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

项目摘要/摘要 生物学中的一个长期目标是确定基因型和表型之间的关系。一位少校 人类基因组计划令人惊讶的是，人类基因组编码的基因如此之少，尽管 例如，构成人脑的细胞类型的复杂性。因此，我们假设 组合基因表达程序是确定特定细胞类型功能的关键 作为神经元。因此，细胞类型特异的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。我们创造了一个新的 表观遗传编辑功能基因组学方法，我们将其命名为CRISPRoff。CRISPROF强健且 特异性地将CpG DNA甲基化(5mC)和抑制性组蛋白修饰写入目标基因座。我们是 建议使用CRISPRoff将受5mC调控的所有基因组调控元件映射到 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 CRISPR用单细胞转录组读数进行筛选，以直接测量5 mC的沉积情况 整个染色体上的CRISPROFF调节基因的表达。这一方法将识别基因 诱导多能干细胞和神经元的关键调控元件，这是了解 组织特异性基因表达受到控制。我们提议的研究将有助于证明 这一方法和动机将这种方法扩展到在整个 人类基因组。拟议研究的结果将作为基本资源和路线图 对于生物医学科学家的广泛社区，并极大地促进我们对人类生物学和 疾病。

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.