High-throughput genetic interaction sequencing in mammalian cells

哺乳动物细胞中的高通量遗传相互作用测序

• 批准号: 9360136
• 负责人: SASHA F LEVY
• 金额: $ 20.04万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-28 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9360136
• 关键词: 4T1; Biological Assay; Biological Process; Biology; Breast Cancer cell line; Breast Epithelial Cells; CRISPR/Cas technology; Cancer Etiology; Cell Culture Techniques; Cell Line; Cells; Chromatin; Complex; DNA; Development; Disease; Drug resistance; Drug usage; ES Cell Line; Engineering; Environment; Future; Gene Deletion; Genes; Genetic; Genome; Goals; Growth; Guide RNA; Haploidy; Heritability; Human; Individual; Lead; Leg; Libraries; Location; MCF10A cells; Malignant Neoplasms; Mammalian Cell; Mammalian Genetics; Maps; Measures; Methodology; Methods; Monitor; Mus; Oncogenes; Other Genetics; Parkinson Disease; Partner in relationship; Performance; Pharmaceutical Preparations; Physiological; Plasmids; RNA library; Reagent; Suppressor Genes; System; Technology; Testing; Time; Tumor Suppressor Genes; Work; Yeasts; base; cell type; combinatorial; embryonic stem cell; fitness; gene interaction; gene product; genetic element; genetic technology; genome-wide; high throughput technology; homologous recombination; in vivo; innovation; insight; knock-down; knockout gene; new therapeutic target; protein complex; protein protein interaction; ras Oncogene; screening; sequencing platform; technology development; three dimensional cell culture; trait; treatment response; tumor; tumor progression; yeast genetics

Project Summary The goal of our R21 project is to develop a powerful platform that will be able to generate and assay millions of combinations of CRISPR/Cas9 single-guide RNAs (sgRNAs) or other genetic perturbagens. Doing so will clear the way for systematic exploration of genetic interactions in mammalian cells. There are many reasons to develop this high-throughput mammalian genetic technology. Currently there is no systematic method to unravel the assortment of genetic interactions that drive specific cancers and determine the variability of individual treatment responses. Nor is there a robust method to study gene interactions in other complex multigenic diseases such as Parkinson's. We are motivated by the tremendous advances recently made in yeast and worms that have resulted from systematic discovery of genetic interactions. Genetic interaction maps in yeast have revealed functional relationships within and between protein complexes orders of magnitude beyond those revealed by protein- protein interaction screens. Systematic screening 65,000 pairs of genes in worms led to the identification of a class of highly connected `hub' genes encoding chromatin regulators. The technologies behind these discoveries depend on several high-throughput steps, including a dependable gene knockout or knockdown method, a method to deliver two gene knockouts/knockdowns into the same cell and to monitor which cells receive which combinations, and also a reliable assay to measure relative fitness. Our major enabling technology for development of a high-throughput system for mammalian cells is the tandem-integration landing pad that allows two plasmids to be inserted next to each other at a neutral location of the genome. Each plasmid contains a DNA barcode that uniquely identifies the associated genetic perturbagen (e.g. sgRNAs). When both plasmids are integrated into the genome, the two barcodes are in close enough proximity to be sequenced together by paired-end amplicon sequencing. We have established this methodology in yeast and have shown that it can generate a library of >108 double barcoded cells via pooled sequential plasmid transformation and integration. The fitness of large double barcode libraries can then be measured using the fit-seq approach that we pioneered: pooled growth and double barcode amplicon sequencing over several time points accurately measures the relative fitness of each double barcoded cell in the pool. In yeast, Genetic interaction Sequencing (GiSeq) promises to be a cheaper and higher-throughput alternative to the commonly used synthetic-genetic array technology. In mammalian cells, GiSeq promises to be a major leap forward over existing technologies: not only will genome-scale interaction libraries become practical, but negligible work will be needed to repeat a screen in a different cells or different conditions. For this proposal, we will establish the utility of GiSeq in mammalian cells (Aims 1 and 2), and prepare reagents to perform genetic interaction screens in vivo (Aim 3).

项目摘要 我们R21项目的目标是开发一个功能强大的平台，该平台能够生成和分析 CRISPR/CAS9单固定RNA（SGRNA）或其他遗传性扰动的数百万组合。正在做 因此，将清除对哺乳动物细胞中遗传相互作用的系统探索的道路。有许多 开发这种高通量哺乳动物遗传技术的原因。目前没有系统 揭示驱动特定癌症并确定的遗传相互作用的分类方法 个体治疗反应的变异性。也没有一种强大的方法来研究其他人的基因相互作用 复杂的多基因疾病，例如帕金森氏症。 我们受到最近在酵母和蠕虫中取得的巨大进步的动力 遗传相互作用的系统发现。酵母中的遗传相互作用图显示了功能 蛋白质复合物之间和之间的关系超出了蛋白质揭示的数量级 蛋白质相互作用筛选。系统筛选蠕虫中的65,000对基因，导致鉴定 编码染色质调节剂的高度连接的“集线器”基因。这些背后的技术 发现取决于几个高通量步骤，包括可靠的基因敲除或敲除 方法，一种将两个基因敲除/敲低输送到同一细胞并监视哪些细胞的方法 接收哪些组合，也可以接受可靠的测定法，以测量相对健身。 我们为开发哺乳动物细胞高通量系统开发的主要能力技术是 串联综合着陆垫，允许在中立位置互相插入两个质粒 基因组。每个质粒都包含一个DNA条形码，该条形码独特地识别相关的遗传 贝特堡（例如SGRNA）。当两个质粒整合到基因组中时，两个条形码在接近 足够的接近度可以通过成对的末端扩增子测序进行测序。我们已经建立了这个 酵母中的方法论，并表明它可以通过汇总生成> 108个双条形码单元格的库 顺序质粒转化和整合。然后可以是大型双条形码库的适合度 使用我们开创的Fit-Seq方法测量：合并的生长和双条形码扩增子 在几个时间点上进行测序准确测量每个双条形码单元中每个双条形码的相对适应性 游泳池。在酵母中，遗传相互作用测序（Giseq）有望更便宜，更高 替代常用的合成遗传阵列技术。在哺乳动物细胞中，Giseq有望 在现有技术上取得重大飞跃：基因组规模的互动库不仅会成为 在不同的单元格或不同条件下重复屏幕需要实用但可以忽略的工作。 对于此建议，我们将在哺乳动物细胞中建立Giseq的实用性（目标1和2），并准备 在体内执行遗传相互作用筛选的试剂（AIM 3）。