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配件和监管综合体遗传学核心概括为了支持所有HARC中心项目，遗传学核心将为 HIV宿主蛋白复合物在原代细胞中的发现和功能表征（即激活主要的CD4+ T细胞，静止的原发性CD4+ T细胞和一级巨噬细胞）。与蛋白质组学，结构生物学和计算核心，我们将开发HARC内源性蛋白发现和功能表征不同HIV宿主蛋白的结构（HEP）平台通过在基因组基因座上插入亲和力标签以允许内源性蛋白复合物的亲和力纯化来复合物直接来自HIV感染的原代细胞。使用我们的突破性CRIS淘汰赛（CRISPRKO），激活（CRISPRA），干扰（CRISPRI）和敲入（CRISPRKI）技术在原代人T细胞中，我们将（1）生成前病毒和抗病毒HIV互动宿主因子的系统图，（2）功能验证新型 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鉴定关键的宿主 - 病原体相互作用界面，（4）引入/更换针对目标的小型DNA 基因组位点研究特定遗传变异的功能。总的来说，这些遗传研究将加强中央HARC的使命是了解HIV配件蛋白与宿主因素之间的相互作用并开放广泛的艾滋病毒研究界的新实验和治疗途径。总之，遗传学核心将开发一个工具箱，用于遗传扰动，以补充创新的结构生物学和蛋白质组学努力。