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.

项目摘要 ENCODE项目制作了高分辨率、高质量的“规则组”组成部分图， 一组组织和细胞系，鉴定推定的调控元件的集合。我们的提案旨在测试 这些推定元件与高通量合并CRISPR筛选的功能相关性。这 强大的平台将允许我们上调，下调或突变特定的调控元件，然后 探索这些扰动对正常生长条件下细胞存活的影响，以及各种压力 条件（氧化应激，蓖麻毒素毒性和营养剥夺），产生不同的敏感性基因 表情为了建立我们的目标，我们将使用ENCODE数据与其他联盟生成的数据 使用综合分析管道，其利用元件活性和基因之间的相关性 表达和更高阶的染色质相互作用，以连接功能元件与潜在的靶基因。为 对于我们已经观察到扰动影响增殖的~3000个基因，我们将产生多个 约100，000个指导RNA的文库，用于冗余干扰与这些基因连接的20，000个增强子， 我们的分析。在通过对引导RNA文库在测序之前和之后进行测序来鉴定该筛选中的“命中”之后， 在我们的测试条件下观察增殖减少的具体指南，我们还将测试组合 的增强子元件，可能会在合作或冗余的方式，探索功能联系， 一个特别的重点是超级增强器和他们的子元素。有了这些表型验证数据，我们 将通过选择100个单独的元素和50种组合来进行分子机制验证， 元件，以产生具有元件的工程化基因消融的稳定细胞系，并进行基因组- 可及染色质的广泛分子表征（ATAC-seq）、染色质环（HiChIA，一种新型的、高分子量的 效率染色质成环测定）和基因表达（RNA-seq）。我们还将产生和评估50个 线烧蚀整个超级增强器和单个超级增强器元件-无论是单独在 组合.对于这些细胞系的一个子集，我们将进行单细胞ATAC-seq，以阐明对变异的影响。 开放的染色质。我们还将比较来自合并CRISPR表达的结果， 报告基因测定与我们在天然基因组背景下产生编辑的方法相关联。分析这些数据，使用 以ENCODE数据为支架的强大的综合分析方法将生成全球地图， 在染色质水平上缺失的分子后果和基因表达变化。这些数据将 将迅速向社区发布，所有技术和细胞系将提供给ENCODE 和整个基因组学社区。这个项目将提供一个巨大的语料库的功能 将调控元件与基因联系起来的数据，以及这些基因的一个子集的广泛分子表征， 调节元件，提供理解功能元件的类别和逻辑的支架，验证 计算预测，并提供广泛扩展到其他细胞类型和组织的技术。