Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China; Kunming Biomed International and National Engineering Research Center of Biomedicine and Animal Science, Kunming 650500, China.
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China.
Cell. 2014 Feb 13;156(4):836-43. doi: 10.1016/j.cell.2014.01.027. Epub 2014 Jan 30.
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-γ 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.
猴子是研究人类疾病和开发治疗策略的重要模式生物，但由于难以在期望的靶位点对动物进行基因修饰，猴子在生物医学研究中的应用受到了显著阻碍。在这里，我们首次将 CRISPR/Cas9 系统（一种用于编辑不同生物基因的多功能工具）应用于猴基因组。通过将 Cas9 mRNA 和 sgRNA 共注射到单细胞期胚胎中，我们成功地在食蟹猴中实现了精确的基因靶向。我们还表明，该系统能够一步同时敲除两个靶基因（Ppar-γ 和 Rag1），且通过全面分析未检测到脱靶突变。因此，将 Cas9 mRNA 和 sgRNA 共注射到单细胞期胚胎是一种高效、可靠的基因修饰食蟹猴产生方法。
