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

摘要 细胞衰老是一种与衰老有关的现象，即细胞停止增殖和分泌 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)，已被发现与衰老和细胞衰老有关，它仍然 尚不确定CpGme的累积变化是否仅与衰老相关，或者它们是否 在驱动它的过程中扮演一个因果角色。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屏幕，以阐明 基因组中的cpGme位点与细胞衰老。 在目标1中，我们将用高通量来评估感兴趣的CpGme位点对细胞衰老的影响 CRISPR屏幕。使用能够沉积CpGme的CRISPR分子，可以产生群体 每个细胞的CpGme在特定的位置被修饰。通过表征这些CpGme修饰是如何 影响进入衰老的速率，我们将找出影响衰老的CpGme位点。 在目标2中，我们将剖析最活跃的部位是如何影响衰老的。这将通过诱导 并观察这如何影响基因组CpGme和基因表达谱， 这将提供对我们所做的扰动如何影响细胞功能的机械理解。我们会 开发更广泛地应用CpGme编辑来控制细胞衰老的方法。这项工作将照亮 一个与衰老相关的重要过程，并将提供新的工具来理解表观遗传学 影响生物功能，以及我们如何在未来设计细胞以优化健康。

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.