Core 2

核心2

• 批准号: 10506984
• 负责人: JENNIFER A DOUDNA
• 金额: $ 34.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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/10506984
• 关键词: Address; Affect; Affinity; Affinity Chromatography; CD4 Positive T Lymphocytes; 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; Investigational Therapies; Knock-in; Knock-out; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Methods; Mission; Pathogenesis; Pathway interactions; Point Mutation; Primary Infection; Property; Proteins; Proteomics; Research; Rest; Ribonucleoproteins; Role; Single-Stranded DNA; Site; Stretching; T-Lymphocyte; Technology; Tertiary Protein Structure; Testing; Time; Validation; Viral Proteins; Virus; Work; cell type; exhaustion; genetic variant; genome editing; genome-wide; genomic locus; innovation; knock-down; macrophage; mutation screening; novel; pathogen; protein complex; protein structure; repaired; structural biology

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：艾滋病毒附件和调节复合物 遗传学核心 总结 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（即活化的 原代CD 4 + T细胞、静息原代CD 4 + T细胞和原代巨噬细胞）。在与 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 用于发现和功能表征不同HIV宿主蛋白的HEPS平台 通过在基因组基因座处插入亲和标签以允许内源蛋白质复合物的亲和纯化 直接从HIV感染的原代细胞中分离使用我们突破性的CRISPR敲除（CRISPRko）， （CRISPRa），干扰（CRISPRi）和敲入（CRISPRki）技术在原代人类T细胞中，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谱，（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， 关键的宿主-病原体相互作用界面，以及（4）在靶向位置引入/替换小段DNA 基因组位点来研究特定遗传变异的功能。总的来说，这些基因研究将加强 HARC的中心使命是了解HIV辅助蛋白和宿主因子之间的相互作用， 为更广泛的艾滋病毒研究界提供新的实验和治疗途径。总之，遗传学 核心将开发一个遗传扰动工具箱，以补充创新的结构生物学和蛋白质组学 努力