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 我们的R21项目的目标是开发一个强大的平台，能够生成和分析 数以百万计的CRISPR/Cas9单向导RNA（sgRNA）或其他遗传干扰物的组合。做 这将为系统地探索哺乳动物细胞中的遗传相互作用扫清道路。有很多 开发这种高通量哺乳动物遗传技术的理由。目前没有系统的 一种方法来解开驱动特定癌症的遗传相互作用的分类，并确定 个体治疗反应的变异性。也没有一个强大的方法来研究基因相互作用在其他 复杂的多基因疾病，如帕金森氏症。 我们的动机是最近在酵母和蠕虫方面取得的巨大进展， 系统地发现遗传相互作用。酵母中的遗传相互作用图谱揭示了 蛋白质复合物内和蛋白质复合物之间的关系超出了蛋白质- 蛋白质相互作用筛选。系统地筛选了蠕虫中的65，000对基因， 编码染色质调节因子的一类高度连接的“枢纽”基因。这些背后的技术 这些发现依赖于几个高通量的步骤，包括可靠的基因敲除或敲除 方法，一种将两种基因敲除/敲低递送到同一细胞中并监测哪些细胞 接收哪些组合，以及测量相对适合度的可靠测定。 我们开发哺乳动物细胞高通量系统的主要技术是 串联整合着陆垫，允许两个质粒在中性位置彼此相邻插入 的基因组。每个质粒都含有一个DNA条形码，可以唯一识别相关的遗传基因。 干扰原（例如sgRNA）。当两个质粒都整合到基因组中时，两个条形码是紧密的。 足够接近以通过配对末端扩增子测序一起测序。我们已经建立了这个 该方法在酵母中进行，并且已经显示其可以通过合并的细胞库产生>108个双条形码化细胞的文库。 连续质粒转化和整合。大型双条形码文库的适合性可以是 使用我们开创的fit-seq方法测量：合并生长和双条形码扩增子 在几个时间点上的测序准确地测量每个双条形码化细胞在细胞中的相对适合度。 泳池在酵母中，基因相互作用测序（GiSeq）有望成为一种更便宜和更高通量的方法。 替代常用的合成基因阵列技术。在哺乳动物细胞中，GiSeq承诺 这将是对现有技术的一次重大飞跃：不仅基因组规模的相互作用库将成为 在不同的细胞或不同的条件下重复筛选将需要实际但可忽略的工作。 对于该提议，我们将建立GiSeq在哺乳动物细胞中的效用（目的1和2），并制备 用于进行体内遗传相互作用筛选的试剂（目的3）。