Functional Genomics Laboratory (FGL)

功能基因组学实验室（FGL）

基本信息

批准号：
10919714
负责人：
Anton Simeonov
金额：
$ 154.3万
依托单位：
NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10919714
关键词：
3-Dimensional Acceleration Adaptive Immune System Address Adverse effects Algorithmic Analysis Area Automation Awareness Bacteria Bacteriophages Binding Biological Assay Biological Process Biology Biotechnology CRISPR interference CRISPR library CRISPR screen CRISPR-mediated transcriptional activation CRISPR/Cas technology Cells Cellular biology Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Communicable Diseases Complex DNA DNA Double Strand Break DNA Sequence DNA biosynthesis Data Data Analyses Databases Development Devices Diabetes Mellitus Disease Drug Targeting Ebola virus Education and Outreach Effectiveness Enhancers Fragile X Syndrome Gene Expression Genes Genetic Recombination Genetic Screening Genetic Transcription Genome Goals HIV Health Hepatitis C virus Human Incidence Informatics Intramural Research Program Knock-in Laboratories Libraries Life Malignant Neoplasms Methodology Methods MicroRNAs Mission Molecular Target Mutation National Center for Advancing Translational Sciences Nematoda Neoplasm Metastasis Parkinson Disease Pathway Analysis Pathway interactions Pharmaceutical Preparations Phenotype Play Private Sector Process Prokaryotic Cells PubChem RNA RNA Interference RNA Sequences RNA interference screen Reading Research Personnel Resistance Resource Sharing Robotics Role Science Scientist Small Interfering RNA System Techniques Technology Testing Time United States National Institutes of Health United States National Library of Medicine Validation Virus Diseases Virus Replication Work Zika Virus assay development cellular imaging computerized tools experimental study functional genomics gene function genetic corepressor genetic element genome editing genome-wide genomic locus inhibitor innovation interest knock-down knockout gene loss of function new technology nuclease pre-clinical public database recruit repaired reverse genetics screening small hairpin RNA small molecule tool

项目摘要

RNA interference was discovered in nematodes in the late 1990s, which blocks the activity of genes by using small interfering (siRNA) or small hairpin RNA (shRNA) molecules. RNAi has emerged as a powerful tool used in thousands of laboratories worldwide to understand gene function. By knocking down a genes function, RNAi can tell us about the role of any gene in maintaining health or causing disease, an invaluable step in identifying potential drug targets. In tests called genome-wide RNAi screens, scientists use automation to introduce siRNA/shRNAs into human cells to knock down the activity of each gene, one at a time. This process can produce a complete list of all genes involved in a particular biological function or disease process. Scientists also can use these techniques to understand what roles genes play in drug effectiveness. The CRISPR/Cas9 system is a form of acquired immune system found in prokaryotes, which helps bacteria resist foreign genetic elements such as bacterial phages. CRISPR RNAs (crRNAs) express in the host genomes bind to the Cas9 nuclease and guide the complex to its target DNA sequence adjacent to PAM (photospacer adjacent motifs). The CRISPR complex then cut both strains of foreign DNA to destroy the invader. In 2012, the bacterial CRISPR/Cas9 system was transformed into a genome-editing tool. Using the CRISPR/Cas9 technology, scientists are now able to modify any genome by either generating random mutations through the error-prone repair mechanism or supplying the DNA template of their choosing to knock in any gene of their interests or correct a mutation by non-homologous recombination. CRISPR/Cas9 system has also been developed as a robust reverse-genetic screening platform. In genetic screening, unlike RNAi knocks down gene expression, CRISPR/Cas9 generates completely loss-off-function phenotypes which can serve as a complementary tool for RNAi. RNAi and CRISPR/Cas9s potential usefulness in genetic screening has been limited by the lack of expertise to perform genome-scale screens, the lack of methodologies that can properly interpret these experiments and the absence of comprehensive RNAi data in public databases for researchers to reference. To address these problems, NCATS operates a state-of-the-art functional genomic screening facility known as the Functional Genomics Laboratory (FGL), and NCATS staff assists NIH intramural investigators with all stages of project planning and execution. The initiative provides public access to functional genomic screening data generated from these experiments through the National Library of Medicines PubChem database. In addition, siRNA/CRISPR RNA sequence information is available from private-sector biotechnology partners. For instance, researchers can access Life Technologies Silencer Select siRNA library, which includes 65,000 siRNA sequences that target more than 20,000 human genes. FGL, administered by NCATS Division of Pre-Clinical Innovation staff, offers a robotic platform with integrated, automated devices for conducting all aspects of screening assays (tests), including manipulating chemicals and cells, reading the results and imaging the cells. Offline (non-robotic) devices can perform smaller-scale work from assay optimization through medium-scale screening. Investigators have the option of using several different siRNA/CRISPR libraries and other small molecules involved. For data analysis, the facility offers powerful computational tools. In addition to enabling collaborations on specific projects, FGL staff work on developing methods that advance the science of functional genomic screening, data analysis algorithms and gene perturbation technologies for exploring gene function. As a result, they recently implemented both pooled CRISPR interference (CRISPRi) and activation (CRISPRa) screening platforms to their facility pipeline. Unlike CRISPR/Cas9 which knocks out gene expression by generating mutations, CRISPRi and CRISPRa technologies do not modified the genome. CRISPRi/a modulates gene expression by recruiting dead Cas9 fused with transcriptional co-repressor/activator onto specific genomic loci, which eliminated the adverse effects cause by DNA double-strand breaks produced by the Cas9 nuclease. In FGL, project areas include cancer (drug enhancer/resistance screens, development of 3D metastasis screens, molecular targets in cancer, and cancer-related pathways), infectious diseases (viral infection and replication such as Zika virus, HIV, Ebola virus, and Hepatitis C virus), fundamental cell biology (DNA replication and reprogramming/differentiation), and other disease-related phenotypes (Parkinsons disease, diabetes, and fragile X syndrome).

