Unlocking the Hidden Kinome

本提案的目标是进一步发展一种独特的遗传相互作用技术，以促进揭示人类基因组中特征不明显的成员的功能。我们的最终目标是产生改变游戏规则的试剂和方案，并阐明基因组的“暗物质”。我们的策略是基于超高通量基因相互作用，这是我们为哺乳动物细胞开发的一项技术。基因相互作用是研究基因功能的黄金标准，是蛋白质相互作用和其他标准协议的补充。我们提出的技术代表了使用CRISPR和RNA干扰的尖端方法的新进展。我们随时准备参与这个项目，因为我们已经建立了一个良好的技术基础，最近发布了该方法。我们将池方法描述为易于扩展的技术。这一提议描述了我们为筛选围绕人类基因组的大量相互作用所做的努力。这将使我们深入了解那些特征不明显的基因的功能和途径。一个主要目标是创建定制的“双文库”，其中518个人类激酶基因中的每一个都与基因组中的每个基因共同消融。该文库包含数百万个基因相互作用对，并在生物反应器中进行高覆盖率筛选。我们最初的计划是探索导致与增殖丧失和细胞死亡相关的基本表型的信号通路。然而，我们保留使用综合荧光：抗生素报告基因对特定信号通路进行额外筛选的选择。这些资源是可扩展的，适用于几乎任何人类基因和几乎所有细胞类型。未来的筛选可能会扩展到探索高阶相互作用，并扩展到kinome之外（即gpcr，核受体和离子通道）。

DESCRIPTION: The goal of this proposal is to further a unique genetic interaction technology to facilitate the unveiling of the functions of the poorly characterized members of the human kinome. Our end goal is to generate game-changing reagents and protocols, and illuminate the "Dark Matter" of the genome. Our strategy is based on ultra-high throughput gene interaction, a technology that we have developed for mammalian cells. Genetic interaction is a gold standard for shedding functional insight into gene function and is complimentary to protein:protein interaction and other standard protocols. Our proposed technologies represent a novel advance on cutting-edge approaches that use CRISPR and RNA interference. We stand ready to engage the project as we have already established a sound technical foundation having recently published the methodology. We describe pooled approaches as a readily scalable technology. This proposal describes our efforts to screen immense numbers of interactions surrounding the human kinome. This will give us insight into functions and pathways for the poorly characterized genes. A major goal is to create customized 'doubles libraries' where each of the 518 human kinase genes are co-ablated against every gene in the genome. This library constitutes millions of gene interaction pairs and is screened at high coverage in bioreactors. Our initial plan is to probe signaling pathways that lead to fundamental phenotypes that relate to loss of proliferation and cell death. However we reserve the option to perform additional screens for specific signaling pathways, using integrated fluorescent:antibiotic reporter genes. These resources are scalable and applicable to nearly any human gene and nearly every cell type. Future screens may scale to explore higher-order interactions, and extend beyond the kinome (i.e., GPCRs, nuclear receptors, and ion channels).