Defining regulators of immunity to acute infection using CRISPR screens

这个U19计划的首要目标是使用小鼠的正向遗传筛查来促进我们的 了解对病原体的免疫反应。该U19计划是由我们最近推出的 一种使用体内CRISPR介导的基因缺失的基因筛查平台的开发 功能基因筛选，以确定积极或消极地调节人的命运和功能的基因 免疫细胞。我们将把这一新方法与单细胞基因图谱和系统水平相结合。 急性感染期间先天细胞和适应性细胞提名候选细胞的计算模型 基因筛查中的功能紊乱。然后，我们将在Pooled中测试数千个潜在的监管机构 基于CRISPR-Cas9的小鼠正向基因筛查，并验证新的候选调控因子 小鼠感染模型和人类细胞。我们建议的U19计划包括两个高度互动的项目 项目，由4核支持。项目1(海宁/库奇鲁/夏普)将进行向前遗传 筛选调控CD8+和CD4+T细胞命运和功能的基因 急性感染。项目2(哈科恩/卡根)将进行筛选，以确定控制DC的基因 对病原体、病原体成分和T细胞作出反应的激活。管理核心A (夏普/海宁)将提供行政和科学协调，并实施我们的试点项目 程序。数据管理和生物信息学核心B(Regev)将开发、应用和传播 免疫细胞反应的单细胞rna-seq分析的前沿方法和工具 在CRISPR筛选中选择和排序用于遗传操作的候选基因。核心B也将 建立和维护共享数据、分析和方法的公共门户和软件管道。 CRISPR库核心C(道奇)将设计和生成定制的单引导RNA(SgRNA)文库 需要进行前向基因筛查和验证候选监管者。核心C也将 分析从混合筛选获得的细胞中的基因组DNA，执行测序和测序 去卷积以确定引起感兴趣表型的sgRNA。鼠标扰动核心D (夏普/海宁)将提供执行CRISPR-CAS9筛选和验证的统一平台 小鼠感染模型的实验。使用标准化的实验方法和 核心的计算工具将使比较和整合不同领域的结果成为可能 环境和疾病模型。我们预计我们体内的前向基因筛查和系统水平 单细胞基因组分析将确定引导 对感染的先天性和获得性免疫反应。这些发现将为新的 传染病的疫苗接种策略以及过敏和自身免疫的治疗。

The overarching goal of this U19 Program is to use forward genetic screens in mice to advance our understanding of immune responses to pathogens. This U19 Program is driven by our recent development of a genetic screening platform that uses pooled, in vivo CRISPR-mediated loss-of- function genetic screens to identify genes that positively or negatively regulate the fate and function of immune cells. We will couple this novel approach with single-cell gene profiling and systems-level computational modeling of innate and adaptive cells during acute infection to nominate candidates to perturb functionally in genetic screens. We will then test thousands of potential regulators in pooled CRISPR-Cas9 based forward genetic screens in mice, and validate novel candidate regulators in mouse infection models and human cells. Our proposed U19 Program consists of 2 highly interactive Projects, supported by 4 Cores. Project 1 (Haining/Kuchroo/Sharpe) will conduct forward genetic screens to identify genes that regulate the fate and function of CD8+ and CD4+ T cells responding to acute infection. Project 2 (Hacohen/Kagan) will conduct screens to identify genes that control DC activation in response to pathogens, pathogen components and T cells. Administrative Core A (Sharpe/Haining) will provide administrative and scientific coordination, and implement our Pilot Project program. Data Management and Bioinformatics Core B (Regev) will develop, apply and disseminate cutting-edge methods and tools for single cell RNA-seq analysis of immune cell responses and for selecting and ranking candidate genes for genetic manipulation in CRISPR screens. Core B will also establish and maintain a public portal and software pipelines for sharing data, analyses and methods. CRISPR Library Core C (Doench) will design and generate custom single guide RNA (sgRNA) libraries needed to conduct forward genetic screens and to validate candidate regulators. Core C will also analyze genomic DNA from cells obtained pooled screens, performing sequencing and sequence deconvolution to identify sgRNAs that caused the phenotype of interest. Mouse Perturbation Core D (Sharpe/Haining) will provide a uniform platform to execute CRISPR-Cas9 screens and validation experiments in mouse infection models. The use of standardized experimental methods and computational tools by the cores will make it possible to compare and integrate results in different settings and disease models. We expect that our in vivo forward genetic screens and systems level single-cell genomic analyses will identify the central molecules, pathways and mechanism that guide innate and adaptive immune responses to infection. These findings will lay the foundation for new vaccination strategies for infectious diseases and therapies for allergy and autoimmunity.