Modular Platform for Combinatorial Epigenome Manipulation

用于组合表观基因组操作的模块化平台

基本信息

批准号：
10592628
负责人：
Mark D ADAMS
金额：
$ 73.47万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10592628
关键词：
Address Adoption Affect Amino Acid Sequence Biological Biological Assay Biological Models Biological Process Cell Line Cell model Cell physiology Cells ChIP-seq Clustered Regularly Interspaced Short Palindromic Repeats Communities Custom DNA Deposition Development Developmental Process Disease Ecosystem Elements Engineering Enhancers Enzymes Epigenetic Process Etiology Gene Expression Gene Expression Regulation Genes Genomics Goals Guide RNA Health Histones In Situ Individual Knock-out Libraries Link Maps Methodology Modification Molecular Open Reading Frames Pathway interactions Phenotype Physiology Plasmids Play Proteins Protocols documentation Publishing RNA library Reagent Registries Regulator Genes Regulatory Element Reporter Research Personnel Resources Scientist Shapes Site Specificity Stretching System Techniques Technology Therapeutic Intervention Transgenes Translating Validation base case-by-case basis cell type combinatorial computational pipelines design developmental disease epigenetic regulation epigenome epigenome editing epigenomics experimental study functional genomics gene regulatory network genome-wide genome-wide analysis genomic locus histone modification human embryonic stem cell in vivo induced pluripotent stem cell innovation insight interest next generation next generation sequencing novel personalized medicine precision medicine programs promoter repository self-renewal side effect tool transcriptome sequencing

项目摘要

Epigenetic modifications of histone and DNA control gene expression and critically shape phenotypes and cell states. These modifications are tightly controlled by interactions with a constellation of trans-acting regulatory factors, and are dysregulated in a plethora of diseases. Next-generation sequencing (NGS) technologies have allowed genome-wide profiling of these modifications in diverse cell types, normal and disease conditions as well as across individuals. However, these strategies have yielded mostly associative and/or correlative insight into relationships between epigenetic state, gene expression and phenotype with limited power to establish the causality of individual epigenetic modifications that is fundamental to understanding normal physiology and diseases. Existing techniques to investigate phenotypic consequences of epigenetic gene regulation irreversibly delete stretches of genomic sequence that can have multiple functions, or perturb protein factors that control thousands of genes, confounding accurate conclusion. A scalable toolbox allowing in situ and in vivo combinatorial modifications of epigenetic states would offer unprecedented opportunities for both mechanistic studies and unbiased discovery of novel functional elements at the genomic scale. This project will respond to this need by developing an expandable molecular toolkit to precisely and reversibly manipulate defined epigenetic modifications (e.g., H3K27ac) at defined genomic addresses based on our innovative CRISPR/Casilio platform. There are three Specific Aims in this project. Aim 1 is focused on the development of a comprehensive set of epigenetic editing modules with which Casilio can achieve multiplexed and combinatorial edits, whereby different epigenetic modifications can be elicited simultaneously at distinct genomic loci while multiple modifications can also be induced in each locus. Aim 2 will deliver Casilio-enabled cell lines with which scientists can achieve epigenome editing with unprecedented ease by delivering short RNA guides. Aim 3 will center on the development of genome-wide guide libraries targeting genomic regulatory elements such as enhancers and insulators with which scientists can perform reverse epigenetic screens to discover novel elements and epigenetic modifications causative to phenotype. This toolkit, once developed, will transform the ways we study epigenetics by providing a fine and scalable technique to directly edit epigenetic states at defined targets, to investigate the underlying causes of gene regulatory changes observed in biological processes and diseases. To truly benefit the field of functional genomics, we will share our methodology and reagents at every stage of platform development with the scientific community. Through the establishment of standards and a module registry as well as reagent sharing via an open repository, we hope to create a sustainable ecosystem of Casilio module users and developers to apply and expand the Casilio toolbox for epigenetic editing. Due to the programmability and precision, epigenetic editing enabled by Casilio may provide new means and tools for powering personalized and precision medicine.

组蛋白和 DNA 的表观遗传修饰控制基因表达并关键地塑造表型和细胞州。这些修改是通过与一系列反式作用监管的相互作用来严格控制的。因素，并且在多种疾病中失调。新一代测序（NGS）技术已允许对不同细胞类型、正常和疾病条件下的这些修饰进行全基因组分析以及跨个体。然而，这些策略产生的大部分是关联和/或相关的见解研究表观遗传状态、基因表达和表型之间的关系，但建立的能力有限个体表观遗传修饰的因果关系对于理解正常生理学和疾病。研究表观遗传基因调控表型后果的现有技术不可逆地删除具有多种功能的基因组序列片段，或扰乱蛋白质因子控制着数千个基因，混淆了准确的结论。一个可扩展的工具箱，允许在原位和在表观遗传状态的体内组合修饰将为两者提供前所未有的机会在基因组规模上进行机制研究和公正地发现新的功能元件。该项目将通过开发可扩展的分子工具包来精确和可逆地操纵来满足这一需求基于我们的创新，在定义的基因组地址定义表观遗传修饰（例如，H3K27ac） CRISPR/Casilio 平台。该项目有三个具体目标。目标 1 的重点是发展一套全面的表观遗传编辑模块，卡西里奥可以用它实现多重和组合编辑，可以在不同的位置同时引发不同的表观遗传修饰基因组基因座，同时每个基因座也可以诱导多种修饰。 Aim 2 将提供支持 Casilio 的科学家们可以通过提供短的细胞系以前所未有的轻松方式实现表观基因组编辑 RNA 指南。目标 3 将集中于开发针对基因组的全基因组指导库调节元件，例如增强子和绝缘子，科学家可以利用它们进行反向表观遗传筛选以发现导致表型的新元素和表观遗传修饰。这个工具包，一次开发的，将通过提供精细且可扩展的技术来直接改变我们研究表观遗传学的方式在定义的目标处编辑表观遗传状态，以研究基因调控变化的根本原因在生物过程和疾病中观察到。为了真正造福功能基因组学领域，我们将分享我们与科学界在平台开发的每个阶段的方法和试剂。通过建立标准和模块注册表以及通过开放存储库共享试剂，我们希望创建一个由Casilio模块用户和开发者组成的可持续生态系统来应用和扩展用于表观遗传编辑的 Casilio 工具箱。由于可编程性和精确性，表观遗传编辑通过卡西利奥可能会提供新的手段和工具来推动个性化和精准医疗。