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：
10671756
负责人：
Lei Stanley Qi
金额：
$ 44.24万
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/10671756
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 MYC gene Machine Learning Malignant Neoplasms Maps Mediating Mediator Messenger RNA Methods Modeling Myeloid Leukemia Oncogenes Pattern Penetrance Phase Physical condensation Play Promoter Regions Proteins RNA Regulation Regulatory Element Resolution Role Single Nucleotide Polymorphism Stretching Techniques Testing Time Transcription Coactivator Translating Untranslated RNA Variant Work 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 ultra high resolution virulence gene

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％。非编码调节元件的协调在哺乳动物中，基因组在控制基因表达中起关键作用。都是实验性和计算研究表明，涉及复杂疾病的致病基因，包括癌基因，是受大量增强剂的调节，这意味着存在复杂的相互依存调节增强子网络调节和维持这些基因的表达。全基因组关联研究（GWAS）表明，非编码调节元件（包括增强剂）是遗传的热点疾病易感性。确定染色质结构与基因之间的因果关系需要转录，生物系统中的扰动。基于CRISPR的基因组的最新进展工程和现场成像技术已启用了超高分辨率询问的新技术各种基因组调节元件的功能及其与基因表达的关系。在初步在我们实验室的研究中，我们进行了针对性的CRISPR干扰（CRISPRI）屏幕，以研究7如何 K562细胞中存在的MYC增强剂共同调节这种癌基因。我们创建了一个库 > 87,000对针对MYC增强剂的GRNA，以了解基因调节的上皮网络 MYC的基础表达。我们发现，当将增强子对的子集靶向在一起时表现出比预期降低增长率更为戏剧性的。我们开发了一个分裂MYC的模型增强子分为两层，与不同程度的效率一起工作，以共同调节MYC的表达 K562细胞。在这里，我们试图扩大这些初步结果，以检查其他致癌物并执行这些实验在其他细胞类型中。此外，我们将将癌基增强剂的扰动与基于CRISPR的活细胞成像（称为CRISPR LiveFish），可以动态成像活细胞中的基因组基因局，mRNA和蛋白质成分。在AIM 1中，我们将发展超高分辨率多路复用CRISPRI/A平铺屏幕平台，以剖析不同癌症基因的增强子相互作用癌细胞系。我们将执行多重CRISPRI/CRISPRA屏幕以抑制或激活增强器对具有超高空间分辨率（〜20bp），可控制四个癌基因（MYC，CCND，BCL2，PDE4DIP）和HeLa细胞。在AIM 2中，我们将表征转录的动态实时相互作用 CRISPRI/A介导的同时激活者，介体，多个增强剂，启动子和RNA转录扰动。我们将监视不同增强剂，启动子，RNA转录的实时动力学和转录共激活因子蛋白BRD4，IRF1和GATA4使用LiveFish带有和没有增强剂的LiveFish 扰动。总的来说，我们试图在实验室中运用新的CRISPR技术来创建一个模型如何在多种癌基因和多种类型的癌症中动态调节致癌基因细胞。