Large-Scale In Vivo Functional Characterization of the Human Cistrome

人类 Cistrome 的大规模体内功能表征

• 批准号: 9131776
• 负责人: MYLES A BROWN
• 金额: $ 73.5万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9131776
• 关键词: Binding; Biological Assay; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cell Line; Cell Survival; Cells; ChIP-seq; Chromatin; Cis Tests; Computing Methodologies; DNA-Protein Interaction; DNase I hypersensitive sites sequencing; Data; Development; Disease; Elements; Embryo; Enhancers; Environment; Essential Genes; Exons; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genetic Variation; Genomic DNA; Green Fluorescent Proteins; Growth; Guide RNA; Health; Human; Human Cell Line; Human Genome; In Situ; In Vitro; Internet; Investigation; Knock-out; Knowledge; Libraries; Maps; Measurement; Modeling; Mus; Nucleic Acid Regulatory Sequences; Phenotype; Physiology; Population; Publishing; Quantitative Trait Loci; Regulatory Element; Reporter; Resources; Screening Result; Somatic Mutation; Statistical Models; Techniques; Technology; Testing; Time; Tissues; Transcriptional Regulation; Untranslated RNA; Variant; Work; cell growth; cell type; computerized tools; design and construction; epigenome; epigenomics; functional genomics; genome editing; genome-wide; genome-wide analysis; improved; in vivo; in vivo Model; mouse model; novel; promoter; screening; transcription factor; transcriptome

 DESCRIPTION (provided by applicant): The in vitro functions of thousands of cis-elements have been characterized using massively parallel reporter assays. However, the in vivo characterization of cis-element functions has been limited to a handful of mammalian enhancers using mouse models. Recently, genome-wide CRISPR/Cas9 knockout screening has proven to be an efficient approach for assessing the function of genomic DNAs and presenting an opportunity to study in vivo cis-element functions on a large scale. We have developed a statistical model to predict gRNA efficiency for the optimization of CRISPR/Cas9 screens, as well as a computational pipeline for the analysis of the screen data. In addition we have performed pilot genome- wide CRISPR/Cas9 knockout screens in several cell lines and have identified known genes essential cell growth. We have demonstrated expertise in studying gene transcriptional regulation through genome-wide analysis of protein-DNA interactions and chromatin accessibility. In this proposal, we hypothesize that the simultaneous lentiviral delivery of two guide RNAs (gRNA) flanking a cis-regulatory element can efficiently knockout (KO) the element, and that CRISPR/Cas9 KO screens are an efficient approach for the large-scale in vivo functional characterization of human cis-elements. We propose to develop the experimental and computational approaches for high-throughput cistrome CRISPR/Cas9 deletion screens to elucidate the regulatory mechanisms of mammalian cis-element in vivo functions and expand our knowledge on transcriptional regulation in normal physiology and diseases. Specifically we propose to 1) use CRISPR/Cas9 knockout screens to identify transcription factors and chromatin regulators in eight human cell lines that have strong effect on cell growth; 2) conduct CRISPR/Cas9 knockout screens on putative cis-regulatory elements to identify elements with strong effects on gene expression and cell growth or survival; 3) computationally model in vivo cistrome function, experimentally validate the model and create a Cistrome annotation web server. At the conclusion of these studies, we will have developed the experimental techniques and computational tools for continued investigation of in vivo cis-element functions, and expanded our knowledge on the mechanism of cell-specific gene transcriptional regulation. The resulting resource will improve interpretation of the function of disease associated somatic mutations or germline variants in the non-coding regions.

 描述（由申请人提供）：已使用大规模平行报告基因试验表征了数千种顺式元件的体外功能。然而，在体内表征的顺式元件功能已被限制到少数哺乳动物增强子使用小鼠模型。最近，全基因组CRISPR/Cas9敲除筛选已被证明是评估基因组DNA功能的有效方法，并提供了大规模研究体内顺式元件功能的机会。我们已经开发了一种统计模型来预测用于优化CRISPR/Cas9筛选的gRNA效率，以及用于分析筛选数据的计算管道。此外，我们已经在几种细胞系中进行了试验性全基因组CRISPR/Cas9敲除筛选，并鉴定了已知的细胞生长必需基因。我们已经展示了通过全基因组分析蛋白质-DNA相互作用和染色质可及性来研究基因转录调控的专业知识。在这个提议中，我们假设同时的慢病毒递送 在一些实施方案中，侧接顺式调节元件的两个引导RNA（gRNA）的组合可以有效地敲除（KO）元件，并且CRISPR/Cas9 KO筛选是用于人顺式元件的大规模体内功能表征的有效方法。我们希望通过建立高通量顺式CRISPR/Cas9缺失筛选的实验和计算方法，阐明哺乳动物顺式元件在体内的功能调控机制，并扩展我们对正常生理和疾病中转录调控的认识。具体而言，我们提出1）使用CRISPR/Cas9敲除筛选来鉴定八种人类细胞系中对细胞生长具有强烈影响的转录因子和染色质调节因子; 2）对推定的顺式调节元件进行CRISPR/Cas9敲除筛选，以鉴定对基因表达和细胞生长或存活具有强烈影响的元件; 3）对体内顺反子函数进行计算建模，实验验证模型，并建立顺反子标注Web服务器。在这些研究的结论，我们将开发的实验技术和计算工具，在体内顺式元件功能的继续调查，并扩大我们的知识细胞特异性基因转录调控的机制。由此产生的资源将改善对非编码区疾病相关体细胞突变或种系变异的功能的解释。