CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein

被引：173

作者：

Tang, Lichun ^{[1
,2
]}

Zeng, Yanting ^{[3
]}

Du, Hongzi ^{[3
]}

Gong, Mengmeng ^{[1
]}

Peng, Jin ^{[1
]}

Zhang, Buxi ^{[1
]}

Lei, Ming ^{[3
]}

Zhao, Fang ^{[4
]}

Wang, Weihua ^{[5
]}

Li, Xiaowei ^{[6
]}

Liu, Jianqiao ^{[3
]}

机构：

[1] Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, 27 Taiping Rd, Beijing 100850, Peoples R China

[2] Natl Ctr Prot Sci Beijing, Life Sci Pk, Beijing 102206, Peoples R China

[3] Guangzhou Med Univ, Affiliated Hosp 3, Ctr Reprod Med, Guangzhou 510150, Guangdong, Peoples R China

[4] Guangxi Med Univ, Collaborat Innovat Ctr Targeting Tumor Diag & The, Natl Ctr Int Res Biol Targeting Diag & Therapy, Guangxi, Guangxi 530021, Peoples R China

[5] Houston Fertil Inst, Houston, TX 77063 USA

[6] Nanjing Univ Chineses Med, Dept Cardiol, Bayi Hosp, Nanjing 210002, Jiangsu, Peoples R China

来源：

MOLECULAR GENETICS AND GENOMICS | 2017年 / 292卷 / 03期

基金：

中国博士后科学基金;

关键词：

CRISPR/Cas9; Homology-directed repair (HDR); Cas9; protein; Gene modification; Human zygotes; ONE-STEP GENERATION; BREAK REPAIR; GENOME; MUTATIONS; SYSTEMS; MICE; DNA; RIBONUCLEOPROTEINS; ELECTROPORATION; PATHWAY;

D O I：

10.1007/s00438-017-1299-z

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

引用

页码：525 / 533

页数：9

共 26 条

[1] The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo [J].

Capmany, Gemma ;

Taylor, Alison ;

Braude, Peter R. ;

Bolton, Virginia N. .

MOLECULAR HUMAN REPRODUCTION, 1996, 2 (05) :299-306

[2] Playing the End Game: DNA Double-Strand Break Repair Pathway Choice [J].

Chapman, J. Ross ;

Taylor, Martin R. G. ;

Boulton, Simon J. .

MOLECULAR CELL, 2012, 47 (04) :497-510

[3] High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases [J].

Chen, Fuqiang ;

Pruett-Miller, Shondra M. ;

Huang, Yuping ;

Gjoka, Monika ;

Duda, Katarzyna ;

Taunton, Jack ;

Collingwood, Trevor N. ;

Frodin, Morten ;

Davis, Gregory D. .

NATURE METHODS, 2011, 8 (09) :753-U96

[4] Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes [J].

Chen, Sean ;

Lee, Benjamin ;

Lee, Angus Yiu-Fai ;

Modzelewski, Andrew J. ;

He, Lin .

JOURNAL OF BIOLOGICAL CHEMISTRY, 2016, 291 (28) :14457-14467

[5] Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9-sgRNA Ribonucleoproteins [J].

Cho, Seung Woo ;

Lee, Jihyun ;

Carroll, Dana ;

Kim, Jin-Soo ;

Lee, Junho .

GENETICS, 2013, 195 (03) :1177-+

[6] 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

[7] Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system [J].

Du, CS ;

Ren, XQ ;

Chen, LM ;

Jiang, WY ;

He, YS ;

Yang, M .

HUMAN HEREDITY, 1999, 49 (03) :133-138

[8] Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse [J].

Hashimoto, Masakazu ;

Yamashita, Yukiko ;

Takemoto, Tatsuya .

DEVELOPMENTAL BIOLOGY, 2016, 418 (01) :1-9

[9] THE LENGTH OF HOMOLOGY REQUIRED FOR GENE TARGETING IN EMBRYONIC STEM-CELLS [J].

HASTY, P ;

RIVERAPEREZ, J ;

BRADLEY, A .

MOLECULAR AND CELLULAR BIOLOGY, 1991, 11 (11) :5586-5591

[10] Regulation of Homologous Recombination in Eukaryotes [J].

Heyer, Wolf-Dietrich ;

Ehmsen, Kirk T. ;

Liu, Jie .

ANNUAL REVIEW OF GENETICS, VOL 44, 2010, 44 :113-139

← 1 2 3 →