Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3,4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

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We are grateful to M. Kay, Z. Y. Chen, G. Lemke and P. G. Burrola for sharing experimental materials; J. Naughton, L. Lisowski and J. Marlett for AAV production; C. Fine, J. Olvera, E. O’Connor and K. E. Marquez for cell sorting; D. Okamura and M. Jacobs for mouse surgery and histology processing; D. Skowronska-Krawczyk for rat experiments; N. V. Gohad, T. Whitfield, I. M. Verma, J. Ogawa, T. Hara, U. Manor and J. Santini for imaging; L. Greg, Y. S. Kida and F. Osakada for valuable discussions; D. O’Keefe for proofreading the manuscript and M. Schwarz for administrative help. Core Facilities were utilized at the Salk Institute (support from: NIH-NCI CCSG: P30 014195, NINDS R24NS092943, and NEI P30 EY019005) and UCSD Neuroscience core grant P30 NS047101. R.H.B. was supported by a CONACYT fellowship of Mexico. J.Z. and T.J. were supported by 973 Program (2013CB967504, 2015CB964600) and 863 Program (2014AA021604). T.H. was partially supported by a Nomis Foundation Fellowship. E.J.K. is a Biogen-IDEC Fellow of the Life Science Research Foundation. M.Y. was partially supported by the Salk Women & Science Special Award. X.F. was supported by NSFC (No. 81601872). G.H.L. and J.Q. were supported by the National Basic Research Program of China (973 Program; 2015CB964800, 2014CB910503, 2013CB967504), National Natural Science Foundation of China (81625009, 81371342, 81271266), the National High Technology Research and Development Program of China (2015AA020307, 2014AA021604), and Program of Beijing Municipal Science and Technology Commission (Z151100003915072). F.M. was supported by RIKEN funding for Development and Regeneration. Ku.Z. was supported by NIH grant R01HL123755. P.J.M. and J.C.I.B. were supported by the King Abdullah University of Science and Technology (KAUST) Office of Sponsored Research (OSR) under award no. OSR-2015-CRG4-2631. Work in the laboratory of J.C.I.B. was supported by The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), the G. Harold and Leila Y. Mathers Charitable Foundation, NIH (R01HL123755), The McKnight Foundation, The Moxie Foundation, Fundacion Dr. Pedro Guillen and Universidad Católica San Antonio de Murcia (UCAM).

Author information

Author notes

    • Keiichiro Suzuki
    • , Yuji Tsunekawa
    • , Reyna Hernandez-Benitez
    •  & Jun Wu

    These authors contributed equally to this work.


  1. Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA

    • Keiichiro Suzuki
    • , Reyna Hernandez-Benitez
    • , Jun Wu
    • , Fumiyuki Hatanaka
    • , Mako Yamamoto
    • , Toshikazu Araoka
    • , Masakazu Kurita
    • , Tomoaki Hishida
    • , Mo Li
    • , Emi Aizawa
    • , April Goebl
    • , Rupa Devi Soligalla
    • , Concepcion Rodriguez Esteban
    •  & Juan Carlos Izpisua Belmonte
  2. Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan

    • Yuji Tsunekawa
    •  & Fumio Matsuzaki
  3. 4700 King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia

    • Reyna Hernandez-Benitez
    •  & Pierre Magistretti
  4. Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain

    • Jun Wu
    • , Toshikazu Araoka
    • , Jeronimo Lajara
    • , Estrella Nuñez-Delicado
    •  & Pedro Guillen
  5. Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, China

    • Jie Zhu
    • , Xin Fu
    •  & Kang Zhang
  6. Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA

    • Jie Zhu
    • , Tingshuai Jiang
    • , Xin Fu
    • , Maryam Jafari
    •  & Kang Zhang
  7. Systems Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA

    • Euiseok J. Kim
    •  & Edward M. Callaway
  8. Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA

    • Zhe Li
    • , Shicheng Guo
    • , Song Chen
    •  & Kun Zhang
  9. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

    • Jing Qu
  10. University of Chinese Academy of Sciences, Beijing 100049, China

    • Jing Qu
    •  & Guang-Hui Liu
  11. Guangzhou EliteHealth Biological Pharmaceutical Technology Company Ltd, Guangzhou 510005, China

    • Tingshuai Jiang
  12. Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA

    • W. Travis Berggren
  13. Fundación Dr. Pedro Guillen, Investigación Biomedica de Clinica CEMTRO, Avenida Ventisquero de la Condesa, 42, 28035 Madrid, Spain

    • Pedro Guillen
  14. Hospital Clinic, University of Barcelona, IDIBAPS, 08036 Barcelona, Spain

    • Josep M. Campistol
  15. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

    • Guang-Hui Liu
  16. Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou 510632, China

    • Guang-Hui Liu
  17. Beijing Institute for Brain Disorders, Beijing 100069, China

    • Guang-Hui Liu
  18. Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, China

    • Kang Zhang
  19. Veterans Administration Healthcare System, San Diego, California 92093, USA

    • Kang Zhang


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K.S., Y.T., R.H.B., J.W. and J.C.I.B. conceived the project and designed experiments. K.S., Y.T. and E.A. constructed plasmids. K.S., R.H.B., M.Y. and M.L. generated minicircle DNA vectors. K.S., Y.T., R.H.B., M.L., E.A., A.G. and R.D.S. performed work on HEK293 cells. K.S., Y.T., R.H.B. and E.A. performed bisulfite sequencing. K.S., Y.T. and R.H.B. measured intracellular localization of dCas9. K.S. and Y.T. performed the Surveyor assay. R.H.B. performed work on primary neurons. K.S., Y.T., R.H.B., E.A. and A.G. performed work on human ES-cell-derived pan neurons. Y.T. and F.M. performed work on in utero electroporation. J.Z., T.J., X.F., M.J. and Ka.Z. performed work on RCS rats. E.J.K. and E.M.C. performed work on adult mouse brain. F.H., T.A., M.K. and T.H. performed in vivo mouse electroporation. F.H., M.Y. and T.A. performed AAV IV and IM injection in neonatal or adult mice. Z.L., S.G., S.C. and Ku.Z. performed deep sequencing and analysed data. K.S. and E.A. performed single-cell genotyping. J.W., J.Q., C.R.E, W.T.B., J.L., E.N.D., P.G., J.M.C., G.H.L., P.M. and J.C.I.B. supervised the project or related experiments. K.S., Y.T., J.W. and J.C.I.B. wrote the manuscript with input from all the authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

Reviewer Information Nature thanks M. Porteus and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains the uncropped gels for Figures 3d, 4c and Extended Data Figures 1b, 2a, 3d, 4f, 5c, f, 7d

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