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

被引：864

作者：

Suzuki, Keiichiro ^{[1
]}

Tsunekawa, Yuji ^{[2
]}

Hernandez-Benitez, Reyna ^{[1
,3
]}

Wu, Jun ^{[1
,4
]}

Zhu, Jie ^{[5
,6
]}

Kim, Euiseok J. ^{[7
]}

Hatanaka, Fumiyuki ^{[1
]}

Yamamoto, Mako ^{[1
]}

Araoka, Toshikazu ^{[1
,4
]}

Li, Zhe ^{[8
]}

Kurita, Masakazu ^{[1
]}

Hishida, Tomoaki ^{[1
]}

Li, Mo ^{[1
]}

Aizawa, Emi ^{[1
]}

Guo, Shicheng ^{[8
]}

Chen, Song ^{[8
]}

Goebl, April ^{[1
]}

Soligalla, Rupa Devi ^{[1
]}

Qu, Jing ^{[9
,10
]}

Jiang, Tingshuai ^{[6
,11
]}

Fu, Xin ^{[5
,6
]}

Jafari, Maryam ^{[6
]}

Esteban, Concepcion Rodriguez ^{[1
]}

Berggern, W. Travis ^{[12
]}

Lajara, Jeronimo ^{[4
]}

Nunez-Delicado, Estrella ^{[4
]}

Guillen, Pedro ^{[4
,13
]}

Campistol, Josep M. ^{[14
]}

Matsuzaki, Fumio ^{[2
]}

Liu, Guang-Hui ^{[15
,16
,17
]}

Magistretti, Pierre ^{[3
]}

Zhang, Kun ^{[8
]}

Callaway, Edward M. ^{[7
]}

Zhang, Kang ^{[5
,6
,18
,19
]}

Belmonte, Juan Carlos Izpisua ^{[1
]}

机构：

[1] Salk Inst Biol Studies, Gene Express Lab, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA

[2] RIKEN, Ctr Dev Biol, Lab Cell Asymmetry, Chuo Ku, 2-2-3 Minatojima Minamimachi, Kobe, Hyogo 6500047, Japan

[3] 4700 King Abdullah Univ Sci & Technol KAUST, Thuwal 239556900, Saudi Arabia

[4] Univ Catolica San Antonio Murcia UCAM, Campus Jeronimos 135, Murcia 30107, Spain

[5] Guangzhou Med Univ, Guangzhou Women & Childrens Med Ctr, Guangzhou 510623, Guangdong, Peoples R China

[6] Univ Calif San Diego, Shiley Eye Inst, Inst Genom Med, Inst Engn Med, 9500 Gilman Dr 0946, La Jolla, CA 92023 USA

[7] Salk Inst Biol Studies, Syst Neurobiol Lab, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA

[8] Univ Calif San Diego, Bioengn, 9500 Gilman Dr,MC0412, La Jolla, CA 92093 USA

[9] Chinese Acad Sci, Inst Zool, State Key Lab Stem Cell & Reprod Biol, Beijing 100101, Peoples R China

[10] Univ Chinese Acad Sci, Beijing 100049, Peoples R China

[11] Guangzhou EliteHlth Biol Pharmaceut Technol Co Lt, Guangzhou 510005, Guangdong, Peoples R China

[12] Salk Inst Biol Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA

[13] Fdn Dr Pedro Guillen, Invest Biomed Clin CEMTRO, Ave Ventisquero de la Condesa 42, Madrid 28035, Spain

[14] Univ Barcelona, Hosp Clin, IDIBAPS, E-08036 Barcelona, Spain

[15] Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Natl Lab Biomacromol, Beijing 100101, Peoples R China

[16] Jinan Univ, Inst Aging & Regenerat Med, Key Lab Regenerat Med, Minist Educ, Guangzhou 510632, Guangdong, Peoples R China

[17] Beijing Inst Brain Disorders, Beijing 100069, Peoples R China

[18] Sichuan Univ, West China Hosp, Mol Med Res Ctr, Chengdu 610041, Peoples R China

[19] Vet Adm Healthcare Syst, San Diego, CA 92093 USA

来源：

NATURE | 2016年 / 540卷 / 7631期

基金：

中国国家自然科学基金; 国家高技术研究发展计划(863计划);

关键词：

LEBERS CONGENITAL AMAUROSIS; MINICIRCLE DNA VECTORS; GENE-THERAPY; TRANSGENE EXPRESSION; STEM-CELLS; IPSCS; ELECTROPORATION; RECOMBINATION; PROGENITORS; MECHANISM;

D O I：

10.1038/nature20565

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 inefficient(1), 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 disorders(2). 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.

