shRNAmir and CRISPR sgRNA Library Construction Core

shRNAmir 和 CRISPR sgRNA 文库构建核心

基本信息

批准号：
10488582
负责人：
Matthew Eugene Pipkin
金额：
$ 14.01万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10488582
关键词：
B-Lymphocytes Biological CD8-Positive T-Lymphocytes CRISPR/Cas technology Cell Maintenance Cell Surface Receptors Cell physiology Cells Chromatin Cloning Clustered Regularly Interspaced Short Palindromic Repeats Complement Core Facility Custom DNA DNA Library Data Development Doctor of Philosophy Evolution Follow-Up Studies Gene Targeting Gene Transfer Generations Genes Goals Guide RNA Human Immune response Immune system In Vitro Individual Infection Laboratories Lentivirus Vector Libraries Mediating Memory Methods MicroRNAs Mus Phenotype Publishing RNA Interference Reagent Regulation Research Institute Retroviral Vector System T memory cell T-Lymphocyte Technology Time Tissues acute infection analysis pipeline base differential expression effector T cell experience flexibility gene function genome-wide in vivo innovation loss of function novel novel strategies plasmid DNA small hairpin RNA tool transcription factor tumor vector virtual

项目摘要

PROJECT SUMMARY / ABSTRACT Core B (Pipkin) Short hairpin RNAs in microRNA contexts (shRNAmirs) and CRISPR-Cas9 guide RNA (sgRNA) are powerful tools for experimental RNA interference (RNAi) and gene-disruption in the immune system, respectively. However, virtually all current approaches require pre-activation of naive cells to render them susceptible for gene transfer, typically with retroviral or lentiviral vectors, and cause constitutive RNAi or immediate gene-disruption. These issues complicate studying genes that function prior to retroviral delivery, and clarifying the temporal requirements of genes at different stages of immune responses. In Core B, we describe development of multiple innovative tools and methods that substantially expands the utility of shRNAmirs and sgRNAs to study gene function. We demonstrate generation of unmanipulated naive CD4 and CD8 T cells carrying conditionally regulated retroviral constructs that enable conducting inducible and reversible RNAi in vivo, and extend this system to a novel approach that facilitates large in vivo pooled screens using inducible shRNAmirs in untouched naive cells. We also demonstrate CRISPR-Cas9 guide RNA (sgRNA) gene disruption in primary B and T cells, and its use during in vivo immune responses to analyze B and T cell function. The combination of these shRNAmir and sgRNA approaches are synergistic and constitute an innovative and rigorous experimental platform for rapidly dissecting gene function in T and B cells during in vivo immune responses. Core B builds on multiple existing libraries of high-fidelity shRNAmir clones, which include those that target all genes encoding mammalian chromatin regulator factors (312 genes, ~1,700 shRNAmirs), and all mammalian transcription factors (2,083 genes, ~8,000 shRNAmirs). The overarching goal of Core B is to provide essential tools for executing the proposed in vivo loss-of-function studies proposed in Projects 1-3 of this P01. Our specific objectives are to sub- clone pooled shRNAmir libraries using an inducible vector for conditional pooled screens in naive T cells that are proposed in Projects 2 and 3 (Aim 1); to produce high-quality, ready-to-transfect retroviral plasmid DNA from cloned libraries of shRNAmirs and sgRNAs for Projects 1-3 (Aim 2); and to re-apply existing arrayed shRNAmir libraries and build custom shRNAmir and gRNA clone sets in appropriate vectors based on gene targets prioritized during the evolution of Projects 1-3 to facilitate detailed follow-up studies (Aim 3).

项目概要/摘要核心B（皮普金） microRNA 环境中的短发夹 RNA (shRNAmirs) 和 CRISPR-Cas9 引导 RNA (sgRNA) 非常强大分别用于免疫系统中实验性 RNA 干扰 (RNAi) 和基因破坏的工具。然而，实际上所有当前的方法都需要预先激活初始细胞，使它们对基因敏感通常使用逆转录病毒或慢病毒载体进行转移，并引起组成型 RNAi 或立即基因破坏。这些问题使研究在逆转录病毒传递之前起作用的基因变得复杂，并阐明了时间免疫反应不同阶段的基因需求。在核心 B 中，我们描述了多种开发创新工具和方法，大大扩展了 shRNAmir 和 sgRNA 在基因研究中的效用功能。我们证明了未经操作的原始 CD4 和 CD8 T 细胞的生成，这些细胞有条件地携带调控的逆转录病毒构建体能够在体内进行可诱导和可逆的 RNAi，并扩展此系统采用一种新方法，可在未接触过的情况下使用诱导型 shRNAmir 促进大型体内混合筛选幼稚细胞。我们还展示了原代 B 和 T 细胞中的 CRISPR-Cas9 引导 RNA (sgRNA) 基因破坏，及其在体内免疫反应中用于分析 B 和 T 细胞功能的用途。这些的组合 shRNAmir 和 sgRNA 方法具有协同作用，构成了创新且严格的实验用于在体内免疫反应期间快速剖析 T 细胞和 B 细胞基因功能的平台。核心 B 构建于多个现有的高保真 shRNAmir 克隆文库，其中包括针对所有编码基因的文库哺乳动物染色质调节因子（312 个基因，约 1,700 个 shRNAmir）和所有哺乳动物转录因子（2,083 个基因，约 8,000 个 shRNAmir）。 Core B 的总体目标是提供执行以下任务所需的基本工具：本 P01 项目 1-3 中提出的体内功能丧失研究。我们的具体目标是使用诱导型载体克隆汇集 shRNAmir 文库，在初始 T 细胞中进行条件汇集筛选，在项目 2 和 3（目标 1）中提出；生产高质量、可立即转染的逆转录病毒质粒 DNA 来自项目 1-3（目标 2）的 shRNAmir 和 sgRNA 克隆文库；并重新应用现有的阵列 shRNAmir 文库并根据基因在适当的载体中构建定制 shRNAmir 和 gRNA 克隆集项目 1-3 发展过程中优先考虑的目标，以促进详细的后续研究（目标 3）。