F31 Childcare Supplement: Optimization of a massively parallel genome editing approach to link regulatory elements to their target genes during mouse germ layer formation

项目总结 细胞命运多样化是哺乳动物发育的重要组成部分。随着人类基因组计划的完成以及随后对基因组功能特征的大规模研究，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。由DNA元素百科全书(ENCODE)项目和其他类似项目进行的高通量、主要是描述性的分析，促进了与细胞类型特定转录和表观遗传计划相关的DNA和RNA调控元件的日益精选的清单。然而，尽管据估计有100万个顺式调控元件(可能主要是增强子)调节蛋白质编码基因组，但它们的身份和它们调节的基因在很大程度上仍不清楚。 在这里，我建议将高度可伸缩的函数方法应用于与开发相关且易于处理的系统中的候选增强子元素。具体地说，我正在进一步开发一个框架，称为“大规模并行基因组编辑”或MPGe，它利用CRISPR/Cas9筛选方法和单细胞RNA测序(scRNA-seq)来全局捕获基因表达的扰动。我将把这种方法应用于小鼠胚胎干细胞来源的生殖层，这对早期发育的身体计划组装是必不可少的。总之，这项研究设计将验证候选增强子元件在其天然背景下的有效性，同时还将确定它们调控的目标基因(S)。这种生殖层特异性增强子-基因对的功能验证将有助于深入了解细胞命运是如何在早期发育中出现的。

PROJECT SUMMARY 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.