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. !

！ 项目概要 基因表达的精确调控对于发育和细胞身份至关重要。监管不当 这种严格控制的过程可能会导致癌症或神经系统疾病等疾病。 独特的表观遗传标记（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 以非针对性的方式发生变化，从而可以改变整个过程中的表观基因组特征 基因组。借助 RNA 引导的 Cas9/CRISPR 复合物，我们现在拥有一种工具，可以轻松地、 精确靶向基因组中的 20 bp 序列。我们和其他人已经制定了有针对性的 基于效应域与催化失活域融合的表观遗传调节因子 dCas9。我们已经证明 dCas9 与各种表观遗传效应域融合 (epi-dCas9) 可以有针对性地调控转录。然而，必须解决两大挑战 在我们能够有效地使用这些工具之前，我们需要克服以下问题：1）表观遗传调节因子的效率是 取决于基因组位置。预先存在的染色质环境和三- 使目标位点适合持久目标表观基因组的维度相互作用 编辑尚未理解。 2）高效实现持久的因素和途径 靶向基因沉默尚未明确定义。显然，需要更好地理解 可靶向的表观基因组，易于持续基因沉默并了解 适应持续基因沉默的途径。我们将获得这些基础 通过确定适合持久目标基因的启动子特征来获得见解 通过 epi-dCas9 沉默（目标 1），并确定持久目标所需的途径 使用创新的 epi-dCas9/knockdown 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 80 个启动子上测试几种 epi-dCas9 融合。 表达水平和表观遗传状态，然后识别允许或抵抗的特征 持续的沉默。在平行目标中，我们将 Epi-dCas9 阻遏物与基因组结合起来 广泛的 CRISPR/Cas9 筛选，以识别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造有针对性的持久性的能力 表观遗传变化对于疾病的研究和治疗也提供了基础 深入了解从一种表观遗传状态转变为另一种表观遗传状态所需的机械步骤。 ！