High-throughput systematic characterization of regulatory element function

调控元件功能的高通量系统表征

• 批准号: 9247643
• 负责人: MICHAEL C BASSIK
• 金额: $ 131.18万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9247643
• 关键词: ATAC-seq; Ablation; Affect; Atlases; Binding Sites; Biological Assay; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell Survival; Cells; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Chromatin Loop; Chromatin Structure; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Coupled; Data; Data Analyses; Data Set; Dissection; Elements; Engineering; Enhancers; Ensure; Epigenetic Process; Excision; Exhibits; Foundations; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Engineering; Genetic Enhancer Element; Genetic Transcription; Genome; Genomics; Growth; Guide RNA; Hand; Individual; International; Investigation; K562 Cells; Knock-out; Libraries; Link; Logic; Maps; Measurement; Measures; Mediating; Methods; Modification; Molecular; Mutate; Nuclear; Oxidative Stress; Phenotype; Population; Proxy; RNA; RNA library; Reagent; Recruitment Activity; Regulator Genes; Regulatory Element; Reporter; Repression; Resolution; Ricin; Series; Signal Transduction; Site; Stress; Techniques; Technology; Testing; Time; Tissues; Toxic effect; Transcription Repressor/Corepressor; Validation; Variant; Work; base; cell type; cohesin; combinatorial; embryonic stem cell; experimental study; genome-wide; insertion/deletion mutation; multimodality; mutant; novel; nutrient deprivation; preference; response; scaffold; screening; single cell analysis; stable cell line; tool; transcriptome sequencing

Project Summary The ENCODE project has produced high-resolution, high-quality maps of components of the `regulome' in a set of tissues and cell lines, identifying a collection of putative regulatory elements. Our proposal aims to test the functional relevance of these putative elements with high-throughput, pooled CRISPR screens. This powerful platform will allow us to up-regulate, down-regulate, or mutate specific regulatory elements, and then probe the effects of these perturbations on cell survival under normal growth conditions, and a variety of stress conditions (oxidative stress, ricin toxicity, and nutrient deprivation) that produce differential sensitivities to gene expression. To establish our targets, we will use ENCODE data in concert with other consortia-generated data using an integrative analysis pipeline that leverages both correlation between element activity and gene expression, and higher order chromatin interactions to link functional elements with potential target genes. For ~3000 genes for which we have observed that perturbation affects proliferation, we will generate multiple libraries of ~100,000 guide RNAs for redundantly perturbing 20,000 enhancers linked to these genes through our analysis. After identifying “hits” in this screen by sequencing the guide RNA libraries before and after proliferation under our test conditions to observe a reduction in specific guides, we will also test combinations of enhancer elements that may act in a cooperative or redundant fashion, exploring the functional linkages with a specific focus on superenhancers and their sub-elements. With these phenotypic validation data in hand, we will carry out molecular mechanistic validation by choosing 100 individual elements, and 50 combinations of elements to generate stable cell lines with engineered genetic ablation of elements, and carry out genome- wide molecular characterization of accessible chromatin (ATAC-seq), chromatin looping (HiChIA, a novel, high- efficiency chromatin looping assay), and gene expression (RNA-seq). We will also generate and assess 50 lines ablating entire superenhancers and individual superenhancer elements – both individually in in combination. For a subset of these lines, we will carry out single cell ATAC-seq to unravel effects on variations of open chromatin within the population of cells. We will also compare results from pooled CRISPR expression reporter assays to our method of generating edits in the native genomic context. Analyzing these data using powerful, integrative analysis methods, scaffolded from ENCODE data, will generate global maps of the molecular consequences of deletions at the level of chromatin and gene expression changes. These data will be rapidly released to the community, and all techniques and cell lines will be made available to the ENCODE consortium and to the genomics community at large. This project will deliver an immense corpus of functional data linking regulatory elements to genes, as well as extensive molecular characterization of a subset of these regulatory elements, providing a scaffold for understanding classes and logic of functional elements, validating computational predictions, and providing techniques that broadly extensible to other cell types and tissues.

项目概要 ENCODE 项目已经生成了高分辨率、高质量的“调节组”成分图 一组组织和细胞系，识别一组假定的调控元件。我们的建议旨在测试 这些假定元件与高通量、汇集的 CRISPR 筛选的功能相关性。这 强大的平台将使我们能够上调、下调或突变特定的监管要素，然后 探究这些扰动对正常生长条件下的细胞存活以及各种应激的影响 对基因产生不同敏感性的条件（氧化应激、蓖麻毒素毒性和营养缺乏） 表达。为了确定我们的目标，我们将结合使用 ENCODE 数据和其他联盟生成的数据 使用综合分析管道，利用元素活性和基因之间的相关性 表达，以及将功能元件与潜在靶基因连接起来的高阶染色质相互作用。为了 我们观察到扰动影响增殖的约 3000 个基因，我们将生成多个 约 100,000 个引导 RNA 的文库，用于冗余干扰与这些基因相关的 20,000 个增强子 我们的分析。通过对之前和之后的向导 RNA 文库进行测序来识别此屏幕中的“命中”后 在我们的测试条件下增殖以观察特定指南的减少，我们还将测试组合 可能以合作或冗余方式起作用的增强子元件，探索与 特别关注超级增强剂及其子元素。有了这些表型验证数据，我们 将通过选择100个单独元素和50种组合进行分子机制验证 元件以通过元件的工程遗传消除来产生稳定的细胞系，并进行基因组- 可及染色质的宽分子表征（ATAC-seq）、染色质环（HiChIA，一种新型的、高通量的） 效率染色质环化分析）和基因表达（RNA-seq）。我们还将生成并评估 50 消融整个超级增强器和单个超级增强器元件的线——两者都单独在 组合。对于这些细胞系的子集，我们将进行单细胞 ATAC-seq 以揭示对变异的影响 细胞群内开放染色质的数量。我们还将比较汇集的 CRISPR 表达的结果 记者对我们在本地基因组环境中生成编辑的方法进行了分析。使用分析这些数据 强大的综合分析方法，以编码数据为基础，将生成全球地图 染色质水平缺失和基因表达变化的分子后果。这些数据将 迅速向社区发布，所有技术和细胞系都将提供给 ENCODE 联盟和整个基因组学界。该项目将提供一个巨大的功能语料库 将调控元件与基因联系起来的数据，以及其中一部分的广泛分子特征 监管元素，为理解功能元素的类和逻辑提供支架，验证 计算预测，并提供可广泛扩展到其他细胞类型和组织的技术。