Unlocking the Hidden Kinome

产品说明：该提案的目标是进一步发展一种独特的遗传相互作用技术，以促进揭示人类激酶组中特征不佳的成员的功能。我们的最终目标是产生改变游戏规则的试剂和协议，并照亮基因组的“暗物质”。我们的策略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技术。遗传相互作用是将功能性洞察转移到基因功能的金标准，并且是对蛋白质：蛋白质相互作用和其他标准方案的补充。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。我们随时准备参与该项目，因为我们已经建立了良好的技术基础，最近公布了方法。我们描述池的方法作为一个容易扩展的技术。这个提议描述了我们努力筛选围绕人类激酶组的大量相互作用。这将使我们深入了解功能和途径，为穷人的特点基因。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一个基因共同消融。该文库由数百万个基因相互作用对组成，并在生物反应器中以高覆盖率进行筛选。我们最初的计划是探测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，我们保留使用整合的荧光：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每种细胞类型。未来的屏幕可能会扩展到探索更高阶的相互作用，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).