Defining regulators of immunity to acute infection using CRISPR screens

这个U19项目的首要目标是在小鼠中使用正向遗传筛选来推进我们的研究。 了解对病原体的免疫反应。这个U19计划是由我们最近的 开发了一个遗传筛选平台，该平台使用合并的体内CRISPR介导的基因缺失， 功能遗传筛选，以确定基因，积极或消极调节的命运和功能， 免疫细胞我们将把这种新方法与单细胞基因谱分析和系统水平的 急性感染期间先天和适应性细胞的计算建模，以提名候选者， 在基因筛选中的功能干扰。然后，我们将测试数千个潜在的监管机构， 在小鼠中进行基于CRISPR-Cas9的正向遗传筛选，并在小鼠中验证新型候选调节剂。 小鼠感染模型和人类细胞。我们建议的U19计划包括2个高度互动的 由4个核心支持的项目。项目1（海宁/Kuchroo/Sharpe）将进行正向遗传学 筛选以鉴定调节响应于CD 8+和CD 4 + T细胞的命运和功能的基因。 急性感染项目2（Hacohen/Kagan）将进行筛选，以确定控制DC的基因 在一些实施方案中，免疫调节剂可以响应于病原体、病原体组分和T细胞而激活。行政核心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 用于免疫细胞应答的单细胞RNA-seq分析以及用于 在CRISPR筛选中选择和排序用于遗传操作的候选基因。核心B还将 建立和维护公共门户网站和软件管道，以分享数据、分析和方法。 CRISPR Library Core C（Doench）将设计和生成定制的单向导RNA（sgRNA）文库 需要进行遗传筛查和验证候选监管机构。核心C也将 分析来自合并筛选获得的细胞的基因组DNA，进行测序和测序 通过解卷积来鉴定引起感兴趣的表型的sgRNA。小鼠扰动核心D （夏普/海宁）将提供一个统一的平台来执行CRISPR-Cas9筛选和验证 在小鼠感染模型中的实验。使用标准化的实验方法， 计算工具的核心将有可能比较和整合结果在不同的 设置和疾病模型。我们希望我们的体内正向遗传筛选和系统水平 单细胞基因组分析将确定指导细胞凋亡的中心分子、途径和机制。 对感染的先天性和适应性免疫反应。这些发现将为新的 针对传染病的疫苗接种策略以及针对过敏和自身免疫的疗法。

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.