CRISPRi基因表达沉默系统在小鼠精原干细胞自我维持与增殖的分子机制研究中的应用

Spermatogonial stem cells（SSC）have the ability to self-renew and differentiate. They play important roles during mammalian spermatogenesis. The molecular mechanisms that regulate SSC remain unclear due to their rarity in the testis and complexity of the niche. During previous work on mouse SSC gene expression profile analysis, we found the expression of akt1s1 about 12 times higher than that of somatic cell in the testis. AKT1S1 is a proline-rich protein substrate of AKT, negatively regulate the expression of mTORC1 which regulates cell growth and survival in response to nutrient and hormonal signals. CRISPR interference (CRISPRi), a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system, is easy to design and operate. CRISPRi sgRNA-guided gene silencing is highly speci?c and do not have signi?cant off-target effects. It can simultaneously silence multiple genes without influence each other. In this study, akt1s1 and its interacting genes, including irs1,akt3，foxo3，hoxb13 which are predicted from bioinformatic analysis are going to be silenced in SSC cell line using CRISPRi technology, revealing mechanism of AKT1S1 function, providing a deep understanding to the molecular mechanisms of SSC maintenance and proliferation.

精原干细胞作为雄性生殖细胞的唯一来源，在哺乳动物精子发生的过程中起着至关重要的作用。目前，精原干细胞自我维持和增殖的分子机制尚未明确。前期工作中，我们对小鼠精原干细胞的基因表达谱进行了分析，发现akt1s1的表达比体细胞高约12倍。AKT1S1是AKT信号通路的底物，负调节mTORC1的表达，而mTORC1信号通路和细胞的生长、增殖、分化相关。CRISPRi是近期发展的RNA导向的基因沉默技术，该系统设计简洁，操作方便，特异性好，可同时沉默多个基因而不相互干扰，具有广泛的应用前景。本项目拟通过生物信息学方法预测与AKT1S1相互作用的蛋白，采用CRISPRi技术，利用精原干细胞体外培养系统，进行基因特异性沉默，揭示akt1s1生物学功能的作用机理，解析akt1s1在PI3K/AKT和mTORC1信号通路中与精原干细胞生长、增殖和维持的关系，加深对精原干细胞自我维持与增殖的分子机制的理解。