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Core 2

核心2

基本信息

批准号：
10666656
负责人：
JENNIFER A DOUDNA
金额：
$ 32.58万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10666656
关键词：
Address Affect Affinity Affinity Chromatography CD4 Positive T Lymphocytes CRISPR interference CRISPR screen CRISPR-mediated transcriptional activation CRISPR/Cas technology Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Communities Complement Complex DNA Data Dependence Electroporation Genes Genetic Genetic study Genomics Goals Guide RNA HIV HIV Infections Human Infection Integration Host Factors Knock-in Knock-out Macromolecular Complexes Macrophage Maps Mass Spectrum Analysis Methods Mission Pathogenesis Pathway interactions Point Mutation Property Proteins Proteomics Research Rest Ribonucleoproteins Role Single-Stranded DNA Site Stretching T-Lymphocyte Technology Tertiary Protein Structure Testing Therapeutic Time Validation Viral Viral Proteins Virus Work cell type genetic variant genome editing genome-wide genomic locus innovation knock-down mutation screening novel pathogen protein complex protein structure repaired structural biology

项目摘要

THE HARC CENTER: HIV ACCESSORY AND REGULATORY COMPLEXES GENETICS CORE SUMMARY To support all HARC Center Projects, the Genetics Core will provide innovative high-throughput methods for the discovery and functional characterization of HIV-host protein complexes in primary cells (i.e. activated primary CD4+ T cells, resting primary CD4+ T cells, and primary macrophages). Working in tandem with the Proteomics, Structural Biology, and Computational Cores, we will develop the HARC endogenous protein structure (HEPS) platform for the discovery and functional characterization of different HIV-host protein complexes by inserting affinity tags at genomic loci to allow affinity purification of endogenous protein complexes directly from HIV infected primary cells. Using our breakthrough CRISPR knock-out (CRISPRko), activation (CRISPRa), interference (CRISPRi), and knock-in (CRISPRki) technologies in primary human T cells, we will (1) generate a systematic map of proviral and antiviral HIV-interacting host factors, (2) functionally validate novel HIV-host interactions, (3) undertake endogenous deep mutational scanning of specific protein domains to identify critical host-pathogen interaction interfaces, and (4) introduce/replace small stretches of DNA at targeted genomic sites to study the function of specific genetic variants. Collectively, these genetic studies will strengthen the central HARC mission to understand interactions between HIV accessory proteins and host factors and open new experimental and therapeutic avenues for a broader HIV research community. In summary, the Genetics Core will develop a toolbox for genetic perturbations to complement innovative structural biology and proteomic efforts.

HARC 中心：HIV 配件和调节复合体遗传学核心概括为了支持所有 HARC 中心项目，遗传学核心将提供创新的高通量方法原代细胞中 HIV 宿主蛋白复合物的发现和功能表征（即激活的原代 CD4+ T 细胞、静息原代 CD4+ T 细胞和原代巨噬细胞）。与蛋白质组学、结构生物学和计算核心，我们将开发HARC内源蛋白结构 (HEPS) 平台，用于不同 HIV 宿主蛋白的发现和功能表征通过在基因组位点插入亲和标签来亲和纯化内源蛋白复合物直接来自HIV感染的原代细胞。使用我们突破性的 CRISPR 敲除 (CRISPRko)、激活人类原代 T 细胞中的 (CRISPRa)、干扰 (CRISPRi) 和敲入 (CRISPRki) 技术，我们将 (1) 生成前病毒和抗病毒 HIV 相互作用宿主因子的系统图，(2) 功能验证新的 HIV-宿主相互作用，(3) 对特定蛋白质结构域进行内源性深度突变扫描以识别关键的宿主-病原体相互作用界面，以及 (4) 在目标位置引入/替换一小段 DNA 基因组位点来研究特定遗传变异的功能。总的来说，这些遗传学研究将加强 HARC 的核心使命是了解 HIV 辅助蛋白和宿主因子之间的相互作用，并开放为更广泛的艾滋病毒研究界提供新的实验和治疗途径。综上所述，遗传学 Core 将开发一个用于遗传扰动的工具箱，以补充创新的结构生物学和蛋白质组学努力。