Mapping cellular communication at single-cell resolution through novel CRISPR systems

项目摘要/摘要 通过新的CRISPR系统绘制单小区分辨率的蜂窝通信图 费尔曼实验室专注于解码疾病进展和治疗中的细胞信号原理，以及 CRISPR-CAS和RNA干扰(RNAi)系统的先驱。十多年来，我的研究主要集中在 关于更好地了解RNA引导的免疫系统和建立解剖疾病的创新策略， 其中许多在科学界得到了广泛的应用。我的实验室目前正在研究 CaS酶和DNA修复途径之间的相互作用，以开发新的基因组编辑和 精准医学。我们应用这些定量的、高通量的方法来研究信号的可塑性 健康和疾病方面的网络，目标是将见解转化为患者的突破性疗法。 生物学中的一个长期挑战是以细胞特有的方式记录分子信息，而不中断 正在研究的系统。为了克服这一点，我们提出了变革性的CRISPR平台来跟踪宿主-病原体 人类疾病中的相互作用和MAP途径失控。方法依赖于Cas9的开发 并引导对1)病原体特异性蛋白水解酶(“ProCas9”)或2)哺乳动物作出反应的RNA系统 细胞信号事件(“CRISPR-Capture”)，从而能够通过刻录标记来记录细胞的历史 在基因组中预定的位置。作为对新出现的病毒威胁的快速反应，我们将开发ProCas9 它可以自动记录SARS-CoV-2感染并标记各自的细胞。我们将利用这一战略来 剖析病毒感染对心肌细胞的长期后果。启用蜂窝网络的常规映射 我们提出了一种新的数据记录方法，称为CRISPR-Capture，它将 在RNA聚合酶II启动子控制下的sgRNAs的表达，而不是常规使用的 RNA Pol-III启动子。由于哺乳动物的信号输出很大程度上是基于转录因子调节的 POL-II启动子，CRISPR-Capture将CA活动调整到细胞状态，提供独特的细胞图谱能力 以单细胞分辨率发送信号，并首次监测细胞生命周期内的关键生物事件。 最终，我们将利用CRISPR-Capture在四个维度(空间-时间)监控单个细胞 疾病进展和治疗，以揭示组织异质性的组织原则并建立 为患者制定新的治疗策略。我与Jennifer Doudna博士在基因组编辑方面的多学科培训 (加州大学伯克利分校)，冷泉港实验室Scott Lowe博士的功能基因组学， 和人类疾病的体内动物模型，让我在基础生物学和以患者为中心之间架起了桥梁 研究。此外，我作为一家成功的初创公司的联合创始人和首席科学官的经历 给我上了宝贵的一课，这对我指导和完成拟议的研究很有帮助。 重要的是，我坚定地致力于指导和成为一群才华横溢、多元化的 下一代科学家，并为所有人提供平等机会。

Project Summary/Abstract Mapping cellular communication at single-cell resolution through novel CRISPR systems The Fellmann lab focuses on decoding principles of cellular signaling in disease progression and therapy, and pioneering of CRISPR-Cas and RNA interference (RNAi) systems. For over a decade, my research has centered on better understanding RNA-guided immune systems and establishing innovative strategies to dissect disease, many of which have found broad application among the scientific community. My lab currently studies the interplay between Cas enzymes and DNA repair pathways to develop novel approaches for genome editing and precision medicine. We apply these quantitative, high-throughput methods to study the plasticity of signaling networks in health and disease, with the goal of translating insights into breakthrough therapies for patients. A persisting challenge in biology is recording molecular information in a cell-specific manner without disrupting the system under study. To overcome this, we propose transformative CRISPR platforms to track host-pathogen interactions and map pathway deregulation in human disease. The approaches rest on the development of Cas9 and guide RNA systems that are responsive to 1) pathogen-specific proteases (“ProCas9s”) or 2) mammalian cell-signaling events (“CRISPR-capture”), thereby enabling the recording of a cell’s history by inscribing marks at predetermined loci in the genome. As rapid response to emerging viral threats, we will develop a ProCas9 that can autonomously record SARS-CoV-2 infections and label respective cells. We will use this strategy to dissect long-term consequences of viral infection in cardiomyocytes. To enable general mapping of cellular communication, we propose a novel data recording methodology termed CRISPR-capture that places the expression of sgRNAs under the control of RNA polymerase II promoters, rather than the conventionally used RNA Pol-III promoters. Since mammalian signaling outputs are largely based on transcription factors regulating Pol-II promoters, CRISPR-capture tunes Cas activity to a cell’s state, providing the unique ability to map cellular signaling at single-cell resolution and monitor key biological events over the lifetime of a cell for the first time. Ultimately, we will leverage CRISPR-capture to monitor individual cells in four dimensions (space-time) during disease progression and treatment, to uncover organizational principles of tissue heterogeneity and establish new therapeutic strategies for patients. My multidisciplinary training in genome editing with Dr. Jennifer Doudna (University of California, Berkeley), functional genomics with Dr. Scott Lowe (Cold Spring Harbor Laboratory), and in-vivo animal models of human disease, allows me to bridge fundamental biology and patient-centered research. Moreover, my experience as co-founder and Chief Scientific Officer of a successful start-up company taught me invaluable lessons that will serve me well in directing and completing the proposed studies. Importantly, I am deeply committed to mentoring and serving as a role model for a talented and diverse group of next-generation scientists, and to providing equal opportunities to all.