Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Published online:


The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

  • Subscribe to Nature Biotechnology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


Primary accessions



  1. 1.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  2. 2.

    & Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

  3. 3.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  4. 4.

    , & Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

  5. 5.

    & CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

  6. 6.

    et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl. Acad. Sci. USA 98, 641–645 (2001).

  7. 7.

    et al. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nat. Biotechnol. 25, 903–910 (2007).

  8. 8.

    et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010).

  9. 9.

    et al. Functional repair of CFTR by CRISPR-Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

  10. 10.

    et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

  11. 11.

    et al. Prevention of muscular dystrophy in mice by CRISPR-Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

  12. 12.

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

  13. 13.

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

  14. 14.

    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).

  15. 15.

    , & Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

  16. 16.

    et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

  17. 17.

    et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225–230 (2008).

  18. 18.

    State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316–328 (2011).

  19. 19.

    et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

  20. 20.

    et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA 107, 1864–1869 (2010).

  21. 21.

    et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172–176 (2010).

  22. 22.

    et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J. Am. Chem. Soc. 134, 6948–6951 (2012).

  23. 23.

    et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

  24. 24.

    et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

  25. 25.

    et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 7300–7306 (2015).

  26. 26.

    , , & A fast and sensitive alternative for β-galactosidase detection in mouse embryos. Development 139, 4484–4490 (2012).

  27. 27.

    et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197, 1069–1080 (2014).

  28. 28.

    et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364 (2015).

  29. 29.

    et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

  30. 30.

    et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2014).

  31. 31.

    et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584–586 (2015).

  32. 32.

    et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

  33. 33.

    et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011).

  34. 34.

    et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

  35. 35.

    et al. Canonical and atypical E2Fs regulate the mammalian endocycle. Nat. Cell Biol. 14, 1192–1202 (2012).

  36. 36.

    , , , & Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998).

  37. 37.

    et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010).

  38. 38.

    , , , & Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014).

  39. 39.

    , & Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

  40. 40.

    et al. Mfge8 is critical for mammary gland remodeling during involution. Mol. Biol. Cell 16, 5528–5537 (2005).

  41. 41.

    et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

  42. 42.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  43. 43.

    et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).

  44. 44.

    et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 11, 237 (2010).

  45. 45.

    Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Front. Biol. 10, 289–296 (2015).

  46. 46.

    , , & CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. PLoS One 9, e108424 (2014).

Download references


We thank M. Grompe, S. Levine, T. Jacks, P. Sharp, E. Sontheimer, C. Mello, P. Zamore, M. Moore, T. Flotte, T. Tammela, F. Sanchez-Rivera, T. Papagiannakopoulos, D. Wang, J. Moore and A. Vegas for discussions and for sharing reagents, S. Hough for technical assistance and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512 and Worcester Foundation (to W.X.). H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH Centers for Cancer Nanotechnology Excellence and the Harvard-MIT Center of Cancer Nanotechnology Excellence). Y.D. acknowledges support from the National Institute of Biomedical Imaging and Bioengineering for his postdoctoral fellowship 1F32EB017625. V.K. acknowledges support from the Russian scientific fund, grant number 14-34–00017. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center for technical support. We thank C. Wang at Boston Children's Hospital Viral Core for AAV prep (supported by core grant 5P30EY012196-17). The authors acknowledge the service to the MIT community of the late S. Collier.

Author information


  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Hao Yin
    • , Joseph R Dorkin
    • , Qiongqiong Wu
    • , Junghoon Yang
    • , Sneha Suresh
    • , Stephen Walsh
    • , Roman L Bogorad
    • , Robert Langer
    •  & Daniel G Anderson
  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Angela Park
    • , Aizhan Bizhanova
    •  & Wen Xue
  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Lihua J Zhu
    •  & Wen Xue
  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Joseph R Dorkin
  5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu
    • , Ankit Gupta
    • , Mehmet F Bolukbasi
    • , Scot A Wolfe
    •  & Wen Xue
  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua J Zhu
    •  & Zhiping Weng
  7. Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P.R. China.

    • Yingxiang Li
  8. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet F Bolukbasi
    •  & Scot A Wolfe
  9. Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Guangping Gao
  10. College of Pharmacy, the Ohio State University, Columbus, Ohio, USA.

    • Yizhou Dong
  11. Skolkovo Institute of Science and Technology, Skolkovo, Russia.

    • Victor Koteliansky
  12. Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Russia.

    • Victor Koteliansky
  13. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  14. Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson
  15. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer
    •  & Daniel G Anderson


  1. Search for Hao Yin in:

  2. Search for Chun-Qing Song in:

  3. Search for Joseph R Dorkin in:

  4. Search for Lihua J Zhu in:

  5. Search for Yingxiang Li in:

  6. Search for Qiongqiong Wu in:

  7. Search for Angela Park in:

  8. Search for Junghoon Yang in:

  9. Search for Sneha Suresh in:

  10. Search for Aizhan Bizhanova in:

  11. Search for Ankit Gupta in:

  12. Search for Mehmet F Bolukbasi in:

  13. Search for Stephen Walsh in:

  14. Search for Roman L Bogorad in:

  15. Search for Guangping Gao in:

  16. Search for Zhiping Weng in:

  17. Search for Yizhou Dong in:

  18. Search for Victor Koteliansky in:

  19. Search for Scot A Wolfe in:

  20. Search for Robert Langer in:

  21. Search for Wen Xue in:

  22. Search for Daniel G Anderson in:


H.Y., W.X. and D.G.A. designed the study. H.Y. and W.X. directed the project. H.Y., C.-Q.S., J.R.D., L.J.Z., Y.L., Q.W., J.Y., S.S., A.B., A.G., M.F.B., A.P., S.W. and R.L.B. performed experiments and analyzed data. G.G., Z.W., Y.D., V.K., S.A.W. and R.L. provided reagents and conceptual advice. H.Y., W.X. and D.G.A. wrote the manuscript with comments from all authors.

Competing interests

D.G.A., H.Y., J.R.K. and W.X. have applied for patents on the subject matter of this paper. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Corresponding authors

Correspondence to Wen Xue or Daniel G Anderson.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Sequences

  2. 2.

    Supplementary Table 7

    sgRNA2 GUIDE-seq +&- strand peaks

  3. 3.

    Supplementary Table 8

    sgRNA2 GUIDE-seq merged peaks

  4. 4.

    Supplementary Table 9

    Deep sequencing of off-target sites