Determinants of Persistence in Epigenetic Editing

表观遗传编辑持久性的决定因素

• 批准号: 9920184
• 负责人: DAVID J SEGAL
• 金额: $ 18.72万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-23 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920184
• 关键词: 3-Dimensional; CRISPR screen; CRISPR/Cas technology; Cardiovascular Diseases; Cells; Chromatin; Complex; Coupled; DNA Methylation; Data; Decitabine; Development; Disease; ERBB2 gene; Engineering; Environment; Epigenetic Process; Experimental Designs; Flow Cytometry; Foundations; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genome; Genomics; Guide RNA; Knock-out; Lead; Location; Malignant Neoplasms; Mental disorders; Modification; Pathway interactions; Pharmaceutical Preparations; Process; Regulator Genes; Regulatory Element; System; Testing; Vorinostat; base; epigenome; epigenome editing; genome-wide; histone modification; human disease; innovation; insight; knock-down; nervous system disorder; nuclease; promoter; screening; tool

! PROJECT SUMMARY Precise regulation of gene expression is critical for development and cell identity. Misregulation of this tightly controlled process can lead to disease such as cancer or neurological disorders. Distinct epigenetic marks (DNA methylation and post-translational histone modifications) have been associated with expressed or silenced genes as well as with regulatory elements in the genome. Traditionally, drugs (e.g., decitabine, vorinostat) are used to induce epigenetic changes in an untargeted manner and thus can alter epigenome signatures throughout the genome. With the RNA-guided Cas9/CRISPR complex, we now have a tool that can easily and precisely target a 20-bp sequence in the genome. We and others have developed targeted epigenetic regulators that are based on fusions of effector domains to the catalytically inactive dCas9. We have shown that dCas9 fused to various epigenetic effector domains (epi-dCas9) can regulate transcription in a targeted manner. However, two major challenges have to be overcome before we can use these tools efficiently: 1) Efficiency of epigenetic regulators is dependent on the genomic locations. Pre-existing chromatin environment and three- dimensional interactions that make a target locus amenable to persistent targeted epigenome editing are not yet understood. 2) The factors and pathways to efficiently achieve persistent targeted gene silencing are not well defined. Clearly, a better understanding is needed of the targetable epigenome that is amenable to persistent gene silencing and an understanding of the pathway(s) to accommodate persistent gene silencing. We will gain these foundational insights by determining promoter features amenable to persistent targeted gene silencing by epi-dCas9 (Aim 1), and identifying pathway(s) required for persistent target gene silencing using an innovative epi-dCas9/knockdown editing screening system (Aim 2). In the first Aim, we will test several epi-dCas9 fusions on 80 promoters representing different expression levels and epigenetic states, then identify features that are permissive or resistive to persistent silencing. In a parallel Aim, we will combine an epi-dCas9 repressor with a genome- wide CRISPR/Cas9 screen to identify cellular genes involved in epigenetic persistence. Not only will this information advance the capabilities of us and others to create targeted persistent epigenetic changes for the study and treatment of disease, it will also provide fundamental insights into the mechanistic steps required to transition from one epigenetic state to another. !

! 项目摘要 基因表达的精确调控对于发育和细胞特性至关重要。误调节 这种严格控制的过程可能导致癌症或神经系统疾病。 不同的表观遗传标记（DNA甲基化和翻译后组蛋白修饰） 与表达或沉默的基因以及调控元件有关， 基因组传统上，药物（例如，地西他滨、伏立诺他）用于诱导表观遗传 以非靶向的方式改变，因此可以改变整个表观基因组签名。 基因组有了RNA引导的Cas9/CRISPR复合物，我们现在有了一种工具，可以轻松地 精确地瞄准基因组中的20个碱基的序列。我们和其他人已经开发了针对性的 表观遗传调节剂，其基于效应结构域与催化失活结构域的融合 dCas9。我们已经表明，dCas 9与各种表观遗传效应结构域（epi-dCas 9）融合， 可以靶向调节转录。然而，两个主要挑战必须是 在我们能够有效地使用这些工具之前，我们必须克服：1）表观遗传调节因子的效率是 取决于基因组的位置。先存在的染色质环境和三个- 三维相互作用，使目标基因座适合于持久的靶向表观基因组 编辑还不了解。2)有效实现持久性的因素和途径 靶向基因沉默没有很好地定义。显然，需要更好地了解 靶向表观基因组，这是经得起持久的基因沉默和理解的， 途径以适应持续的基因沉默。我们将获得这些基础 通过确定适合持续靶基因的启动子特征的见解 通过epi-dCas 9（Aim 1）沉默，并鉴定持久靶向所需的途径 使用创新的epi-dCas 9/敲低编辑筛选系统进行基因沉默（Aim 2）。在第一个目标中，我们将在代表不同的基因的80个启动子上测试几种epi-dCas 9融合体。 表达水平和表观遗传状态，然后确定允许或抵抗的特征。 持续沉默在平行的Aim中，我们将联合收割机将epi-dCas 9阻遏物与基因组结合， 广泛的CRISPR/Cas9筛选，以鉴定参与表观遗传持久性的细胞基因。不仅 这些信息是否会提高我们和其他人创建有针对性的持久 对于研究和治疗疾病的表观遗传变化，它也将提供基本的 深入了解从一种表观遗传状态转变到另一种表观遗传状态所需的机械步骤。 !