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 编码项目已经生成了“调节组”组成部分的高分辨率，高质量的地图 一组组织和细胞系，确定一系列推定的调节元件。我们的建议旨在测试 这些推定的元素与高通量，合并的CRISPR屏幕的功能相关性。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 探测这些扰动对正常生长条件下细胞存活的影响以及多种应力 对基因敏感的差异敏感性的疾病（氧化应激，ricin毒素毒性和营养剥夺） 表达。为了建立我们的目标，我们将与其他宪法的数据一起使用编码数据 使用利用元素活动和基因之间相关性的集成分析管道 表达和高阶染色质相互作用将功能元件与潜在靶基因联系起来。为了 〜3000个基因，我们观察到扰动会影响增殖，我们将产生多个 约100,000个指南RNA的图书馆，用于削减与这些基因相关的20,000个增强子 我们的分析。通过对导向RNA库进行测序，在此屏幕中识别“命中”之后 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 可以以合作或多余的方式起作用的增强子元素 针对超级女性及其子元素的特定关注。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个组合来执行分子机械验证 元素以产生稳定的细胞系具有工程元素的遗传消融，并进行基因组 - 可访问的染色质（ATAC-SEQ），染色质环（Hichia）（一种新型，高 - 高）的广泛分子表征 效率染色质循环测定）和基因表达（RNA-SEQ）。我们还将生成和评估50 排在整个超级女主角和单个超级元素的线条 - 都单独 组合。对于这些线的子集，我们将执行单细胞ATAC-SEQ，以解散变化的影响 细胞种群中的开放染色质。我们还将比较汇总CRISPR表达的结果 记者探讨我们在天然基因组环境中生成编辑的方法。使用 从编码数据脚手架的功能强大，集成的分析方法将生成 缺失在染色质和基因表达水平上的分子后果变化。这些数据将 迅速向社区释放，所有技术和细胞系都将提供给编码 财团和整个基因组学界。该项目将提供巨大的功能语料库 将调节元素与基因联系起来的数据，以及这些子集的广泛分子表征 监管元素，为了解功能元素的课程和逻辑提供了脚手架，验证 计算预测，并提供对其他细胞类型和组织广泛扩展的技术。