Protein tagging at scale to enable functional genomic studies

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.