Defining regulators of immunity to acute infection using CRISPR screens

使用 CRISPR 筛选定义急性感染免疫调节因子

• 批准号: 9380804
• 负责人: Arlene H. Sharpe
• 金额: $ 246.29万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-05 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9380804
• 关键词: Acute; Autoimmunity; Bioinformatics; Boston; CD4 Positive T Lymphocytes; CD8B1 gene; CRISPR library; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communicable Diseases; Complement; Computer Simulation; Computer software; Custom; Dana-Farber Cancer Institute; Data Analyses; Dendritic Cells; Development; Disease; Disease model; Foundations; Gene Expression; General Hospitals; Genes; Genetic Screening; Genomic DNA; Genomics; Goals; Guide RNA; Hematopoietic; Hospitals; Human; Hypersensitivity; Immune; Immune response; Immune system; Immunity; Immunosuppressive Agents; Immunotherapy; Infection; Institutes; Massachusetts; Mediating; Methods; Modeling; Molecular Profiling; Mus; Pathway interactions; Pediatric Hospitals; Phenotype; Pilot Projects; RNA library; Regimen; Research Personnel; Research Project Grants; Standardization; System; T-Lymphocyte; Testing; Translations; Vaccine Adjuvant; Vaccines; Validation; Woman; adaptive immune response; base; computerized tools; data management; data sharing; design; experimental study; genetic manipulation; immune function; immunoregulation; in vivo; innovation; insight; interest; loss of function; medical schools; novel; novel strategies; pathogen; programs; response; single cell analysis; tool; transcriptome sequencing; vaccination strategy; validation studies

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.

该 U19 计划的总体目标是利用小鼠的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 计划是由我们最近推动的 开发一个基因筛选平台，该平台使用汇集的体内 CRISPR 介导的缺失 功能遗传筛选，以确定积极或消极调节命运和功能的基因 免疫细胞。我们将把这种新方法与单细胞基因分析和系统级结合起来 急性感染期间先天性和适应性细胞的计算模型，以提名候选者 扰乱遗传筛选中的功能。然后，我们将测试数千个潜在的监管机构 基于 CRISPR-Cas9 的小鼠正向遗传筛选，并验证新的候选调控因子 小鼠感染模型和人类细胞。我们提出的 U19 计划包括 2 个高度互动的项目 由 4 核支持的项目。项目1（海宁/Kuchroo/Sharpe）将进行正向遗传 筛选以确定调节 CD8+ 和 CD4+ 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 针对病原体、病原体成分​​和 T 细胞的激活。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 用于免疫细胞反应的单细胞 RNA-seq 分析的尖端方法和工具 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 建立和维护公共门户和软件管道，以共享数据、分析和方法。 CRISPR Library Core C (Doench) 将设计和生成定制单引导 RNA (sgRNA) 文库 需要进行正向遗传筛选并验证候选监管者。核心C也将 分析来自合并筛选的细胞的基因组 DNA，进行测序和排序 反卷积以识别引起感兴趣表型的 sgRNA。鼠标扰动核心 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 小鼠感染模型实验。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 设置和疾病模型。我们期望我们的体内前向遗传筛选和系统水平 单细胞基因组分析将确定指导的中心分子、途径和机制 对感染的先天和适应性免疫反应。这些发现将为新的研究奠定基础 传染病的疫苗接种策略以及过敏和自身免疫的治疗。