High-throughput systematic characterization of regulatory element function
调控元件功能的高通量系统表征
基本信息
- 批准号:9247643
- 负责人:
- 金额:$ 131.18万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ATAC-seqAblationAffectAtlasesBinding SitesBiological AssayCRISPR libraryCRISPR screenCRISPR/Cas technologyCell LineCell NucleusCell SurvivalCellsChromatinChromatin Interaction Analysis by Paired-End Tag SequencingChromatin LoopChromatin StructureChromosomesClustered Regularly Interspaced Short Palindromic RepeatsCollectionCommunitiesCoupledDataData AnalysesData SetDissectionElementsEngineeringEnhancersEnsureEpigenetic ProcessExcisionExhibitsFoundationsGene ExpressionGene Expression ProfilingGene Expression RegulationGene TargetingGenerationsGenesGenetic EngineeringGenetic Enhancer ElementGenetic TranscriptionGenomeGenomicsGrowthGuide RNAHandIndividualInternationalInvestigationK562 CellsKnock-outLibrariesLinkLogicMapsMeasurementMeasuresMediatingMethodsModificationMolecularMutateNuclearOxidative StressPhenotypePopulationProxyRNARNA libraryReagentRecruitment ActivityRegulator GenesRegulatory ElementReporterRepressionResolutionRicinSeriesSignal TransductionSiteStressTechniquesTechnologyTestingTimeTissuesToxic effectTranscription Repressor/CorepressorValidationVariantWorkbasecell typecohesincombinatorialembryonic stem cellexperimental studygenome-wideinsertion/deletion mutationmultimodalitymutantnovelnutrient deprivationpreferenceresponsescaffoldscreeningsingle cell analysisstable cell linetooltranscriptome sequencing
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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MICHAEL C BASSIK其他文献
MICHAEL C BASSIK的其他文献
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{{ truncateString('MICHAEL C BASSIK', 18)}}的其他基金
High-throughput development and characterization of compact tools for transcriptional and chromatin perturbations
用于转录和染色质扰动的紧凑工具的高通量开发和表征
- 批准号:
10632140 - 财政年份:2021
- 资助金额:
$ 131.18万 - 项目类别:
Project 3: Systematic characterization of factors controlling breast cancer progression and resistance
项目3:控制乳腺癌进展和耐药因素的系统表征
- 批准号:
10704691 - 财政年份:2021
- 资助金额:
$ 131.18万 - 项目类别:
Project 3: Systematic characterization of factors controlling breast cancer progression and resistance
项目3:控制乳腺癌进展和耐药因素的系统表征
- 批准号:
10272391 - 财政年份:2021
- 资助金额:
$ 131.18万 - 项目类别:
Project 3: Systematic characterization of factors controlling breast cancer progression and resistance
项目3:控制乳腺癌进展和耐药因素的系统表征
- 批准号:
10911510 - 财政年份:2021
- 资助金额:
$ 131.18万 - 项目类别:
High-throughput development and characterization of compact tools for transcriptional and chromatin perturbations
用于转录和染色质扰动的紧凑工具的高通量开发和表征
- 批准号:
10276866 - 财政年份:2021
- 资助金额:
$ 131.18万 - 项目类别:
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:
10238366 - 财政年份:2020
- 资助金额:
$ 131.18万 - 项目类别:
Development of novel protein-based therapeutics for lung cancer
开发基于蛋白质的新型肺癌疗法
- 批准号:
10373026 - 财政年份:2018
- 资助金额:
$ 131.18万 - 项目类别:
Development of novel protein-based therapeutics for lung cancer
开发基于蛋白质的新型肺癌疗法
- 批准号:
10133002 - 财政年份:2018
- 资助金额:
$ 131.18万 - 项目类别:
Development of novel protein-based therapeutics for lung cancer
开发基于蛋白质的新型肺癌疗法
- 批准号:
9894638 - 财政年份:2018
- 资助金额:
$ 131.18万 - 项目类别:
Using Protein Interaction Networks and Combinatorial Screens to target KRAS driven cancer
使用蛋白质相互作用网络和组合筛选来靶向 KRAS 驱动的癌症
- 批准号:
9315124 - 财政年份:2015
- 资助金额:
$ 131.18万 - 项目类别:
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