CRISPR Knockout screens for the identification of X-linked genes that drive sex differences in mouse embryonic stem cells. CRISPR Knockout screens for the identification of X-linked genes that drive sex differences in mouse embryonic stem cells

Dosage imbalance for X-chromosomal genes contributes to sex differences, in particular during early development, when both X chromosomes are active in females. X-encoded inhibitors of the differentiation-promoting MAP kinase (MAPK) signalling pathway slow down development, increase levels of naive pluripotency factors, and decrease MAPK target gene expression. Through a hierarchical CRISPR screening approach in murine embryonic stem cells(mESC) we have comprehensively identified X-linked genes that modulate MAPK signalling, pluripotency factor expression, and differentiation. Overall design: 1. Primary knockout screen for the identification of genes that modulate MAPK target genes expression using an sgRNA library targeting all X-linked genes (SRE-Elk screen, GeCKOx sgRNA library). 2. Secondary knockout screens on top hits from the primary screen (GeCKOxs sgRNA library) for the identification of genes modulating pluripotency factor expression (Nanog screen), differentiation kinetics (Esrrb screen) and Mek phosphorylation (pMek screen).

X染色体基因的剂量失衡可引发性别差异，该效应在发育早期尤为突出——此时雌性个体的两条X染色体均处于激活状态。X染色体编码的、可抑制促分化丝裂原活化蛋白激酶（MAPK）信号通路的抑制剂，能够延缓发育进程、提升初始多能性因子的水平，并降低MAPK靶基因的表达量。我们通过在小鼠胚胎干细胞（mESC）中开展分层CRISPR筛选实验，全面鉴定出了能够调控MAPK信号通路、多能性因子表达以及细胞分化的X连锁基因。整体实验设计如下：
1. 一级敲除筛选：使用靶向所有X连锁基因的sgRNA文库（SRE-Elk筛选实验，GeCKOx sgRNA文库），以筛选鉴定调控MAPK靶基因表达的相关基因。
2. 二级敲除筛选：针对一级筛选得到的候选基因，采用GeCKOxs sgRNA文库开展二级筛选，分别用于鉴定调控多能性因子表达的基因（Nanog筛选实验）、细胞分化动力学的基因（Esrrb筛选实验）以及Mek磷酸化水平的基因（pMek筛选实验）。