Editing CG and non-CG DNA methylation to identify genomic elements that regulate gene expression

编辑 CG 和非 CG DNA 甲基化以识别调节基因表达的基因组元件

• 批准号: 10487529
• 负责人: Hani Goodarzi
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487529
• 关键词: Address; Biology; Brain; CRISPR screen; Cell division; Cells; Chimeric Proteins; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Correlative Study; CpG Islands; DNA; DNA Methylation; DNA Methylation Regulation; DNA Sequence; Deposition; Development; Disease; Elements; Enhancers; Epigenetic Process; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Silencing; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Genomic approach; Genomics; Genotype; Goals; Histones; Human; Human Biology; Human Chromosomes; Human Genome; Human Genome Project; Maps; Measures; Methods; Methylation; Modeling; Names; Neurons; Nucleic Acid Regulatory Sequences; Outcome Measure; Phenotype; Pluripotent Stem Cells; Proteins; Regulation; Regulator Genes; Regulatory Element; Research; Resolution; Resources; Sequence-Specific DNA Binding Protein; Specific qualifier value; Technology; Testing; Tissue-Specific Gene Expression; Untranslated RNA; Writing; base; biomedical scientist; cell type; combinatorial; epigenetic memory; epigenome editing; experimental study; functional genomics; gene repression; genetic regulatory protein; histone modification; human disease; human pluripotent stem cell; induced pluripotent stem cell; programs; promoter; stem cell differentiation; stem cells; tool; transcription factor; transcriptome

PROJECT SUMMARY / ABSTRACT A long-standing goal in biology is to define the relationship between genotype and phenotype. A major surprise of the human genome project was that the human genome encodes so few genes despite the complexity of cell types that compose for example, the human brain. As such it is assumed that combinatorial gene expression programs are key for specifying the function of specialized cell types such as neurons. Cell type specific gene expression programs therefore must be encoded by cis- and trans- non-coding regulatory DNA elements whose function is regulated by the epigenetic code and key proteins such as transcription factors. Elucidating how non-coding regulatory elements function to program cells will transform our understanding of human biology, development and disease. CRISPR/dCas9 technologies enable us to move beyond correlative studies, by editing the epigenome and determining the direct effect of epigenetic alterations on gene expression. We have created a new epigenetic editing functional genomics approach that we have named CRISPRoff. CRISPRoff robustly and specifically writes CpG DNA methylation (5mC) and repressive histone modifications to target loci. We are proposing to use CRISPRoff to map all genomic regulatory elements that are regulated by 5mC across an entire human chromosome. In the proposed experiments we will use perturb-seq, which combines pooled CRISPR screens with a single cell transcriptome readout, to directly measure how deposition of 5mC by CRISPRoff across an entire chromosome modulates gene expression. This approach will identify genetic regulatory elements key for induced pluripotent stem cells and neurons, a key step to understanding how tissue-specific gene expression is controlled. Our proposed research will serve to demonstrate the utility of this approach and motivate extending this approach to map gene regulatory elements across the entire human genome. The results of the proposed research will serve as a fundamental resource and roadmap for a broad community of biomedical scientists and greatly inform our understanding of human biology and disease.

项目概要/摘要 生物学的一个长期目标是定义基因型和表型之间的关系。一个专业 人类基因组计划的令人惊讶之处在于，人类基因组编码的基因如此之少，尽管 构成人类大脑的细胞类型的复杂性。因此假设 组合基因表达程序是指定特殊细胞类型功能的关键，例如 作为神经元。因此，细胞类型特异性基因表达程序必须由顺式和反式编码 非编码调控DNA元件，其功能受表观遗传密码和关键蛋白调控 例如转录因子。阐明非编码调控元件如何对细胞进行编程将 改变我们对人类生物学、发育和疾病的理解。 CRISPR/dCas9 技术使我们能够超越相关研究，通过编辑表观基因组和 确定表观遗传改变对基因表达的直接影响。我们创建了一个新的 表观遗传编辑功能基因组学方法，我们将其命名为 CRISPRoff。 CRISPR 稳健地关闭并且 专门将 CpG DNA 甲基化 (5mC) 和抑制性组蛋白修饰写入目标位点。我们是 提议使用 CRISPRoff 来绘制整个基因组中受 5mC 调控的所有基因组调控元件 整个人类染色体。在提议的实验中，我们将使用 perturb-seq，它结合了池化 CRISPR 筛选具有单细胞转录组读数，可直接测量 5mC 的沉积情况 整个染色体上的 CRISPRoff 调节基因表达。该方法将识别遗传 诱导多能干细胞和神经元的关键调控元件，这是了解如何进行的关键一步 组织特异性基因表达受到控制。我们提出的研究将有助于证明 这种方法并激励扩展这种方法来绘制整个基因调控元件的图谱 人类基因组。拟议研究的结果将作为基本资源和路线图 为广泛的生物医学科学家群体提供帮助，极大地增进我们对人类生物学的理解 疾病。