High throughput CRISPR-mediated functional validation of regulatory elements

项目摘要 拟议研究的首要目标是从功能上描述大量的候选人 哺乳动物基因组中的功能元件。Encode项目已经揭示了数百万个假定的 涉及一百多种细胞类型和组织的调控元件。虽然这些地图具有显著的 扩展我们对非编码序列的了解，距离拥有描述性地图仍然有很大差距 了解功能元件，并了解这些元件在基因调控下的生物学。这些 包括：(A)ENCODE实验预测的几个候选功能元件在功能上是 验证；(B)表观基因组学研究没有给出/揭示关于候选目标基因的信息 功能要素。因此，解释非编码DNA的生物学功能仍然是一个挑战 序列。为了解决这些问题，此UM1应用程序的目标是执行大规模功能 候选功能元件在其天然染色质环境中的特征。我们将首先确定 候选调控元件利用编码数据并生成细胞中感兴趣的报告标签基因 利用高吞吐量、自动化平台的生产线。第二，我们将询问候选功能元素 在它们的天然染色质环境中利用两个互补的高通量CRIPSR/Cas9介导 基因组编辑方法。我们预计这些分析将极大地提高我们对 哺乳动物细胞中候选调控区的生物学功能和基因调控。

Project Summary The overarching goal of the proposed study is to functionally characterize a large number of candidate functional elements in the mammalian genome. The ENCODE projects have revealed millions of putative regulatory elements across more than one hundred cell types and tissues. While these maps have significantly expanded our knowledge of non-coding sequences, there are still large gaps between having descriptive maps of functional elements and understanding the biology of these elements underlying gene regulation. These include: (a) few candidate functional elements predicted by the ENCODE experiments are functionally validated; (b) Epigenomic studies have not given/revealed information on the target genes of candidate functional elements. Therefore, it is still a challenge to interpret the biological functions of non-coding DNA sequences. To address these issues, the objective of this UM1 application is to perform large scale functional characterization of candidate functional elements in their native chromatin context. We will first identify candidate regulatory elements utilizing ENCODE data and generate reporter tagged genes of interest in cell lines utilizing a high throughput, automated platform. Second, we will interrogate candidate functional elements in their native chromatin contexts utilizing two complementary high throughput CRIPSR/Cas9 mediated genome editing approaches. We anticipate these analyses will significantly advance our knowledge of the biological functions of candidate regulatory regions and gene regulation in mammalian cells.