shRNAmir and CRISPR sgRNA Library Construction Core

项目总结/摘要 核心B（Pipkin） microRNA背景下的短发夹RNA（shRNAmir）和CRISPR-Cas9指导RNA（sgRNA）是强大的 分别用于实验性RNA干扰（RNAi）和免疫系统基因破坏的工具。 然而，几乎所有目前的方法都需要预活化幼稚细胞以使它们对基因表达敏感。 转移，通常用逆转录病毒或慢病毒载体，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递送之前起作用的基因和澄清逆转录病毒递送的时间复杂化。 在免疫反应的不同阶段对基因的需求。在核心B中，我们描述了多个 创新的工具和方法，大大扩展了shRNAmir和sgRNA的效用，以研究基因 功能我们证明了未经操作的幼稚CD 4和CD 8 T细胞的产生， 调控的逆转录病毒构建体，其能够在体内进行可诱导的和可逆的RNAi，并延长这种 系统的一种新的方法，促进大规模的体内合并筛选使用诱导型shRNAmir在未触及的 幼稚细胞我们还证明了原代B和T细胞中的CRISPR-Cas9引导RNA（sgRNA）基因破坏， 以及其在体内免疫应答期间分析B和T细胞功能的用途。结合这些 shRNAmir和sgRNA方法是协同的，并构成了一个创新和严格的实验 用于在体内免疫应答期间快速剖析T和B细胞中的基因功能的平台。核心B基于 多个现有的高保真shRNAmir克隆文库，其中包括那些靶向所有编码 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）。核心B的首要目标是提供执行 本P01项目1-3中拟定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合并的shRNAmir文库，用于在幼稚T细胞中进行条件性合并筛选， 在项目2和3（目标1）中提出，生产高质量的、可直接转染的逆转录病毒质粒DNA 从项目1-3（Aim 2）的shRNAmir和sgRNA克隆文库中;并重新应用现有的阵列 shRNAmir文库，并基于基因在适当载体中构建定制的shRNAmir和gRNA克隆集。 在项目1-3的发展过程中，优先考虑的目标是促进详细的后续研究（目标3）。

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).