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

• 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
• 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

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.

摘要 细胞衰老是与衰老相关的现象，其中细胞停止增殖并分泌 可能影响周围细胞和组织功能的因素。虽然表观遗传读数，并在 特别是DNA甲基化（CpGme），已经发现与衰老和细胞衰老有关，它仍然存在 尚未确定CpGme的累积变化是否仅与衰老相关，或者它们是否与衰老相关。 为了回答这个问题，我们将结合联合收割机下一代测序技术 沿着基于CRISPR的CpGme编辑和CRISPR筛选，以阐明 基因组中的CpGme位点与细胞衰老 在目标1中，我们将用高通量的免疫印迹技术评估感兴趣的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，有可能产生一个群体， 每个细胞在特定位点都有修饰的CpGme。通过描述这些CpGme修饰 影响进入衰老的速率，我们将鉴定因果地影响衰老的CpGme位点。 在目标2中，我们将剖析最活跃的部位如何影响衰老。这将通过诱导 靶位点的CpGme，并观察这如何影响基因组CpGme和基因表达谱， 这将提供我们对扰动如何影响细胞功能的机械理解。我们将 开发更广泛地应用CpGme编辑来控制细胞衰老的方法。这项工作将阐明 这是一个与衰老相关的重要过程，将为理解表观遗传学如何提供新的工具。 影响生物功能，以及我们如何设计细胞以优化未来的健康。