孤雄单倍体胚胎干细胞携带CRISPR-Cas9文库用于筛选胚胎发育关键因子的研究

Genome-wide recessive genetic screening is a powerful approach to efficiently identify the set of genes underlying biological processes. This strategy has been successful in lower organisms such as yeast Saccharomyces cerevisiae and C. elegans. To date, there remains an unmet need for an efficient, large-scale, loss-of-function screening strategy at organismal level in mammalian systems. In 2012, we generated androgenetic haESCs (AG-haESCs) that could support full-term development of embryos upon injection into mature oocytes (intracytoplasmic AG-haESCs injection, semi-cloned technology), resulting in live animals referred to as semi-cloned (SC) animals. However, the birth rate of SC pups is extremely low. Recently, we successfully derived "sperm-like" AG-haESCs that could stably and efficiently support the generation of SC mice. Meanwhile, the CRISPR-Cas9 system from bacteria has been applied to efficient loss-of-function screening in mouse and human cells. In this proposed study, we will attempt to test whether "sperm-like" AG-haESCs would be used as a fertilization agent, which carrying genome-scale CRISPR-Cas9 knockout library, to generate mutant mice at a large-scale, thus enabling genetic screening at organismal level in mammalians. We will then use this system to screen factors that are critical for embryonic development. We believe that, from this proposed study, we will not only establish a brand-new system for genetic screening at organismal level in mouse, but also reveal some new factors that are critical for embryonic development.

个体水平的遗传筛选是生命科学研究的重要手段，目前只能在低等生物中开展，如何实现哺乳动物个体水平上的遗传筛选是生命科学研究领域的一项空白。2012年，本实验室建立了孤雄单倍体胚胎干细胞(AG-haESC)和将AG-haESC注入卵母细胞获得健康小鼠的半克隆技术；然而，半克隆小鼠出生效率低。最近，我们通过修饰AG-haESCs实现了半克隆小鼠的稳定出生；另外，快速发展CRISPR-Cas9技术使得哺乳动物细胞水平的遗传筛选成为可能。本研究中，我们以AG-haESC为中介体，携带CRISPR-Cas9文库，探讨实现小鼠个体水平遗传筛选的可行性。之后，利用这些技术系统筛选并获得一批影响胚胎发育的关键因子，包括单倍剂量不足基因和纯合突变影响胚胎发育的基因。本研究将建立一个基于小鼠个体水平遗传筛选的研究体系。

本项目以本实验室自主研发的孤雄单倍体胚胎干细胞(AG-haESC)和将AG-haESC注入卵母细胞获得健康小鼠的半克隆技术为基础，结合CRISPR-Cas9技术，将目的基因的sgRNA文库导入AG-haESC，快速并大量获得了携带目的基因突变的半克隆小鼠，实现了哺乳动物个体水平上的遗传筛选，填补了生命科学领域的一项空白。通过本项目的实施，优化并建立了快速敲低基因且能一步获得大量突变小鼠的方法，筛选出一些影响胚胎发育的关键基因，并把该方法拓展到调控小鼠骨发育关键基因的领域，及建立体氨基酸位点的遗传筛选体系。项目获得了一系列原创性的研究成果，建立一个基于小鼠个体水平遗传筛选的研究体系，对生命科学研究的发展起着巨大的促进作用。