Mapping the DNA damage response in human cells with high-resolution functional genomics

项目摘要 DNA是生命的蓝图。它对构建和维护所有生物体的指令进行了编码，来自 原核生物传给人类。尽管如此，DNA经常遭到破坏。事实上，据估计，每个人类 细胞每天可能经历多达105个皮损。为了保护它们的基因组，生物体因此 进化出一套复杂的机制，可以感知、发出信号并修复化学上不同形式的DNA 损伤，当损伤无法修复时，诱导细胞死亡程序。几十年的工作提供了 人类细胞中这些机制的一个广泛的“部分清单”。然而，对我们理解的一个重大挑战 仍然：我们不知道这些机制如何在系统层面协同工作以确保响应 在不同条件下的灵活性或在一种机制发生故障时启用补偿。这种缺乏系统性的 知识是有问题的。它限制了我们对人类疾病的理解，例如DNA修复的癌症 不足，它挑战了我们开发和改进利用反应的医学疗法的能力 活动。我们最近开发了功能基因组学方法，使系统地询问 基因在人类细胞中的功能，利用这些工具，我们建议解决这一知识差距。这个 我们方法背后的基本逻辑很简单。我们配对了基于CRISPR的遗传扰动技术 用可扩展的方法获得高含量的表型，如单细胞RNA测序。这使得 我们将收集细胞行为的丰富读数数据，在这些细胞中，我们已经扰乱了许多 基因。有了这些数据，我们就可以推断基因之间的功能关系，并描绘出遗传途径。 在这里，我建议使用其中的两种技术来绘制人类细胞中的DNA损伤反应机制， 目标是改进基因组编辑技术(项目1)并加深对药物的理解 癌症治疗中的反应(项目2)。为了支持第一个项目，我们演示了一种新方法 将基于CRISPR的遗传筛选与DNA修复连接的深度测序配对以产生高含量 DNA修复读数。我们使用来自化脓性链球菌的Cas9建立了这一方法，并提出了工作 这将成为未来理解基因组编辑技术的路线图。

Project Summary DNA is the blueprint for life. It encodes the instructions for building and maintaining all organisms, from prokaryotes to humans. Despite this, DNA is frequently damaged. In fact, estimates suggest that each human cell may experience as many as ~105 lesions per day. To protect their genomes, organisms have therefore evolved a sophisticated set of mechanisms that sense, signal, and repair chemically diverse forms of DNA damage and, when damage cannot be repaired, induce programs of cell death. Decades of work have provided an extensive ‘parts list’ of these mechanisms in human cells. However, a major challenge to our understanding remains: We do not know how these mechanisms work together at the systems level to ensure response flexibility across conditions or enable compensation when one mechanism fails. This lack of systematic knowledge is problematic. It limits our comprehension of human diseases, such as cancers with DNA repair deficiencies, and it challenges our ability to develop and improve medical therapies that exploit response activities. We have recently developed functional genomics approaches that enable systematic interrogation of gene function in human cells, and with these tools, we propose to address this gap in knowledge. The fundamental logic behind our approaches is simple. We pair CRISPR-based genetic perturbation techniques with scalable methods for obtaining high-content phenotypes, such as single-cell RNA-sequencing. This allows us to collect data rich readouts of cell behavior across cells in which we have perturbed the function of many genes. With such data, we can infer functional relationships between genes and delineate genetic pathways. Here, I propose to use two of these technologies to map DNA damage response mechanisms in human cells, with the goals of improving genome editing technologies (Project 1) and achieving deeper understanding of drug responses during cancer therapy (Project 2). To enable the first project, we demonstrate a new approach that pairs CRISPR-based genetic screens with deep sequencing of DNA repair junctions to generate high-content readouts of DNA repair. We establish this approach using Cas9 from Streptococcus pyogenes and propose work that will serve as a roadmap for understanding genome editing technologies in the future.