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

通过新型 CRISPR 系统以单细胞分辨率绘制细胞通讯图

基本信息

批准号：
10277350
负责人：
Christof Fellmann
金额：
$ 47.25万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-18 至 2022-08-21
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10277350
关键词：
Animal Model Biological Biology California Cardiac Myocytes Cells Clustered Regularly Interspaced Short Palindromic Repeats Communication Communities DNA Polymerase II DNA Polymerase III DNA Repair Pathway Data Development Disease Disease Progression Enzymes Event Four-dimensional Genome Goals Guide RNA Health Heterogeneity Human Immune system In Vitro Individual Label Laboratories Mammalian Cell Maps Mentors Methodology Methods Molecular Monitor Output Pathway interactions Patients Peptide Hydrolases RNA RNA Interference RNA Polymerase II Recording of previous events Research Resolution Rest SARS-CoV-2 infection Scientist Signal Transduction System Talents Time Tissues Training Translating Universities Viral Viral Physiology Virus Diseases base experience functional genomics genome editing human disease human model in vivo innovation insight multidisciplinary next generation novel novel strategies novel therapeutic intervention pathogen patient oriented precision medicine promoter response role model transcription factor

项目摘要

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.

项目摘要/摘要通过新颖的CRISPR系统绘制单细胞分辨率的蜂窝通信 Fellmann实验室的重点是疾病进展和治疗中细胞信号传导的解码原理，以及 CRISPR-CAS和RNA干扰（RNAI）系统的开创性。十多年来，我的研究一直居中更好地了解RNA引导的免疫系统并建立创新策略以剖析疾病，其中许多人在科学界发现了广泛的应用。我的实验室目前正在研究 CAS酶与DNA修复途径之间的相互作用，以开发基因组编辑的新方法和精密医学。我们应用这些定量的高通量方法来研究信号的可塑性健康和疾病的网络，目的是将洞察力转化为患者突破性疗法。生物学的持续挑战是以细胞特异性的方式记录分子信息正在研究的系统。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人类疾病中的相互作用和地图途径放松管制。这种方法取决于CAS9的发展并引导对1）病原体特异性蛋白酶（“ Procas9s”）或2）哺乳动物的RNA系统细胞信号事件（“ CRISPR捕获”），从而通过刻上标记来录制细胞的历史记录在基因组中的预定局部。作为对新兴病毒威胁的快速反应，我们将开发一个Procas9 可以自主记录SARS-COV-2感染和标记相对细胞。我们将使用此策略来剖析心肌细胞病毒感染的长期后果。为了使细胞的一般映射沟通，我们提出了一种新的数据记录方法，称为CRISPR捕获，将其放置在RNA聚合酶II启动子的控制下SGRNA的表达，而不是常规使用的 RNA Pol-III启动子。由于哺乳动物信号传导输出主要基于调节的转录因子 POL-II启动子，CRISPRAPTURE曲调CAS活动到一个细胞的状态，提供了映射细胞的独特能力在单细胞分辨率和监测细胞生命周期的关键生物学事件时，信号首次传导。最终，我们将利用CRISPR捕获来监视在四个维度（时空）的单个单元疾病的进展和治疗，以发现组织异质性和建立的组织原理针对患者的新治疗策略。我与Jennifer Doudna博士进行基因组编辑的多学科培训（加利福尼亚大学伯克利分校），与斯科特·洛（Scott Lowe）博士（冷泉港实验室）的功能基因组学，和体内人类疾病动物模型，使我能够桥接基本生物学和以患者为中心的生物学研究。此外，我作为成功的创业公司的联合创始人兼首席科学官的经验教会了我宝贵的课程，这些课程将在指导和完成拟议的研究方面很好地为我服务。重要的是，我非常致力于心理和作为一个有才华和潜水员的榜样下一代科学家，为所有人提供平等的机会。