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. !

呢 项目摘要 基因表达的精确调节对于发育和细胞身份至关重要。正直 在这种严格控制的过程中，可能导致疾病，例如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）具有 与表达或沉默的基因以及与调节元素有关 基因组。传统上，使用药物（例如，去足替滨，伏地）来诱导表观遗传学 以非目标的方式变化，因此可以改变整个整个整个表观基因组的特征 基因组。使用RNA引导的Cas9/CRISPR综合体，我们现在拥有一个可以轻松而可以的工具 精确靶向基因组中的20 bp序列。我们和其他人开发了目标 基于效应域融合到催化性无活性的表观遗传调节剂 DCAS9。我们已经表明，DCAS9融合到各种表观遗传效应子域（EPI-DCAS9） 可以以目标方式调节转录。但是，必须面临两个主要挑战 在我们有效使用这些工具之前要克服：1）表观遗传调节剂的效率是 取决于基因组位置。先前存在的染色质环境和三 尺寸相互作用使目标位点可及时靶向表观基因组 编辑尚未理解。 2）有效实现持久性的因素和途径 靶向基因沉默尚未很好地定义。显然，需要更好地理解 可以持续的基因沉默和对 途径可容纳持续的基因沉默。我们将获得这些基础 通过确定启动子特征可符合持续的靶向基因来见解 通过EPI-DCAS9（AIM 1）进行沉默，并识别持续目标所需的途径 使用创新的EPI-DCAS9/敲低编辑筛选系统的基因沉默（AIM 2）。在第一个目的中，我们将对代表不同的80个发起人测试几个EPI-DCAS9融合 表达水平和表观遗传状态，然后确定允许或电阻的特征 持续的沉默。在平行目的中，我们将将EPI-DCAS9抑制剂与基因组结合在一起 广泛的CRISPR/CAS9筛选，以鉴定与表观遗传持久性有关的细胞基因。不仅 这些信息是否会推动我们和其他人创建目标持久性的能力 研究和治疗疾病的表观遗传变化，它也将提供基本 对从一个表观遗传状态过渡到另一个表观遗传状态所需的机械步骤的见解。 呢