Probing relationships between DNA methylation and cellular senescence with high-throughput CRISPR-based epigenetic editing

利用基于 CRISPR 的高通量表观遗传编辑探索 DNA 甲基化与细胞衰老之间的关系

基本信息

批准号：
10593233
负责人：
Lei Stanley Qi
金额：
$ 19.38万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10593233
关键词：
Acceleration Active Sites Address Affect Age Aging Automobile Driving Biological Biological Process Biology CRISPR screen CRISPR/Cas technology Cell Aging Cell physiology Cells Clustered Regularly Interspaced Short Palindromic Repeats Coupled DNA DNA Methylation Data Deposition Disease Elements Engineering Epigenetic Process Exhibits Frequencies Future Gene Expression Gene Silencing Genome Genomics Gold Guide RNA Health Human In Vitro Individual Inflammation Libraries Link Mesenchymal Stem Cells Methodology Methods Methylation Modeling Modification Molecular Conformation Morphology Organism Outcome Output Phenotype Play Population Process Proliferating Proteins Publishing RNA library Role Signal Induction Site Sorting Specific qualifier value System Techniques Technology Testing Tissues Untranslated RNA Validation Work Writing aged bisulfite sequencing cell age cell behavior cell type cellular development cellular engineering combinatorial epigenome epigenomics genome-wide genomic locus high throughput screening insight interest methylome next generation sequencing nuclease response senescence standard measure technology platform tool transcriptome sequencing whole genome

项目摘要

ABSTRACT Cellular senescence is a phenomenon associated with aging, wherein cells stop proliferating and secrete factors which may impact the function of surrounding cells and tissues. Although epigenetic readouts, and in particular DNA methylation (CpGme), have found to be associated with aging and cellular senescence, it remains undetermined whether accumulated changes in CpGme are merely correlated with senescence or whether they play a causal role in driving it. To answer this question, we will combine next-generation sequencing techniques along with CRISPR-based CpGme editing and CRISPR screens to elucidate the causative associations between CpGme sites across the genome and cellular senescence. In Aim 1, we will assess CpGme sites of interest for effect on cellular senescence with a high-throughput CRISPR screen. Using a CRISPR molecule capable of depositing CpGme, it is possible to generate a population of cells, each with CpGme modified at a particular site. By characterizing how these CpGme modifications influence the entry rate into senescence, we will identify CpGme sites that causally affect senescence. In Aim 2, we will dissect how the most active sites operate to affect senescence. This will be done by inducing CpGme at the target sites and observing how this influences genomic CpGme and gene expression profiles, which will provide mechanistic understanding of how the perturbations we make affect cellular function. We will develop methods to more broadly apply CpGme editing to control cellular senescence. The work will illuminate an important process associated with aging and will provide new tools to for understanding how epigenetics impacts biological function, as well as how we can engineer cells to optimize health in the future.

抽象的细胞衰老是一种与衰老相关的现象，其中细胞停止增殖和分泌影响周围细胞和组织功能的因素。尽管表观遗传读数，并且在特定的 DNA 甲基化 (CpGme) 已被发现与衰老和细胞衰老有关，但它仍然存在尚未确定 CpGme 的累积变化是否仅与衰老相关，或者是否与衰老相关对其起到因果作用。为了回答这个问题，我们将结合下一代测序技术以及基于 CRISPR 的 CpGme 编辑和 CRISPR 筛选，以阐明之间的因果关系跨基因组和细胞衰老的 CpGme 位点。在目标 1 中，我们将通过高通量评估感兴趣的 CpGme 位点对细胞衰老的影响。 CRISPR 筛选。使用能够沉积 CpGme 的 CRISPR 分子，可以产生群体细胞，每个细胞的特定位点都经过 CpGme 修饰。通过表征这些 CpGme 修饰的方式影响进入衰老的速度，我们将确定因果影响衰老的 CpGme 位点。在目标 2 中，我们将剖析最活跃的位点如何影响衰老。这将通过诱导来完成目标位点的 CpGme 并观察这如何影响基因组 CpGme 和基因表达谱，这将从机制上理解我们所做的扰动如何影响细胞功能。我们将开发更广泛地应用 CpGme 编辑来控制细胞衰老的方法。工作将会照亮与衰老相关的重要过程，将为理解表观遗传学如何提供新工具影响生物功能，以及我们如何改造细胞以优化未来的健康。