Optimization of a massively parallel genome editing approach to link regulatory elements to their target genes during mouse germ layer formation

细胞命运多样化是哺乳动物发育的重要组成部分。随着人类的完成 基因组计划和随后的大规模努力，以确定基因组的功能，我们现在知道 编码和非编码DNA相互依赖地影响细胞命运的决定。高吞吐量，主要是 由DNA成分百科全书(ENCODE)项目和其他类似项目进行的描述性分析， 促进了与细胞类型相关的DNA和RNA调控元件的日益精选的清单 特定的转录和表观遗传程序。然而，尽管据估计有100万顺应监管 元素，可能主要是增强子，调节蛋白质编码的基因组，它们的身份和 他们调控的基因在很大程度上仍不为人所知。 在这里，我建议将高度可伸缩的函数方法应用于 与发展相关且易于处理的系统。具体地说，我正在进一步开发一个框架，称为 作为“大规模并行基因组编辑”或MPGe，利用CRISPR/Cas9筛选方法和 单细胞RNA测序(scRNA-seq)，以全局捕获基因表达的扰动。我会申请的 这种方法可以用于小鼠胚胎干细胞来源的生殖层，这是组装身体计划所必需的 处于早期开发阶段。总而言之，这项研究设计将验证其原生的候选增强子元素 同时识别他们调控的目标基因(S)。生殖层特异性的这种功能验证 增强子-基因对将深入了解细胞命运是如何在早期发育中出现的。

Cell fate diversification is part and parcel to mammalian development. With the completion of the Human Genome Project and subsequent large-scale efforts to functionally characterize the genome, we now know that coding and non-coding DNA interdependently affect cell fate decisions. High-throughout, largely descriptive assays carried out by the Encyclopedia of DNA Elements (ENCODE) Project and others like it, have contributed to an increasingly curated list of DNA and RNA regulatory elements associated with cell-type specific transcriptional and epigenetic programs. However, while it is estimated that 1 million cis-regulatory elements, presumably mostly enhancers, regulate the protein-coding genome, their identity and the genes they regulate remain largely unknown. Here, I propose to apply highly scalable functional approaches to candidate enhancer elements in a developmentally relevant and tractable system. Specifically, I am further developing a framework, known as 'massively parallel genome editing' or MPGE, that utilizes a CRISPR/Cas9 screening approach and single-cell RNA sequencing (scRNA-seq) to globally capture perturbations to gene expression. I will apply this method to mouse embryonic stem cell-derived germ layers, which are essential for body plan assembly in early development. Together, this study design will validate candidate enhancer elements in their native context while also identifying the target gene(s) that they regulate. This functional validation of germ layer-specific enhancer-gene pairs will yield insights into how cell fates emerge in early development.