RNA 干扰是 20 世纪 90 年代末在线虫中发现的，它通过使用小干扰 (siRNA) 或小发夹 RNA (shRNA) 分子来阻断基因的活性。 RNAi 已成为全球数千个实验室用于了解基因功能的强大工具。通过敲低基因功能，RNAi 可以告诉我们任何基因在维持健康或引起疾病方面的作用，这是识别潜在药物靶点的宝贵一步。在称为全基因组 RNAi 筛选的测试中，科学家利用自动化技术将 siRNA/shRNA 引入人体细胞，一次一个地降低每个基因的活性。这个过程可以产生参与特定生物功能或疾病过程的所有基因的完整列表。科学家还可以利用这些技术来了解基因在药物有效性中发挥的作用。 CRISPR/Cas9系统是原核生物中发现的一种获得性免疫系统，可帮助细菌抵抗细菌噬菌体等外来遗传元件。宿主基因组中表达的 CRISPR RNA (crRNA) 与 Cas9 核酸酶结合，并引导复合物到达与 PAM（光间隔相邻基序）相邻的目标 DNA 序列。然后，CRISPR 复合物切割两种外源 DNA 菌株以消灭入侵者。 2012年，细菌CRISPR/Cas9系统被改造为基因组编辑工具。使用 CRISPR/Cas9 技术，科学家们现在能够通过容易出错的修复机制生成随机突变，或提供他们选择的 DNA 模板来敲入他们感兴趣的任何基因，或通过非非校正方法纠正突变，从而修改任何基因组。同源重组。 CRISPR/Cas9 系统也已被开发为强大的反向遗传筛选平台。在基因筛选中，与 RNAi 敲低基因表达不同，CRISPR/Cas9 产生完全丧失功能的表型，可以作为 RNAi 的补充工具。由于缺乏进行基因组规模筛选的专业知识、缺乏能够正确解释这些实验的方法以及公共数据库中缺乏可供研究人员参考的全面 RNAi 数据，RNAi 和 CRISPR/Cas9 在遗传筛查中的潜在用途受到限制。为了解决这些问题，NCATS 运营着一个最先进的功能基因组筛查设施，称为功能基因组实验室 (FGL)，NCATS 工作人员协助 NIH 校内研究人员完成项目规划和执行的所有阶段。该计划通过国家药物图书馆 PubChem 数据库向公众提供对这些实验生成的功能基因组筛选数据的访问。此外，siRNA/CRISPR RNA 序列信息可从私营部门生物技术合作伙伴处获得。例如，研究人员可以访问 Life Technologies Silencer Select siRNA 库，其中包括针对 20,000 多个人类基因的 65,000 个 siRNA 序列。 FGL 由 NCATS 临床前创新部门管理，提供一个带有集成自动化设备的机器人平台，用于进行筛选分析（测试）的各个方面，包括操作化学品和细胞、读取结果和对细胞成像。离线（非机器人）设备可以执行从测定优化到中等规模筛选的小规模工作。研究人员可以选择使用几种不同的 siRNA/CRISPR 文库和其他相关小分子。对于数据分析，该设施提供了强大的计算工具。除了在特定项目上进行合作外，FGL 工作人员还致力于开发方法，以推进功能基因组筛选、数据分析算法和基因扰动技术的科学发展，以探索基因功能。因此，他们最近在其设施管道中实施了混合 CRISPR 干扰 (CRISPRi) 和激活 (CRISPRa) 筛选平台。与通过产生突变来敲除基因表达的 CRISPR/Cas9 不同，CRISPRi 和 CRISPRa 技术不会修改基因组。 CRISPRi/a 通过将与转录共阻遏物/激活物融合的死亡 Cas9 募集到特定基因组位点上来调节基因表达，从而消除了 Cas9 核酸酶产生的 DNA 双链断裂引起的不利影响。在 FGL 中，项目领域包括癌症（药物增强剂/耐药性筛选、3D 转移筛选的开发、癌症中的分子靶标以及癌症相关途径）、传染病（病毒感染和复制，例如寨卡病毒、艾滋病毒、埃博拉病毒和丙型肝炎病毒）、基础细胞生物学（DNA 复制和重编程/分化）以及其他疾病相关表型（帕金森病、糖尿病和脆性 X 综合征）。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors.

