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

项目摘要 细胞命运的多样化是哺乳动物发育的重要组成部分。随着人类基因组计划的完成和随后对基因组功能特征的大规模努力，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元件百科全书（ENCODE）项目和其他类似项目进行的高通量，主要是描述性的测定，有助于越来越多地策划与细胞类型特异性转录和表观遗传程序相关的DNA和RNA调控元件的列表。然而，虽然估计有100万个顺式调控元件（可能主要是增强子）调控蛋白质编码基因组，但它们的身份及其调控的基因在很大程度上仍然未知。 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的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种称为“大规模并行基因组编辑”或MPGE的框架，该框架利用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衍生的胚层，这对早期发育中的身体计划组装至关重要。总之，这项研究设计将在其天然背景下验证候选增强子元件，同时还确定它们调节的靶基因。这种对胚层特异性增强子-基因对的功能验证将有助于深入了解细胞在早期发育中的命运。

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.