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

细胞命运的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，以功能特征的基因组，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。高通量，大部分 由DNA元件百科全书（ENCODE）项目和其他类似项目进行的描述性测定， 已经促成了越来越多的与细胞类型相关的DNA和RNA调控元件列表， 特定的转录和表观遗传程序。然而，尽管估计有100万个顺式调节基因 元件，大概主要是增强子，调节蛋白质编码基因组，它们的身份和 它们所调控的基因在很大程度上仍是未知的。 在这里，我建议将高度可扩展的功能方法应用于候选增强子元素， 发展相关和易于处理的系统。具体来说，我正在进一步开发一个框架， 作为“大规模并行基因组编辑”或MPGE，它利用CRISPR/Cas9筛选方法， 单细胞RNA测序（scRNA-seq），以全局捕获基因表达的扰动。我会申请 这种方法对小鼠胚胎干细胞衍生的胚层，这是必不可少的身体计划组装 在早期发展中。总之，这项研究设计将验证候选增强子元件在其天然的 背景，同时也确定它们调节的靶基因。胚层特异性的功能验证 增强子-基因对将有助于了解细胞在早期发育中的命运。

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.