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: 10207344
• 负责人: Arlene H. Sharpe
• 金额: $ 229.25万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207344
• 关键词: 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 Models; 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; Immunotherapy; Infection; Institutes; Massachusetts; Mediating; Methods; Modeling; 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; acute infection; 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; single-cell RNA sequencing; tool; 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程序的总体目标是使用小鼠的远期遗传筛选来推进我们的 了解对病原体的免疫反应。这个U19程序是由我们最近的 开发使用汇总的基因筛查平台，体内CRISPR介导的损失 功能遗传筛选以识别积极或负调节命运和功能的基因 免疫细胞。我们将这种新颖的方法与单细胞基因分析和系统级别 急性感染期间先天和自适应细胞的计算模型，以提名候选 在遗传筛选中功能性。然后，我们将测试成千上万的潜在调节器 基于CRISPR-CAS9的小鼠的远期遗传筛选，并验证新颖的候选调节剂 小鼠感染模型和人类细胞。我们提出的U19计划由2个高度互动性组成 项目，由4个核心支持。项目1（Haining/Kuchroo/Sharpe）将进行前遗传 筛选以识别调节CD8+和CD4+ T细胞的命运和功能的基因 急性感染。项目2（Hacohen/Kagan）将进行筛选以识别控制DC的基因 响应病原体，病原体成分​​和T细胞的激活。行政核心a （Sharpe/Haining）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筛选中选择和排名候选基因进行基因操纵。核心也将 建立并维护用于共享数据，分析和方法的公共门户和软件管道。 CRISPR库Core C（DOENCH）将设计和生成自定义的单指南RNA（SGRNA）库 需要进行正向遗传筛选并验证候选调节剂。核心也将 分析来自获得的汇总筛选细胞的基因组DNA，进行测序和序列 识别引起感兴趣表型的SGRNA的反卷积。鼠标扰动核心D （Sharpe/Haining）将为执行CRISPR-CAS9屏幕和验证提供统一的平台 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您可以比较和集成结果 设置和疾病模型。我们预计我们的体内远期遗传筛选和系统水平 单细胞基因组分析将确定指导的中央分子，途径和机制 对感染的先天和适应性免疫反应。这些发现将为新的基础奠定基础 过敏和自身免疫的传染病和疗法的疫苗接种策略。