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

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。一个主要 令人惊讶的是，人类基因组编码的基因如此之少，尽管 复杂的细胞类型，例如，组成人类大脑。因此，假设 组合基因表达程序是指定特定细胞类型功能的关键， 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，其功能由表观遗传密码和关键蛋白质调控 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/dCas 9技术使我们能够超越相关研究，通过编辑表观基因组， 确定表观遗传改变对基因表达的直接影响。我们创造了一种新 表观遗传编辑功能基因组学方法，我们命名为CRISPRoff。CRISPRoff稳健， 专门写CpG DNA甲基化（5 mC）和抑制性组蛋白修饰的目标基因座。我们 建议使用CRISPRoff来绘制所有由5 mC调控的基因组调控元件， 整个人类染色体在拟议的实验中，我们将使用扰动序列，它结合了池化的 CRISPR使用单细胞转录组读数进行筛选，以直接测量5 mC如何沉积。 整个染色体上的CRISPRoff调节基因表达。这种方法将识别遗传 调控元件对诱导多能干细胞和神经元至关重要，这是了解如何 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 这种方法和动机扩展这种方法，以映射整个基因调控元件， 人类基因组拟议研究的结果将作为基本资源和路线图 为生物医学科学家的广泛社区，并大大告知我们对人类生物学的理解， 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.