High resolution dissection of oncogene enhancer networks via CRISPR screening and live-cell imaging.

通过 CRISPR 筛选和活细胞成像对癌基因增强子网络进行高分辨率解剖。

基本信息

批准号：
10522013
负责人：
Lei Stanley Qi
金额：
$ 43.98万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10522013
关键词：
3-Dimensional Architecture BCL2 gene Biology CRISPR interference CRISPR screen CRISPR-mediated transcriptional activation CRISPR/Cas technology Cancer cell line Cells Chromatin Chromatin Structure Clustered Regularly Interspaced Short Palindromic Repeats Color Complex DNA Data Disease Dissection Distant Elements Engineering Enhancers Exhibits GATA4 gene Gene Expression Gene Expression Regulation Genes Genetic Epistasis Genetic Predisposition to Disease Genetic Transcription Genome Genome engineering Genomic approach Growth Guide RNA Hela Cells Human Genome IRF1 gene Image Imaging technology Individual K-562 K562 Cells Leukemic Cell Libraries Light MYC gene Machine Learning Malignant Neoplasms Maps Mediating Mediator of activation protein Messenger RNA Methods Modeling Myeloid Leukemia Oncogenes Pathogenicity Pattern Penetrance Phase Play Promoter Regions Proteins RNA Regulation Regulatory Element Resolution Role Single Nucleotide Polymorphism Stretching Techniques Testing Time Transcription Coactivator Translating Untranslated RNA Variant Work base biological systems cancer cell cancer risk cancer type cell type clinical risk computer studies design disorder risk experimental study gene network genetic association genetic variant genome wide association study genomic locus live cell imaging machine learning model mammalian genome promoter real time monitoring screening

项目摘要

ABSTRACT Non-coding elements comprise 98% of the human genome. The coordination of non-coding regulatory elements in the mammalian genome plays a pivotal role in controlling gene expression. Both experimental and computational studies reveal that pathogenic genes involved in complex diseases, including oncogenes, are regulated by a large number of enhancers, implying the existence of a complex interdependent regulatory network of enhancers in modulating and maintaining expression of these genes. Genome-Wide Association Studies (GWAS) reveal that non-coding regulatory elements, including enhancers, are hotspots for the genetic predisposition to disease. To determine causal relationships between chromatin architecture and gene transcription, perturbation in a biological system is necessary. Recent advances in CRISPR-based genome engineering and live cell imaging technologies have enabled new techniques for ultrahigh resolution interrogation of the function of various genome regulatory elements and how they relate to gene expression. In preliminary studies in our lab, we performed a targeted CRISPR interference (CRISPRi) based screen to study how the 7 MYC enhancers present in K562 cells work together to co-regulate this oncogene. We created a library with >87,000 pairs of gRNAs targeting the MYC enhancers to understand the epistatic network of gene regulation underlying MYC expression. We found that when a subset of enhancer pairs were targeted together, they exhibited a more dramatic than expected reduction in growth rate. We developed a model that divides MYC enhancers into 2 layers that work together with varying degrees of efficiency to co-regulate MYC expression in K562 cells. Here, we seek to expand these preliminary results to examine additional oncogenes and perform these experiments in additional cell types. In addition, we will combine perturbation of oncogene enhancers with CRISPR-based live cell imaging (termed CRISPR LiveFISH), that allows for the dynamic imaging of multiple genomic loci, mRNA, and protein components in living cells. In Aim 1, we will develop an ultrahigh-resolution multiplexed CRISPRi/a tiling screens platform to dissect enhancer interactions of different oncogenes in different cancer cell lines. We will perform multiplexed CRISPRi/CRISPRa screens to inhibit or activate pairs of enhancers with an ultrahigh spatial resolution (~20bp) controlling four oncogenes (MYC, CCND, BCL2, PDE4DIP) in K562 and HeLa cells. In Aim 2, we will characterize the dynamic real-time interactions between transcriptional coactivators, mediators, multiple enhancers, promoters, and RNA transcription during CRISPRi/a-mediated perturbation. We will monitor real-time dynamics of different enhancers, promotors, RNA transcription, and the transcriptional coactivator proteins BRD4, IRF1, and Gata4 using LiveFISH with and without enhancer perturbation. Altogether, we seek to apply new CRISPR technologies developed in our lab to create a model of how oncogene enhancers are dynamically regulated across multiple oncogenes and in multiple types of cancer cells.

抽象的非编码元件占人类基因组的 98%。非编码监管要素的协调在哺乳动物基因组中，在控制基因表达方面发挥着关键作用。既是实验性的又是计算研究表明，涉及复杂疾病的致病基因，包括癌基因，受到大量增强子的调节，这意味着存在复杂的相互依赖的调节调节和维持这些基因表达的增强子网络。全基因组协会研究（GWAS）表明，非编码调控元件，包括增强子，是遗传的热点。患病倾向。确定染色质结构与基因之间的因果关系转录，生物系统中的扰动是必要的。基于 CRISPR 的基因组的最新进展工程和活细胞成像技术使超高分辨率询问新技术成为可能各种基因组调控元件的功能以及它们与基因表达的关系。在初步在我们实验室的研究中，我们进行了基于靶向 CRISPR 干扰 (CRISPRi) 的筛选，以研究 7 K562 细胞中存在的 MYC 增强子共同作用，共同调节该癌基因。我们创建了一个库 >87,000 对靶向 MYC 增强子的 gRNA，用于了解基因调控的上位网络基础 MYC 表达。我们发现，当增强子对的子集一起靶向时，它们增长率下降幅度超出预期。我们开发了一个划分MYC的模型增强子分为两层，以不同程度的效率协同作用，共同调节 MYC 的表达 K562细胞。在这里，我们寻求扩展这些初步结果以检查其他癌基因并进行这些实验是在其他细胞类型中进行的。此外，我们将把癌基因增强子的扰动与基于 CRISPR 的活细胞成像（称为 CRISPR LiveFISH），可对多个细胞进行动态成像活细胞中的基因组位点、mRNA 和蛋白质成分。在目标 1 中，我们将开发一种超高分辨率多重 CRISPRi/a 平铺筛选平台可剖析不同癌基因的增强子相互作用癌细胞系。我们将进行多重 CRISPRi/CRISPRa 筛选以抑制或激活增强子对具有超高空间分辨率 (~20bp) 控制 K562 中的四种癌基因（MYC、CCND、BCL2、PDE4DIP）和海拉细胞。在目标 2 中，我们将描述转录因子之间的动态实时交互。 CRISPRi/a 介导过程中的共激活子、介体、多个增强子、启动子和 RNA 转录扰动。我们将监测不同增强子、启动子、RNA 转录和使用带或不带增强子的 LiveFISH 检测转录共激活蛋白 BRD4、IRF1 和 Gata4 扰动。总之，我们寻求应用我们实验室开发的新 CRISPR 技术来创建一个模型癌基因增强子如何在多种癌基因和多种类型的癌症中动态调节细胞。