystrophin gene therapy for Duchenne muscular dystrophy. Mol. Ther. 26, 2337–2356 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Garvilles, R. G. et al. Dual functions of the RFTS domain of Dnmt1 in replication-coupled DNA methylation and in protection of the genome from aberrant methylation. PLoS ONE 10, e0137509 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat. Neurosci. 13, 423–430 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Inagaki, K. et al. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol. Ther. 14, 45–53 (2006).

    CAS  PubMed  Google Scholar 

  38. Duan, D., Yue, Y. & Engelhardt, J. F. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol. Ther. 4, 383–391 (2001).

    CAS  PubMed  Google Scholar 

  39. Xu, Z. et al. Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency. Hum. Gene Ther. 15, 896–905 (2004).

    CAS  PubMed  Google Scholar 

  40. Van Putten, M. et al. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice. FASEB J. 27, 2484–2495 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Li, D., Yue, Y. & Duan, D. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice. PLoS ONE 5, e15286 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Tuchman, M., Jaleel, N., Morizono, H., Sheehy, L. & Lynch, M. G. Mutations and polymorphisms in the human ornithine transcarbamylase gene. Hum. Mutat. 19, 93–107 (2002).

    CAS  PubMed  Google Scholar 

  43. Treacy, E. P. et al. Analysis of phenylalanine hydroxylase genotypes and hyperphenylalaninemia phenotypes using l-[1-13C]phenylalanine oxidation rates in vivo: a pilot study1. Pediatr. Res. 42, 430–435 (1997).

    CAS  PubMed  Google Scholar 

  44. Hamman, K. et al. Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol. Ther. 12, 337–344 (2005).

    CAS  PubMed  Google Scholar 

  45. Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).

    CAS  PubMed  Google Scholar 

  46. Asico, L. D. et al. Nephron segment-specific gene expression using AAV vectors. Biochem. Biophys. Res. Commun. 497, 19–24 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Foust, K. D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 59–65 (2009).

    CAS  PubMed  Google Scholar 

  48. Mercuri, E. et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. N. Engl. J. Med. 378, 625–635 (2018).

    CAS  PubMed  Google Scholar 

  49. Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Hordeaux, J. et al. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol. Ther. 26, 664–668 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Huang, Q. et al. Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. PLoS ONE 14, e0225206 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Harvey, R. J. & Napper, R. M. Quantitative study of granule and Purkinje cells in the cerebellar cortex of the rat. J. Comp. Neurol. 274, 151–157 (1988).

    CAS  PubMed  Google Scholar 

  53. Vogel, M. W., Sunter, K. & Herrup, K. Numerical matching between granule and Purkinje cells in lurcher chimeric mice: a hypothesis for the trophic rescue of granule cells from target-related cell death. J. Neurosci. 9, 3454–3462 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim, J. Y. et al. Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo. Eur. J. Neurosci. 37, 1203–1220 (2013).

    PubMed  PubMed Central  Google Scholar 

  55. Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E. & Jankowsky, J. L. Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J. Vis. Exp. 91, 51863 (2014).

    Google Scholar 

  56. Hoxha, E., Balbo, I., Miniaci, M. C. & Tempia, F. Purkinje cell signaling deficits in animal models of ataxia. Front. Syn. Neurosci. 10, 6 (2018).

    Google Scholar 

  57. Matilla-Duenas, A. et al. Consensus paper: pathological mechanisms underlying neurodegeneration in spinocerebellar ataxias. Cerebellum 13, 269–302 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Chakrabarty, P. et al. Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain. PLoS ONE 8, e67680 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  PubMed  Google Scholar 

  60. Zinn, E. et al. In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Rep. 12, 1056–1068 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Koch, S. F. et al. Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa. Proc. Natl Acad. Sci. USA 114, 5259–5264 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).

    CAS  PubMed  Google Scholar 

  63. Park, W. D. et al. Identification of 58 novel mutations in Niemann–Pick disease type C: correlation with biochemical phenotype and importance of PTC1-like domains in NPC1. Hum. Mutat. 22, 313–325 (2003).

    CAS  PubMed  Google Scholar 

  64. Praggastis, M. et al. A murine Niemann–Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele. J. Neurosci. 35, 8091–8106 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Yu, T., Shakkottai, V. G., Chung, C. & Lieberman, A. P. Temporal and cell-specific deletion establishes that neuronal Npc1 deficiency is sufficient to mediate neurodegeneration. Hum. Mol. Genet. 20, 4440–4451 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Loftus, S. K. et al. Rescue of neurodegeneration in Niemann–Pick C mice by a prion-promoter-driven Npc1 cDNA transgene. Hum. Mol. Genet. 11, 3107–3114 (2002).

    CAS  PubMed  Google Scholar 

  67. Lopez, M. E., Klein, A. D., Dimbil, U. J. & Scott, M. P. Anatomically defined neuron-based rescue of neurodegenerative Niemann–Pick type C disorder. J. Neurosci. 31, 4367–4378 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Elrick, M. J. et al. Conditional Niemann–Pick C mice demonstrate cell autonomous Purkinje cell neurodegeneration. Hum. Mol. Genet. 19, 837–847 (2010).

