Abstract

Efficient genome editing with Cas9–sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9–sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.

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Acknowledgements

We thank T. Jacks, P. Sharp, T. Tammela, Z. Weng, G. Gao, E. Sontheimer and A. Vegas for discussions and for sharing reagents, Y. Li and A. Park for technical assistance, and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512, DP2HL137167 and P01HL131471 (to W.X.). V.K. acknowledges support from the Russian Scientific Fund, Grant number 14-34-00017. H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH). 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.

Author information

Author notes

    • Chun-Qing Song
    • , Sneha Suresh
    •  & Qiongqiong Wu

    These authors contributed equally to this work.

Affiliations

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

    • Hao Yin
    • , Sneha Suresh
    • , Qiongqiong Wu
    • , Stephen Walsh
    • , Luke Hyunsik Rhym
    • , Kevin Kauffman
    • , Alicia Oberholzer
    • , Junmei Ding
    • , Roman L Bogorad
    • , Robert Langer
    •  & Daniel G Anderson
  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Chun-Qing Song
    • , Haiwei Mou
    • , Suet-Yan Kwan
    •  & Wen Xue
  3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Luke Hyunsik Rhym
    • , Kevin Kauffman
    • , Robert Langer
    •  & Daniel G Anderson
  4. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Esther Mintzer
    • , Mehmet Fatih Bolukbasi
    • , Lihua Julie Zhu
    • , Scot A Wolfe
    •  & Wen Xue
  5. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet Fatih Bolukbasi
    •  & Scot A Wolfe
  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua Julie Zhu
  7. Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Lihua Julie Zhu
    •  & Wen Xue
  8. Center of Translational Biomedicine, Skolkovo Institute of Science and Technology, Skolkovo, Moscow, Russia.

    • Timofei Zatsepin
    •  & Victor Koteliansky
  9. Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.

    • Timofei Zatsepin
  10. Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts, USA.

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

    • Robert Langer
    •  & Daniel G Anderson

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Contributions

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

Competing interests

H.Y., D.G.A. and R.L. have applied for patents related to this study. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Corresponding author

Correspondence to Daniel G Anderson.

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