引用

页码：144 / +

页数：24

共 45 条

[1] Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair [J].

Auer, Thomas O. ;

Duroure, Karine ;

De Cian, Anne ;

Concordet, Jean-Paul ;

Del Bene, Filippo .

GENOME RESEARCH, 2014, 24 (01) :142-153

[2] Effect of gene therapy on visual function in Leber's congenital amaurosis [J].

Bainbridge, James W. B. ;

Smith, Alexander J. ;

Barker, Susie S. ;

Robbie, Scott ;

Henderson, Robert ;

Balaggan, Kamaljit ;

Viswanathan, Ananth ;

Holder, Graham E. ;

Stockman, Andrew ;

Tyler, Nick ;

Petersen-Jones, Simon ;

Bhattacharya, Shomi S. ;

Thrasher, Adrian J. ;

Fitzke, Fred W. ;

Carter, Barrie J. ;

Rubin, Gary S. ;

Moore, Anthony T. ;

Ali, Robin R. .

NEW ENGLAND JOURNAL OF MEDICINE, 2008, 358 (21) :2231-2239

[3] Brains, Genes, and Primates [J].

Belmonte, Juan Carlos Izpisua ;

Callaway, Edward M. ;

Churchland, Patricia ;

Caddick, Sarah J. ;

Feng, Guoping ;

Homanics, Gregg E. ;

Lee, Kuo-Fen ;

Leopold, David A. ;

Miller, Cory T. ;

Mitchell, Jude F. ;

Mitalipov, Shoukhrat ;

Moutri, Alysson R. ;

Movshon, J. Anthony ;

Okano, Hideyuki ;

Reynolds, John H. ;

Ringach, Dario ;

Sejnowski, Terrence J. ;

Silva, Afonso C. ;

Strick, Peter L. ;

Wu, Jun ;

Zhang, Feng .

NEURON, 2015, 86 (03) :617-631

[4] Is Non-Homologous End-Joining Really an Inherently Error-Prone Process? [J].

Betermier, Mireille ;

Bertrand, Pascale ;

Lopez, Bernard S. .

PLOS GENETICS, 2014, 10 (01)

[5] Millisecond-timescale, genetically targeted optical control of neural activity [J].

Boyden, ES ;

Zhang, F ;

Bamberg, E ;

Nagel, G ;

Deisseroth, K .

NATURE NEUROSCIENCE, 2005, 8 (09) :1263-1268

[6] Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo [J].

Chen, ZY ;

He, CY ;

Ehrhardt, A ;

Kay, MA .

MOLECULAR THERAPY, 2003, 8 (03) :495-500

[7] Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver [J].

Chen, ZY ;

Yant, SR ;

He, CY ;

Meuse, L ;

Shen, S ;

Kay, MA .

MOLECULAR THERAPY, 2001, 3 (03) :403-410

[8] Multiplex Genome Engineering Using CRISPR/Cas Systems [J].

Cong, Le ;

Ran, F. Ann ;

Cox, David ;

Lin, Shuailiang ;

Barretto, Robert ;

Habib, Naomi ;

Hsu, Patrick D. ;

Wu, Xuebing ;

Jiang, Wenyan ;

Marraffini, Luciano A. ;

Zhang, Feng .

SCIENCE, 2013, 339 (6121) :819-823

[9] Therapeutic genome editing: prospects and challenges [J].

Cox, David Benjamin Turitz ;

Platt, Randall Jeffrey ;

Zhang, Feng .

NATURE MEDICINE, 2015, 21 (02) :121-131

[10] Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat [J].

D'Cruz, PM ;

Yasumura, D ;

Weir, J ;

Matthes, MT ;

Abderrahim, H ;

LaVail, MM ;

Vollrath, D .

HUMAN MOLECULAR GENETICS, 2000, 9 (04) :645-651

← 1 2 3 4 5 →