Functional Genomics Laboratory (FGL)

RNA干扰是在20世纪90年代末在线虫中发现的，其通过使用小干扰（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。通过敲除基因功能，RNAi可以告诉我们任何基因在维持健康或引起疾病中的作用，这是确定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，科学家使用自动化将siRNA/shRNAs引入人类细胞中，以一次一个地敲低每个基因的活性。这个过程可以产生一个完整的列表，列出所有参与特定生物功能或疾病过程的基因。科学家们也可以利用这些技术来了解基因在药物有效性中扮演的角色。 CRISPR/Cas9系统是在原核生物中发现的一种获得性免疫系统，它可以帮助细菌抵抗外来遗传因子，如细菌梭菌。在宿主基因组中表达的CRISPR RNA（crRNA）与Cas9核酸酶结合，并将复合物引导至其与PAM相邻的靶DNA序列（光间隔区相邻基序）。然后，CRISPR复合物切割两种外来DNA菌株以摧毁入侵者。2012年，细菌CRISPR/Cas9系统被转化为基因组编辑工具。使用CRISPR/Cas9技术，科学家现在能够通过易错修复机制产生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同源重组纠正突变来修改任何基因组。CRISPR/Cas9系统也被开发为一个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。 RNAi和CRISPR/Cas9在遗传筛查中的潜在用途受到缺乏进行基因组规模筛选的专业知识，缺乏可以正确解释这些实验的方法以及公共数据库中缺乏全面的RNAi数据供研究人员参考的限制。为了解决这些问题，NCATS运营着一个最先进的功能基因组筛选设施，称为功能基因组学实验室（FGL），NCATS工作人员协助NIH内部研究人员进行项目规划和执行的所有阶段。该计划通过国家医学图书馆PubChem数据库向公众提供从这些实验中产生的功能基因组筛选数据。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文库，其中包括65，000个siRNA序列，靶向超过20，000个人类基因。FGL由NCATS临床前创新部门的工作人员管理，提供了一个机器人平台，该平台具有集成的自动化设备，用于进行筛选测定（测试）的所有方面，包括操作化学品和细胞，阅读结果和细胞成像。离线（非机器人）设备可以执行从分析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文库和其他相关的小分子。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的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，以探索基因功能。因此，他们最近在其设施管道中实施了合并的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技术不会修饰基因组。CRISPRi/a通过将与转录辅阻遏物/激活物融合的死亡Cas9募集到特定的基因组位点上来调节基因表达，这消除了由Cas9核酸酶产生的DNA双链断裂引起的不利影响。在FGL，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、癌症中的分子靶点和癌症相关途径）、感染性疾病（病毒感染和复制，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）和其他疾病相关表型（帕金森病，糖尿病和脆性X综合征）。

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).