    CAS  PubMed  Google Scholar 

  69. Ko, D. C. et al. Cell-autonomous death of cerebellar Purkinje neurons with autophagy in Niemann–Pick type C disease. PLoS Genet. 1, 81–95 (2005).

    CAS  PubMed  Google Scholar 

  70. Langmade, S. J. et al. Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann–Pick C disease. Proc. Natl Acad. Sci. USA 103, 13807–13812 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Hughes, M. P. et al. AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann–Pick type C1 disease. Hum. Mol. Genet. 27, 3079–3098 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Ling, C. et al. High-efficiency transduction of primary human hematopoietic stem/progenitor cells by AAV6 vectors: strategies for overcoming donor-variation and implications in genome editing. Sci. Rep. 6, 35495 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Nathwani, A. C. et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N. Engl. J. Med. 371, 1994–2004 (2014).

    PubMed  PubMed Central  Google Scholar 

  74. Chandler, R. J. et al. Systemic AAV9 gene therapy improves the lifespan of mice with Niemann–Pick disease, type C1. Hum. Mol. Genet 26, 52–64 (2017).

    CAS  PubMed  Google Scholar 

  75. Xie, C., Gong, X. M., Luo, J., Li, B. L. & Song, B. L. AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease. J. Lipid Res. 58, 512–518 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat. Med. 12, 342–347 (2006).

    CAS  PubMed  Google Scholar 

  78. Habib, N. et al. Massively parallel single-nucleus RNA-Seq with DroNc-Seq. Nat. Methods 14, 955–958 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Li, P. et al. Allele-specific CRISPR–Cas9 genome editing of the single-base P23H mutation for rhodopsin-associated dominant retinitis pigmentosa. CRISPR J. 1, 55–64 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Sommer, C., Strähle, C., Köthe, U. & Hamprecht, F. A. Ilastik: Interactive learning and segmentation toolkit. In Eighth IEEE International Symposium on Biomedical Imaging 230–233 (ISBI, 2011).

  81. Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    PubMed  PubMed Central  Google Scholar 

  82. Tsai, S. Q. et al. CIRCLE-Seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    PubMed  PubMed Central  Google Scholar 

  84. Ullman-Cullere, M. H. & Foltz, C. J. Body condition scoring: a rapid and accurate method for assessing health status in mice. Lab. Anim. Sci. 49, 319–323 (1999).

    CAS  PubMed  Google Scholar 

  85. Foltz, C. & Ullman-Cullere, M. Guidelines for assessing the health and condition of mice. Lab Animal 28, 28–32 (1998).

    Google Scholar 

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. UG3 TR002636, U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062), St. Jude Collaborative Research Consortium, DARPA (HR0011-17-2-0049), Ono Pharma Foundation, Bill and Melinda Gates Foundation and Howard Hughes Medical Institute. We thank the Harvard Center for Biological Imaging for infrastructure and support. We thank F. Zhang, B. Deverman and K. Chan for equipment access, guidance and helpful discussions, M. Doan for image analysis assistance and A. Hamidi for assistance with editing the manuscript.

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Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Jonathan M. Levy, Wei-Hsi Yeh, Jessie R. Davis, Luke W. Koblan & David R. Liu

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Jonathan M. Levy, Wei-Hsi Yeh, Jessie R. Davis, Luke W. Koblan & David R. Liu

  3. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Jonathan M. Levy, Wei-Hsi Yeh, Jessie R. Davis, Luke W. Koblan & David R. Liu

  4. Program in Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, MA, USA

    Wei-Hsi Yeh

  5. Ocular Genomics Institute, Massachusetts Eye and Ear Institute, Boston, MA, USA

    Nachiket Pendse, Erin Hennessey, Rossano Butcher, Jason Comander & Qin Liu

  6. Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

    Nachiket Pendse, Erin Hennessey, Rossano Butcher, Jason Comander & Qin Liu

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Contributions

J.M.L. designed the research, constructed the plasmids, produced the AAV and performed the HEK cell, mouse systemic and CNS injection experiments. W.-H.Y. performed all of the CBE 3T3 experiments, image analysis and off-target analysis. J.R.D. performed the ABE 3T3 experiments. L.W.K. constructed the plasmids and performed the HEK cell experiments. N.P., R.B. and E.H. performed the retinal experiments. J.C. conceived of the retinal experiments and performed the data analysis. Q.L. conceived of and performed the sub-retinal injection experiments and data analysis. D.R.L. designed and supervised the research. J.M.L., W.-H.Y. and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Prime Medicine, Editas Medicine and Pairwise Plants, all of which are companies that use genome editing. D.R.L., J.M.L., W.-H.Y. and L.W.K. have filed patent applications on AAV systems for base editor delivery. The remaining authors declare no competing interests.

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Supplementary Figs. 1–11, Supplementary Tables 1 and 2, Supplementary Methods, Supplementary Code and Supplementary Notes.

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Levy, J.M., Yeh, WH., Pendse, N. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat Biomed Eng 4, 97–110 (2020). https://doi.org/10.1038/s41551-019-0501-5

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