Abstract
The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.
Highlights
Of the ~20,000 protein coding genes within the human genome, fewer than 10% are targets of research and drug discovery programs[1]
The results will demonstrate that CRISPR-mediated HiBiT tagging of endogenous proteins is a broadly applicable strategy that enables the study of numerous proteins in the human proteome, and they will show that cell lines edited to express reporter fusions more accurately capture biology than overexpression techniques
For CRISPR-mediated HiBiT tagging to be considered scalable and broadly applicable for studying endogenous proteins, it is essential that successful tagging and detection be demonstrated across a panel of targets that captures the diversity of the proteome
Summary
Of the ~20,000 protein coding genes within the human genome, fewer than 10% are targets of research and drug discovery programs[1]. Mass spectrometry tends to under-represent low abundance proteins, while antibody-based techniques are restricted by the availability of high quality, specific antibodies[2,3] Of significance, both require cell lysis which prevents real time analysis and disrupts the spatiotemporal dynamics that underlie basic physiology. To circumvent the constraints of mass spectrometry and immunoanalysis, target proteins are often overexpressed as fusions to a reporter This permits functional and quantitative analysis without the need for specific reagents, complex workflows, or cell lysis. The results will demonstrate that CRISPR-mediated HiBiT tagging of endogenous proteins is a broadly applicable strategy that enables the study of numerous proteins in the human proteome, and they will show that cell lines edited to express reporter fusions more accurately capture biology than overexpression techniques
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