In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

Keiichiro Suzuki, Yuji Tsunekawa, Reyna Hernández-Benítez, Jun Wu, Jie Zhu, Euiseok J. Kim, Fumiyuki Hatanaka, Mako Yamamoto, Toshikazu Araoka, Zhe Li, Masakazu Kurita, Tomoaki Hishida, Mo Li, Emi Aizawa, Shicheng Guo, Song Chen, April Goebl, Rupa Devi Soligalla, Jing Qu, Tingshuai JiangXin Fu, Maryam Jafari, Concepcion Rodriguez Esteban, W. Travis Berggren, Jeronimo Lajara, Estrella Nuñez-Delicado, Pedro Guillen, Josep M. Campistol, Fumio Matsuzaki, Guang-Hui Liu, Pierre J. Magistretti, Kun Zhang, Edward M. Callaway, Kang Zhang, Juan Carlos Izpisua Belmonte

Research output: Contribution to journalArticlepeer-review

795 Scopus citations

Abstract

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.
Original languageEnglish (US)
Pages (from-to)144-149
Number of pages6
JournalNature
Volume540
Issue number7631
DOIs
StatePublished - Nov 16 2016

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): OSR-2015-CRG4-2631
Acknowledgements: 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).

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