Protein tagging at scale to enable functional genomic studies

项目总结/摘要： 生物医学研究中的一个重大挑战是理解每个生物体的作用。 人类基因组中成千上万的蛋白质。蛋白质的行为使这一挑战变得复杂 高度依赖于上下文，需要在各种细胞内进行研究， 环境设置。阐明蛋白质功能的一种强有力的方法是使用 蛋白质标签这些标签促进相互作用伴侣的鉴定（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）。尽管它们很有用，但大量的时间和精力 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。 潜力该提案的目标是开发一个系统，用于快速创建数百个 每个细胞系都有一个独特的蛋白质标签，这对实现我们的长期目标至关重要。 从而能够对蛋白质功能进行大规模的平行检测。我们的中心假设是， 细胞固有的非同源末端连接机制，与通用供体结合 模板和强大的选择策略，将使数百个细胞库的创建成为可能。 每一个都含有一个独特的标记蛋白质。这一建议的基本原理是，如果 如果成功，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，为基因功能的系统性询问开辟了新的前沿。我们提供 初步数据，以证明我们的方法的可行性，并概述了以下内容 旨在进一步成熟我们的技术：1）表征脱靶标签插入的速率， 确定战略，以减轻其发生; 2）证明我们的方法的可塑性， 在几种细胞背景中创建数百个标记的细胞系，包括诱导的 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， 具有精确遗传插入的细胞系的产生，打开了全面的 蛋白质功能的表征。这项工作是重要的，因为它代表了两个秩序， 与现有技术相比，在可扩展性方面有很大的改进，并且在以下方面具有直接的应用： 设计者细胞系的产生。这项工作的预期成果是一个高通量 用于产生独特修饰的细胞系（即每种具有不同的 内源性蛋白质标记），以一次数百个的速率。这项工作将产生积极的影响 立即通过提供一种易于适应的方法，同时标记数百个 蛋白质在其天然环境中，并在长期内通过实现必要的第一步， 使蛋白质功能的平行询问成为可能。

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.