shRNAmir and CRISPR sgRNA Library Construction Core

项目摘要/摘要 核心B(管道) MicroRNA环境中的短发夹状RNA(ShRNAmir)和CRISPR-Cas9指南RNA(SgRNA)功能强大 分别用于实验RNA干扰(RNAi)和免疫系统中的基因破坏的工具。 然而，几乎所有目前的方法都需要对幼稚细胞进行预激活，使它们对基因易感。 转移，通常是通过逆转录病毒或慢病毒载体，并导致结构性RNAi或立即基因中断。 这些问题使逆转录病毒传播前功能基因的研究复杂化，并澄清了时间上的 免疫反应不同阶段对基因的要求。在核心B中，我们描述了多个 极大地扩展shRNAmir和sgRNAs用于基因研究的创新工具和方法 功能。我们展示了未经处理的原始CD4和CD8T细胞的产生有条件地携带 受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，并延长这一过程 系统到一种新的方法，该方法促进使用未接触的可诱导shRNAmir的大的活体汇集屏幕 幼稚的细胞。我们还证明了CRISPR-Cas9引导RNA(SgRNA)基因在原代B和T细胞中的破坏， 并在体内免疫反应中用于分析B细胞和T细胞的功能。这些结合在一起 ShRNAmir和sgRNA方法是协同的，构成了一个创新和严格的实验 在体内免疫反应中快速剖析T和B细胞基因功能的平台。核心B构建在 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克隆 哺乳动物染色质调节因子(312个基因，~1,700个shRNAmir)，以及所有哺乳动物转录因子 (2,083个基因，约8,000个shRNAmir)。核心B的首要目标是提供必要的工具来执行 本P01项目1-3中提出的在体功能丧失研究。我们的具体目标是分流 使用可诱导载体在幼稚T细胞中克隆条件池筛选的池化shRNAmir文库 在项目2和项目3(目标1)中提出；生产高质量的、可用于转染的逆转录病毒质粒DNA 从项目1-3的shRNAmir和sgRNA克隆文库(目标2)；并重新应用现有的Arraed ShRNAmir文库，并根据基因在适当的载体中构建定制的shRNAmir和gRNA克隆集 在项目1-3的演变过程中确定目标的优先次序，以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).