SLFN11 会促进 CUL4 对 CDT1 的降解，以响应复制 DNA 损伤，而 SLFN11 的缺失会导致 ATR/CHK1 抑制剂的合成致死作用。

DOI：
发表时间：
2021
期刊：
影响因子：
11.1
作者：
Jo, Ukhyun;Murai, Yasuhisa;Chakka, Sirisha;Chen, Lu;Cheng, Ken;Murai, Junko;Saha, Liton Kumar;Miller Jenkins, Lisa M;Pommier, Yves
通讯作者：
Pommier, Yves

Whole-genome RNAi screen highlights components of the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity.

全基因组 RNAi 筛选强调内质网/高尔基体的成分是免疫毒素介导的细胞毒性的抵抗来源。

DOI：
发表时间：
2015-03-10
期刊：
影响因子：
11.1
作者：
Pasetto, Matteo;Antignani, Antonella;Ormanoglu, Pinar;Buehler, Eugen;Guha, Rajarshi;Pastan, Ira;Martin, Scott E;FitzGerald, David J
通讯作者：
FitzGerald, David J

Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

基因组规模的 RNA 干扰筛选将抗酶 1 (OAZ1) 确定为提高哺乳动物细胞中重组蛋白产量的靶标。

DOI：
发表时间：
2016-11
期刊：
影响因子：
3.8
作者：
Xiao, Su;Chen, Yu Chi;Buehler, Eugen;Mandal, Swati;Mandal, Ajeet;Betenbaugh, Michael;Park, Myung Hee;Martin, Scott;Shiloach, Joseph
通讯作者：
Shiloach, Joseph

Translational in vitro research: integrating 3D drug discovery and development processes into the drug development pipeline.

转化体外研究：将 3D 药物发现和开发流程整合到药物开发流程中。