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 Jiang; Xin 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

doi:10.1038/nature20565

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 journal › Article › peer-review

833 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 language	English (US)
Pages (from-to)	144-149
Number of pages	6
Journal	Nature
Volume	540
Issue number	7631
DOIs	https://doi.org/10.1038/nature20565
State	Published - Nov 16 2016

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10.1038/nature20565

https://repository.kaust.edu.sa/bitstream/10754/622418/1/Suzuki%20et%20al%20final%20submission.docx

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Suzuki, K., Tsunekawa, Y., Hernández-Benítez, R., Wu, J., Zhu, J., Kim, E. J., Hatanaka, F., Yamamoto, M., Araoka, T., Li, Z., Kurita, M., Hishida, T., Li, M., Aizawa, E., Guo, S., Chen, S., Goebl, A., Soligalla, R. D., Qu, J., ... Belmonte, J. C. I. (2016). In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 540(7631), 144-149. https://doi.org/10.1038/nature20565

@article{d4d697472ec24c519d4118d895642027,

title = "In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration",

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.",

author = "Keiichiro Suzuki and Yuji Tsunekawa and Reyna Hern{\'a}ndez-Ben{\'i}tez and Jun Wu and Jie Zhu and Kim, {Euiseok J.} and Fumiyuki Hatanaka and Mako Yamamoto and Toshikazu Araoka and Zhe Li and Masakazu Kurita and Tomoaki Hishida and Mo Li and Emi Aizawa and Shicheng Guo and Song Chen and April Goebl and Soligalla, {Rupa Devi} and Jing Qu and Tingshuai Jiang and Xin Fu and Maryam Jafari and Esteban, {Concepcion Rodriguez} and Berggren, {W. Travis} and Jeronimo Lajara and Estrella Nu{\~n}ez-Delicado and Pedro Guillen and Campistol, {Josep M.} and Fumio Matsuzaki and Guang-Hui Liu and Magistretti, {Pierre J.} and Kun Zhang and Callaway, {Edward M.} and Kang Zhang and Belmonte, {Juan Carlos Izpisua}",

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{\textquoteright}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{\textquoteright}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 Cat{\'o}lica San Antonio de Murcia (UCAM).",

year = "2016",

month = nov,

day = "16",

doi = "10.1038/nature20565",

language = "English (US)",

volume = "540",

pages = "144--149",

journal = "Nature",

issn = "0028-0836",

publisher = "Nature Publishing Group",

number = "7631",

}

Suzuki, K, Tsunekawa, Y, Hernández-Benítez, R, Wu, J, Zhu, J, Kim, EJ, Hatanaka, F, Yamamoto, M, Araoka, T, Li, Z, Kurita, M, Hishida, T, Li, M, Aizawa, E, Guo, S, Chen, S, Goebl, A, Soligalla, RD, Qu, J, Jiang, T, Fu, X, Jafari, M, Esteban, CR, Berggren, WT, Lajara, J, Nuñez-Delicado, E, Guillen, P, Campistol, JM, Matsuzaki, F, Liu, G-H, Magistretti, PJ, Zhang, K, Callaway, EM, Zhang, K & Belmonte, JCI 2016, 'In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration', Nature, vol. 540, no. 7631, pp. 144-149. https://doi.org/10.1038/nature20565

TY - JOUR

T1 - In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

AU - Suzuki, Keiichiro

AU - Tsunekawa, Yuji

AU - Hernández-Benítez, Reyna

AU - Wu, Jun

AU - Zhu, Jie

AU - Kim, Euiseok J.

AU - Hatanaka, Fumiyuki

AU - Yamamoto, Mako

AU - Araoka, Toshikazu

AU - Li, Zhe

AU - Kurita, Masakazu

AU - Hishida, Tomoaki

AU - Li, Mo

AU - Aizawa, Emi

AU - Guo, Shicheng

AU - Chen, Song

AU - Goebl, April

AU - Soligalla, Rupa Devi

AU - Qu, Jing

AU - Jiang, Tingshuai

AU - Fu, Xin

AU - Jafari, Maryam

AU - Esteban, Concepcion Rodriguez

AU - Berggren, W. Travis

AU - Lajara, Jeronimo

AU - Nuñez-Delicado, Estrella

AU - Guillen, Pedro

AU - Campistol, Josep M.

AU - Matsuzaki, Fumio

AU - Liu, Guang-Hui

AU - Magistretti, Pierre J.

AU - Zhang, Kun

AU - Callaway, Edward M.

AU - Zhang, Kang

AU - Belmonte, Juan Carlos Izpisua

N1 - 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).

PY - 2016/11/16

Y1 - 2016/11/16

N2 - 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.

AB - 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.

UR - http://hdl.handle.net/10754/622418

UR - http://dx.doi.org/10.1038/nature20565

UR - http://www.scopus.com/inward/record.url?scp=85000819280&partnerID=8YFLogxK

U2 - 10.1038/nature20565

DO - 10.1038/nature20565

M3 - Article

C2 - 27851729

SN - 0028-0836

VL - 540

SP - 144

EP - 149

JO - Nature

JF - Nature

IS - 7631

ER -

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

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