Rapid and direct control of the proteome through a multiplexed tag system

项目摘要 了解和调节蛋白质组的功能是生物医学科学的主要目标。癌 尤其是生物学，是目前受益于高通量功能分析的一个领域 基因和蛋白质。癌细胞对共同细胞途径的选择性依赖，这种现象被称为 非癌基因成瘾，通常通过高通量筛查方法进行研究，从而发现新的 潜在的治疗方法。不幸的是，基于CRISPR和RNA干扰的现代功能筛查，尽管 信息性的，不能剧烈地影响蛋白质组，因此不仅在技术上是有限的，而且特别是 容易受到补偿机制的影响，这种补偿机制模糊了蛋白质的作用。此外，在建立 基因敲除，许多对生存至关重要的蛋白质丢失，无法测试有趣的表型 以不可行的屏幕格式。为了促进对蛋白质组的更广泛和更全面的研究，我 建议开发一种基于蛋白质的快速和直接调节的新的筛选范式 全基因组的规模。这个筛查平台最初将被开发来发现许多关于非 癌细胞中的致癌成瘾。这项技术基于一个多路细胞库，其中每个基因 用一种配体结合的生物正交蛋白标记，一次一个细胞。小分子配体将是双向的 调节标记蛋白的稳定性。在第一个目标中，我将确定高效大规模所需的规则 蛋白质标签。随后，在第二个目标中，我将构建一个全基因组的癌症多重标签文库 细胞并通过快速降解进行蛋白质重要性筛选，展示了该平台的能力 揭开小说的大热。使用CRISPR和CRISPR等传统遗传扰动工具进行重要性筛选 RNA干扰，预计更容易受到细胞补偿机制的影响，所以我会 直接将从该筛选获得的结果与从其他可比较的重要性获得的结果进行比较 屏幕。在第三个目标中，我将利用开发的平台来全面探索蛋白质平衡的作用 在非癌基因成瘾中。具体地说，我将揭示对蛋白毒性压力的急性反应的亚细胞图谱 在癌细胞和健康细胞中都有。这将通过使用多路复用标签单元库的子集来实现 诱导隔室特异的蛋白质不稳定，这将通过单细胞RNA测序进行分析。在……里面 总而言之，我将开发一种策略，以高通量的方式快速、直接地调节蛋白质组， 促进研究，这将极大地有助于我们理解癌细胞和 导致了新的癌症治疗方法的发展。这个项目的成功将大大得益于它的 环境，提供对设施的访问和来自儿童医院的专家的见解 费城和宾夕法尼亚大学。令人兴奋的就近合作只需一次即可轻松建立 这个平台被开发出来，立即把它的潜力用在最有益的地方。

Project Summary Understanding and regulating the functions of the proteome is a primary goal in the biomedical sciences. Cancer biology, in a particular, is a field that is currently greatly benefiting from high-throughput functional analysis of genes and proteins. The selective reliance of cancer cells on common cell pathways, a phenomenon known as non-oncogene addiction, is often studied via high-throughput screening approaches, thus uncovering new potential therapies. Unfortunately, modern functional screens based on CRISPR and RNA interference, although informative, cannot affect the proteome acutely and are thus not only technically limiting, but also especially susceptible to compensation mechanisms that obscure protein roles. Additionally, in the time it takes to establish genetic knockdown, many proteins essential for viability are lost and cannot be tested for interesting phenotypes in non-viability screen formats. To facilitate a broader and more comprehensive study of the proteome, I propose to develop a new screening paradigm based on rapid and direct modulation of proteins at a genome-wide scale. This screening platform will initially be developed to uncover many new insights on non- oncogenic addiction in cancer cells. The technology is based on a multiplexed cell library where each gene is tagged with a ligand-binding, bioorthogonal protein, one cell at a time. Small-molecule ligands will bidirectionally regulate the stability of tagged proteins. In the first aim, I will determine the rules required for efficient large-scale protein tagging. Subsequently, in the second aim, I will construct a genome-wide multiplexed tag library in cancer cells and perform a protein essentiality screen by rapid degradation, demonstrating the ability of the platform to uncover novel hits. Essentiality screens performed with traditional genetic perturbation tools, like CRISPR and RNA interference, are predicted to be much more susceptible to cell compensation mechanisms, and so I will directly compare the results obtained from this screen to results obtained from other, comparable essentiality screens. In the third aim, I will utilize the developed platform to comprehensively explore the role of proteostasis in non-oncogene addiction. Specifically, I will uncover a subcellular map of acute responses to proteotoxic stress in both cancerous and healthy cells. This will be achieved by using a subset of the multiplexed tag cell library to induce compartment-specific protein destabilization, which will be analyzed by single-cell RNA sequencing. In summary, I will develop a strategy to rapidly and directly modulate the proteome in a high-throughput manner, facilitating studies that will greatly contribute to our understanding of non-oncogenic addiction in cancer cells and leading to the development of novel cancer therapies. The success of this project will be greatly facilitated by its environment, which provides access to facilities and insight from experts from both the Children’s Hospital of Philadelphia and the University of Pennsylvania. Exciting, nearby collaborations can be easily established once this platform is developed, immediately putting its potential to use where it will be most beneficial.