CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
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CRISPR/Cas9介导的人类三核受精卵基因编辑
DOI：
10.1007/s13238-015-0153-5
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发表时间：
2015-05
期刊：
影响因子：
21.1
通讯作者：
Huang, Junjiu
中科院分区：
文献类型：
--
作者：
Liang, Puping;Xu, Yanwen;Zhang, Xiya;Ding, Chenhui;Huang, Rui;Zhang, Zhen;Lv, Jie;Xie, Xiaowei;Chen, Yuxi;Li, Yujing;Sun, Ying;Bai, Yaofu;Songyang, Zhou;Ma, Wenbin;Zhou, Canquan;Huang, Junjiu
关键词：
诸如成簇规则间隔短回文重复序列（CRISPR）相关系统（Cas）等基因组编辑工具已广泛用于修饰动物受精卵和人类细胞等模型系统中的基因，并为基础研究和临床应用带来了巨大前景。迄今为止，我们对人类早期胚胎 DNA 修复机制以及在人类植入前胚胎中使用 CRISPR/Cas9 等技术的效率和潜在脱靶效应的理解仍然存在严重的知识差距。在本报告中，我们使用三原核（3PN）受精卵进一步研究人类细胞中 CRISPR/Cas9 介导的基因编辑。我们发现CRISPR/Cas9可以有效切割内源性β-珠蛋白基因（HBB）。然而，HBB同源重组定向修复（HDR）效率较低，且编辑后的胚胎是嵌合体。 T7E1 测定和全外显子组测序表明，这些 3PN 受精卵中的脱靶切割也很明显。此外，与HBB同源的内源性δ珠蛋白基因（HBD）与外源供体寡核苷酸竞争作为修复模板，导致不良突变。我们的数据还表明，这些胚胎中 HBB 基因座的修复优先通过非交叉 HDR 途径发生。总而言之，我们的工作强调了进一步提高 CRISPR/Cas9 平台的保真度和特异性的迫切需要，这是 CRSIPR/Cas9 介导的编辑的任何临床应用的先决条件。
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) ofHBBwas low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous toHBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of theHBBlocus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
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影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
7
作者：
Hosono, S;Faruqi, AF;Lasken, RS
通讯作者：
Lasken, RS
影响因子：
4
作者：
Kuwayama, M;Vajta, G;Kato, O
通讯作者：
Kato, O
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle