High-throughput genetic interaction sequencing in mammalian cells

项目摘要 我们R21项目的目标是开发一个强大的平台，该平台将能够生成和分析 数以百万计的CRISPR/Cas9单引导RNA(SgRNAs)或其他遗传扰动的组合。vbl.做，做 这将为系统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基因技术的原因。目前还没有一个系统的 方法解开驱动特定癌症的基因相互作用的分类，并确定 个体治疗反应的变异性。也没有一种强有力的方法来研究其他物种的基因相互作用 复杂的多基因疾病，如帕金森氏症。 我们的动力来自于最近在酵母和蠕虫方面取得的巨大进步，这些进步是由 基因相互作用的系统发现。酵母中的遗传相互作用图谱揭示了功能 蛋白质复合体内部和之间的关系超出了蛋白质揭示的数量级- 蛋白质相互作用屏幕。对蠕虫中65,000对基因的系统筛选导致鉴定出一种 一类高度连接的“中心”基因，编码染色质调节剂。这些背后的技术 发现依赖于几个高通量步骤，包括可靠的基因敲除或敲除 方法，一种将两个基因敲除/敲除传递到同一细胞并监控哪些细胞的方法 得到哪些组合，以及衡量相对适合度的可靠方法。 我们为哺乳动物细胞开发高通量系统的主要使能技术是 串联整合着陆垫，允许两个质粒在中性位置彼此相邻插入 在基因组中。每个质粒都包含一个DNA条形码，该条形码唯一地识别相关的基因 扰动因子(如sgRNA)。当两个质粒整合到基因组中时，两个条形码就接近了 足够近的距离，可以通过成对末端扩增子测序来一起测序。我们已经确立了这一点 在酵母中的方法学，并已表明它可以通过池化产生一个包含&gt；108双条码细胞的文库。 序贯转化和整合。然后，大型双重条形码库的适合性可以是 使用我们首创的FIT-SEQ方法进行测量：混合增长和双条形码扩增 在多个时间点上的排序准确地测量每个双条形码单元的相对适合度 泳池。在酵母中，基因相互作用测序(GiSeq)有望成为一种成本更低、产量更高的方法 替代常用的合成基因阵列技术。在哺乳动物细胞中，GiSeq承诺 是现有技术的重大飞跃：不仅基因组规模的交互文库将成为 要在不同的单元格或不同的条件下重复屏幕，实用但可以忽略不计的工作将需要进行。 对于这项提议，我们将建立GiSeq在哺乳动物细胞中的用途(目标1和2)，并准备 在体内进行遗传相互作用筛选的试剂(目标3)。

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).