基于CRISPR/Cas9聚焦型文库筛查和多基因转录激活系统诱导绵羊胚胎干细胞向雄性生殖细胞分化的研究

Inducing embryonic stem cells(ESCs) differentiation into male germ cells in vitro is of great importance for livestock breeding and also exploring the mechanism of germ cell development. However, the functional genes that regulate the differentiation of male germ cells are still not clear, which leads to low differentiation efficiency, and it is difficult to obtain germ cells with function in vitro. The CRISPR/Cas9 system can be edited at specific sites in the genome, making it an important tool for high throughput gene screening and gene function research. Therefore,we firstly used high-throughput sequencing technology to screen mRNA related with in vivo of sheep spermatogenesis, to establish CRISPR lentiviral library. Next, the library will be used to infect Cas9 expressed sheep ESCs, and infected sheep ESCs will be induced into germ cells and subjected to whole genome sequencing, to identify the genes that are essential for germ cells differentiation in vitro. Furthermore, 2-3 key genes will be selected and CRISPR/dCas9 based gene activation system will be used to induce the sheep ESCs differentiation into male germ cells. This project contributes to identify key genes that are essential for sheep ESCs differentaion into male germ cells in vitro, and improve the differentiation efficiency.

建立ES细胞体外诱导为雄性生殖细胞的方法，对家畜优良品种培育及生殖细胞发育机制的研究具有重要意义。然而因调控雄性生殖细胞分化的功能基因仍不明确，导致体外诱导效率低且难以获得功能齐全的生殖细胞。CRISPR/Cas9系统可在基因组特定位点进行编辑，使其成为高通量基因筛选和功能研究的重要工具。本项目拟首先利用转录组测序技术挖掘绵羊体内精子减数分裂前后差异表达基因，构建聚焦型CRISPR慢病毒文库；然后侵染体外稳定表达Cas9的绵羊ES细胞，经Bmp-4诱导后，测序鉴定绵羊ES细胞体外定向为雄性生殖细胞的必需基因；最后选择2-3个关键基因，利用CRISPR/dCas9介导的转录激活系统，建立高效的绵羊ES细胞分化为雄性生殖细胞的新模型。本项目有助于在全基因组水平上鉴定雄性生殖细胞诱导分化过程中的少数必需基因，建立绵羊ES细胞体外向功能性雄性生殖细胞的诱导技术体系，提高干细胞向生殖细胞分化效率。

建立稳定的ES细胞分离培养体系及体外诱导为生殖细胞的方法，对家畜优良品种培育及生殖细胞发育机制的研究具有重要意义。本研究建立了绵羊ES细胞系并诱导其体外向生殖细胞发生转分化。①成功获得了稳定传代至30代以上的绵羊ESCs，分离的ESCs具有与绵羊囊胚 ICM一致的多能性，体外可以形成拟胚体，体内可形成畸胎瘤并可分化为三个胚层；②利用scRNA-seq分析了绵羊 ESCs 中潜在的细胞类群，表明分离的绵羊 ESCs 可能介于原始态和始发态之间的中间状态，即活化/形成（formative）态；③利用Activin A、bFGF、BMP4等因子可将 ESCs 诱导为原始生殖样细胞，证明绵羊 ESCs 具有向生殖细胞诱导分化的能力；④利用单细胞测序技术建立了绵羊睾丸精子发生过程的转录表达图谱，筛选了生精发育过程的关键调控基因，可用于聚焦型CRISPR文库的建立；利用CRISPR/dCas9技术构建了4个关键候选基因的过表达激活载体，验证了载体的有效性，并建立了基于CRISPR系统的绵羊生殖细胞报告载体。通过这些研究，为绵羊雄性生殖细胞的体外分化机理的研究提供新思路，为家畜体外育种策略的实施提供技术支撑。