Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos
Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos
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DOI：
10.1016/j.cell.2014.01.027
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发表时间：
2014-02-13
期刊：
影响因子：
64.5
通讯作者：
Sha, Jiahao
中科院分区：
文献类型：
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作者：
Niu, Yuyu;Shen, Bin;Sha, Jiahao
猴子是研究人类疾病和发展治疗策略的重要模型，但是在生物医学研究中的应用在生产所需目标部位进行基因修饰的动物的困难极大地阻碍了生物医学研究。在这里，我们首先应用了CRISPR/CAS9系统，这是一种用于编辑不同生物体基因的多功能工具，以靶向猴子基因组。通过将Cas9 mRNA和SGRNA共同注射到一个单细胞阶段的胚胎中，我们成功地实现了cynomolgus猴子中的精确基因靶向。我们还表明，该系统可以在一个步骤中同时破坏两个靶基因（PPAR-GAMMA和RAG1），并且通过全面分析未检测到脱靶诱变。因此，与Cas9 mRNA和SGRNA一起将单细胞阶段的胚胎共同注射是一种有效且可靠的方法，用于基因改性的cynomolgus猴子产生。
Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-gamma and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.