生物医学研究所面临的最基本的挑战之一是研发并建立合适的动物模型。靶向基因操作被认为是建立理想动物模型的有效策略。由于遗传和生理上与人相似，猴被认为是当前最理想的模式动物。然而由于技术和多方面因素的限制，靶向基因工程猴的构建非常困难。最近发展起来的CRISPR-Cas9和TALEN系统已经成功应用于不同基因工程动物的构建。在利用上述技术实现猴基因敲除后，我们现在面临的挑战是：实现猴基因组的同源重组，制备靶向基因敲入猴。本课题拟选用食蟹猴为实验动物，以Oct4为靶基因，1、建立CRISPR/Cas9促进同源重组的猴基因敲入策略；2、完成猴的基因敲入，对Oct4的3’末端进行GFP标记，得到基因标记Oct4gfp食蟹猴。考虑到猴的性成熟需要4至5年时间，短期内不能进行功能和机制研究，本项目将在猴基因组同源重组取得突破，从而完善基因敲入猴模型制备策略，使基因工程猴在生物医学得到广泛应用。

One of the fundamental challenges for medical research is to develop appropriate animal models to fully recapitulate human diseases. Monkeys are considered as one of the most important models due to their genetic and physiological similarities to humans. Nevertheless, to generate gene engineering monkey is very difficult because of the difficulty in monkey gene manipulation, which limited the application of monkey models in medical research. Recently-developed CRISPR/Cas9 and TALEN systems have been successfully used for gene targeting in different model animals, which paves the way for precise gene targeting in monkeys. We succeeded in gene targeting in monkeys by using these two approaches. Here we propose to generate knockin monkeys through CRISPR/Cas9-assisted homologous recombination. The cynomolgus monkeys (Macaca fascicularis) will be used as the model animals. Oct4 is selected as candidate gene. With these, we aim, 1. to develop the CRISPR/Cas9-assisted homologous recombination strategy in monkeys; 2. to generate Oct4gfp monkeys by tagging the gfp to C-terminal of Oct4.