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

项目摘要 DNA是生命的蓝图。它编码了建造和维持所有生物体的指令， 原核生物到人类。尽管如此，DNA经常被破坏。事实上，估计表明， 细胞每天可能经历多达105个损伤。为了保护它们的基因组， 进化出了一套复杂的机制，可以感知、发送信号和修复化学上不同形式的DNA 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 这些机制在人类细胞中的一个广泛的“零件清单”。然而，我们对人类认知的一个重大挑战 仍然：我们不知道这些机制如何在系统层面上协同工作，以确保响应 在各种条件下具有灵活性，或者在一个机制失败时能够进行补偿。缺乏系统性 知识有问题。它限制了我们对人类疾病的理解，例如具有DNA修复的癌症 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，能够系统地询问 基因在人类细胞中的功能，并与这些工具，我们建议解决知识的差距。的 我们的方法背后的基本逻辑很简单。我们将基于CRISPR的遗传扰动技术 通过可扩展的方法获得高含量的表型，如单细胞RNA测序。这允许 我们收集数据丰富的读数的细胞行为跨细胞，我们已经扰乱了许多功能， 基因.有了这些数据，我们可以推断基因之间的功能关系，并描绘遗传途径。 在这里，我建议使用其中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。 癌症治疗期间的反应（项目2）。为了实现第一个项目，我们展示了一种新的方法， 将基于CRISPR的基因筛选与DNA修复连接的深度测序配对， DNA修复的读数我们使用来自化脓性链球菌的Cas9建立了这种方法，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.