Administrative Supplement for Direct and rapid control of proteins at scale

用于大规模直接和快速控制蛋白质的行政补充

• 批准号: 10521748
• 负责人: Ophir Shalem
• 金额: $ 15.66万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10521748
• 关键词: Acute; Administrative Supplement; Affect; Binding; Biological; Cells; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Essential Genes; Fluorescence; Genes; Genetic Transcription; Genome; Goals; Growth; Human; Human Genome Project; Introns; Libraries; Ligands; Methods; Plant Roots; Protein Dynamics; Proteins; RNA Interference; Science; Screening procedure; Tertiary Protein Structure; base; cell growth; gene function; genome-wide; novel; protein aggregation; protein degradation; protein function; screening; small molecule; transcriptome

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.

摘要： 自从人类基因组计划完成以来，生物医学科学的主要努力是 利用微扰来理解蛋白质的功能。基于RNAi和RNAi的功能筛选平台 尤其是该研究所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和 非同寻常的优势，即使是今天最先进的平台，其能力仍然有限。二 突出的局限性包括：(1)无法研究对细胞至关重要的基因的功能 生长，以及(2)蛋白质消耗的缓慢动态，使适应机制能够 影响结果。为了克服这些限制，我建议开发一种新的联合筛查 基于在基因组水平上对人类细胞中蛋白质的快速和直接调节的平台。这 平台将基于用HaloTag(一种生物正交蛋白质)对基因进行多路内源标记 能够结合各种小分子配体的结构域，可用于增加或 降低融合蛋白的稳定性。在本提案中，我将优化池中多路传输的内源 使用我们实验室开发的一种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不稳定的影响， 识别易于转录适应的基因，并在无偏倚的 采用不同的荧光配基方式。

项目成果

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics.

汇集内源蛋白标记和招募，以大规模发现诱导邻近疗法的效应器。

• DOI: 10.21203/rs.3.rs-3161717/v1
• 发表时间: 2023
• 期刊: Research square
• 作者: Serebrenik,YevgeniyV;Mani,Deepak;Maujean,Timothé;Burslem,GeorgeM;Shalem,Ophir
• 通讯作者: Shalem,Ophir