Protein tagging at scale to enable functional genomic studies

项目摘要/摘要： 生物医学研究中的重大挑战之一是理解每一个 人类基因组中数以千计的蛋白质。这一挑战因蛋白质行为而变得复杂 高度依赖于上下文，因此有必要在各种细胞和 环境设置。阐明蛋白质功能的一种有效方法是通过使用 蛋白质标签。这些标签有助于识别相互作用的伙伴(通过亲和力或表位 标签)、定位动态(通过荧光标记)和细胞功能(通过小分子- 蛋白质稳定性的调节控制)。尽管它们有用，但相当大的时间和精力 开发内源性标记细胞系的需要限制了我们利用它们的能力 潜力。这项提案的目标是开发一个系统，快速创建数百个 每个细胞系都标记了一种独特的蛋白质，这对于实现我们的长期目标是必不可少的 使蛋白质功能的大规模平行检测成为可能。我们的中心假设是 细胞固有的非同源末端连接机制，与普通供体相结合 模板和强大的选择策略，将使数百个细胞的库得以创建 每一行都包含一种唯一标记的蛋白质。这项提议背后的理由是，如果 成功后，我们将把目前有限的蛋白质标记方法转变为高度可扩展的 技术，为系统地研究基因功能开辟了一个新的前沿。我们提供 初步数据，以证明我们方法的可行性，并概述了以下内容 旨在进一步成熟我们的技术：1)表征偏离目标的标签插入率和 确定减少其发生的策略；2)通过以下方式展示我们方法的可塑性 在几种细胞背景下创建数百个标记的细胞系，包括诱导 多能干细胞。这一建议具有创新性，因为它解决了一个长期存在的瓶颈 用精确的基因插入产生细胞系，打开了全面 蛋白质功能的表征。这项工作意义重大，因为它代表了 在可伸缩性方面比现有技术有了很大的改进，并立即在 设计细胞系的产生。这项工作的预期结果是高吞吐量 用于生成唯一修改的细胞系的库的方法(即，每个具有不同的 内源蛋白)，以一次数百个的速度。这项工作将产生积极的影响 通过提供一种易于适应的方法来同时标记数百个 蛋白质在其自然环境中，并在长期内通过实现基本的第一步 从而能够对蛋白质功能进行整体的平行询问。

PROJECT SUMMARY/ABSTRACT: One of the grand challenges within biomedical research is understanding the role of each of the thousands of proteins in the human genome. This challenge is complicated by protein behavior being highly context-dependent, necessitating its study within a variety of cellular and environmental settings. A powerful approach for elucidating protein function is through the use of protein tags. These tags facilitate the identification of interacting partners (via an affinity or epitope tag), localization dynamics (via a fluorescent marker), and cellular function (via small-molecule- regulated control of protein stability). Despite their utility, the sizable amount of time and effort needed to develop endogenously tagged cell lines has limited our ability to capitalize on their potential. The objective of this proposal is to develop a system for rapidly creating hundreds of cell lines each with a unique protein tagged, which is essential to achieve our long-term goal of enabling the massively parallel examination of protein function. Our central hypothesis is that the cell’s intrinsic non-homologous end joining machinery, in combination with generic donor templates and a robust selection strategy, will enable the creation of libraries of hundreds of cell lines each containing a uniquely tagged protein. The rationale underlying this proposal is that, if successful, we will transform the current, limited approach to protein tagging into a highly scalable technology, opening a new frontier for the systematic interrogation of gene function. We provide preliminary data, to demonstrate the feasibility of our approach and have outlined the following aims for further maturing our technology: 1) characterize the rate of off-target tag insertion and identify strategies to mitigate its occurrence; 2) demonstrate the plasticity of our approach by creating hundreds of tagged cell lines within several cellular backgrounds, including induced pluripotent stem cells. This proposal is innovative because it solves a long-standing bottleneck in the generation of cell lines with precise genetic insertions, opening the door to the comprehensive characterization of protein function. This work is significant as it represents a two order of magnitude improvement in scalability over the state of the art, and has immediate applications in the generation of designer cell lines. The expected outcome of this work is a high-throughput method for generating libraries of uniquely modified cell lines (i.e. each with a different endogenous protein tagged), at a rate of hundreds at once. This work will exert a positive impact immediately by delivering a readily adaptable method for simultaneously tagging hundreds of proteins within their native context, and in the long-term by achieving the essential first step towards enabling the parallel interrogation of protein function en masse.