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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.

摘要