Determinants of Persistence in Epigenetic Editing

好了！ 项目总结 对基因表达的精确调控对发育和细胞特性至关重要。监管不善 这种严格控制的过程可能会导致癌症或神经疾病等疾病。 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基因中的调控元件有关 基因组。传统上，药物(如地他滨、伏立诺)用于诱导表观遗传学 以一种无针对性的方式发生变化，因此可能会改变整个 基因组。有了RNA引导的Cas9/CRISPR复合体，我们现在有了一个工具，可以轻松地和 精确定位基因组中20个碱基对的序列。我们和其他人开发了有针对性的 基于效应结构域与催化失活的融合的表观遗传调节剂 DCas9。我们已经证明了dCas9与不同的表观遗传效应域(epi-dCas9)融合。 可以以有针对性的方式调节转录。然而，两大挑战必须是 在我们可以有效地使用这些工具之前克服：1)表观遗传调节器的效率是 取决于基因组的位置。先前存在的染色质环境和三个- 使靶点服从持久靶向表观基因组的维度相互作用 目前还不了解编辑情况。2)有效实现可持续发展的因素和途径 靶向基因沉默还没有很好的定义。显然，需要更好地理解 可持续基因沉默的靶向表观基因组和对 途径(S)，以适应持续性的基因沉默。我们将获得这些基础 通过确定启动子对持久靶基因的顺应性获得的见解 Epi-dCas9沉默(目标1)，识别持续靶标所需的途径(S) 使用创新的epi-dCas9/基因敲除编辑筛选系统(AIM)的基因沉默 2)。在第一个目标中，我们将在80个启动子上测试几个epi-dCas9融合，代表不同的 表达水平和表观遗传状态，然后识别允许或抵抗 持续静音。在一个平行的目标中，我们将把epi-dCas9抑制子与基因组相结合- 宽CRISPR/Cas9屏幕，以识别与表观遗传持久性有关的细胞基因。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性的能力 表观遗传学的变化，也将为疾病的研究和治疗提供基础 对从一种表观遗传状态转换到另一种表观遗传状态所需的机械性步骤的洞察。 好了！

! 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. !