Administrative Supplement for Direct and rapid control of proteins at scale

摘要： 自人类基因组计划完成以来，生物医学科学的一项重大努力是 利用微扰来理解蛋白质的功能。基于RNAi的功能筛选平台， CRISPR在实现这一目标方面发挥了重要作用。然而，尽管它们被广泛使用， 尽管有着非凡的优势，但即使是今天最先进的平台，其能力仍然有限。两 突出的局限性包括：（1）无法研究细胞生长所必需的基因的功能， 生长，以及（2）蛋白质消耗的缓慢动态，使适应机制， 影响结果。为了克服这些局限性，我建议开发一种新的汇总筛选方法， 该平台基于在基因组规模上快速和直接调节人类细胞中的蛋白质。这 该平台将基于用HaloTag（一种生物正交蛋白质）对基因进行多重内源性标记 结构域，其能够结合多种小分子配体，所述小分子配体可用于增加或 降低融合蛋白的稳定性。在本提案中，我将优化合并的多重内源性 使用我们实验室开发的独特的内含子靶向方法进行基因标记。基因组规模 HaloTag融合文库将用于筛选急性蛋白质降解和去稳定化的影响， 鉴定易于转录适应的基因，并以无偏的方式鉴定聚集蛋白质。 使用不同的荧光配体的方式。

Abstract: Since the completion of the Human Genome Project, a major effort in the biomedical sciences has been to understand the function of proteins using perturbation. Functional screening platforms based on RNAi and CRISPR, in particular, have been instrumental in pursuing this goal. Yet despite their wide use and extraordinary strengths, even todays state-of-the-art platforms are still limited in their capabilities. Two prominent limitations include (1) The inability to study the function of genes that are essential for cellular growth, and (2) the slow dynamics of protein depletion that enable adaptation mechanisms that can influence the results. To overcome these limitations, I propose to develop a novel pooled screening platform based on rapid and direct modulation of proteins in human cells at a genome-scale. This platform will be based on multiplexed endogenous tagging of genes with HaloTag, a bioorthogonal protein domain capable of binding a variety of small molecule ligands that can be used to either increase or decrease the stability of the fusion protein. In this proposal I will optimize pooled multiplexed endogenous gene tagging using a unique intron targeting approach which our lab has developed. Genome-scale HaloTag fusion libraries will be used to screen for effects of acute protein degradation and destabilization, identify genes that are prone for transcriptional adaptation, and identify aggregating proteins in an unbiased manner using a different fluorescence ligands.