Harnessing CRISPR for Targeted and Inducible Epigenomic Reprogramming

描述（由申请人提供）：表观遗传修饰，如DNA甲基化和组蛋白乙酰化/甲基化，在决定细胞的功能和行为中起着关键作用。 一个有机体然而，由于我们操纵染色质修饰的能力受到严重的技术限制，解开表观基因组工作原理的努力受到了限制。现有的方法通常涉及药理学试剂或遗传修饰，并且往往具有多效性。该提案旨在开发一套通用的工具，用于操纵表观遗传修饰，能够将特定的染色质修饰剂靶向任何所需的位点，并精确控制这些修饰的时间和幅度。所提出的平台建立在我们最近展示的CRISPR（重复的规则间隔短回文重复序列）干扰系统上-一种源自CRISPR细菌免疫系统的高效RNA引导的基因组靶向系统。该方法仅需要单一蛋白dCas 9，CRISPR Cas9蛋白的催化失活变体，以及设计的具有与感兴趣基因互补的区域的小向导RNA（sgRNA）。sgRNA引导dCas 9-sgRNA复合物与互补基因组位点结合。首先，我们将开发一系列优化的dCas 9-sgRNA对，其中靶向的DNA位置仅由sgRNA的序列决定，靶向的表观遗传影响由dCas 9募集的效应子结构域决定。然后，这将与能够随意靶向dCas 9的全基因组sgRNA文库偶联到人类基因组中的大约100，000个位置。其次，为了实现诱导型表观遗传控制，我们将dCas 9蛋白融合到可以通过光或药物控制的多种表观遗传修饰结构域和效应结构域的互补物中。表观遗传修饰剂结构域将含有衍生自天然表观遗传调节剂的不同催化和/或调节结构域，包括“写入器”和“擦除器”模块。光遗传学和化学门控相互作用模块将用于实现空间和时间控制。第三，我们将把这些工具应用到一小部分试点机制和生物工程研究中，以测试CRISPR表观遗传工具箱如何被利用。具体而言，我们将修改小鼠Oct 4和Sox 2基因座的表观遗传标记作为内源性实验测试床，并研究修饰剂募集定位和动力学在控制染色质状态中的重要性。最后，我们将开发可编程的CRISPR绝缘子，以控制人类β珠蛋白表达的长距离染色质相互作用。总之，该项目将为表观基因组的精确空间和时间调控提供一个通用的RNA指导平台。我们设想这个平台将成为研究不同基因座上多种表观遗传修饰之间相互作用的重要工具，了解表观基因组编程与疾病之间的关系，并促进重写和重新编程表观遗传标记的治疗方法的发展。

DESCRIPTION (provided by applicant): Epigenetic modifications, such as DNA methylation and histone acetylation/methylation, play a critical role in dictating the function and behavior of an organism. Efforts to unravel the workings of the epigenome, however, are constrained by severe technical limitations in our ability to manipulate chromatin modifications. Existing approaches typically involve pharmacological agents or genetic modification and tend to have pleiotropic effects. This proposal aims to develop a universal set of tools for manipulating epigenetic modifications capable of targeting specific chromatin modifiers to any desired locus and precisely controlling the timing and magnitude of these modifications. The proposed platform is built on our recently demonstrated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference system-a highly efficient RNA- guided genome-targeting system derived from the CRISPR bacterial immune system. The method requires only a single protein, dCas9, a catalytically inactive variant of the CRISPR Cas9 proteins, and a designed small guide RNA (sgRNA) with a region that is complementary to the gene of interest. The sgRNA guides binding of the dCas9-sgRNA complex to the complementary genomic site. We will develop a CRISPR RNA-based toolset for targeted and inducible manipulation of the epigenetic modifications First, we will develop a series of optimized dCas9-sgRNA pairs in which the DNA location of targeting is determined solely by the sequence of the sgRNA and the epigenetic impact of targeting is dictated by the effector domain that is recruited by dCas9. This will then be coupled with a genome-wide library of sgRNAs capable of targeting the dCas9 at will to roughly 100,000 positions in the human genome. Second, to enable inducible epigenetic controls, we will fuse the dCas9 proteins to a complement of diverse epigenetic modifier domains and effector domains that can be controlled by light or drugs. The epigenetic modifier domains will contain different catalytic and/or regulatory domains derived from natural epigenetic regulators, including both "writer" and "eraser" modules. Optogenetically and chemically gated interaction modules will be used to achieve spatial and temporal control. Third, we will apply these tools to a small set of pilot mechanistic and bioengineering studies to test how the CRISPR epigenetic toolbox can be utilized. Specifically, we will modify the epigenetic marks of the murine Oct4 and Sox2 loci as the endogenous experimental testbed, and study the importance of modifier recruitment positioning and dynamics in controlling chromatin state. Finally, we will develop programmable CRISPR insulators to control long-range chromatin interactions of human beta globin expression. Together, this project will provide a universal RNA-guided platform for precise spatial and temporal regulation of the epigenome. We envision this platform will be a critical tool for studying the interplay between multiple epigenetic modifirs at different loci, understanding the relationship between epigenomic programming and disease, and facilitate the development of therapeutic methods to rewrite and reprogram epigenetic marks.