A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.

Area of Science:

• Cellular and Molecular Biology
• Biotechnology
• Genomics

Background:

• Understanding protein function in native cellular environments is crucial for cellular physiology.
• Existing methods like overexpression may not accurately reflect endogenous protein behavior.
• Developing scalable strategies for analyzing protein dynamics in situ is needed.

Purpose of the Study:

• To assess the scalability of CRISPR-mediated HiBiT tagging for endogenous proteins.
• To determine if HiBiT-tagged proteins accurately represent target biology in cellular models.
• To evaluate the utility of endogenous tagging for studying protein dynamics.

Main Methods:

• CRISPR-Cas9 gene editing to integrate the HiBiT luminescent peptide tag into endogenous gene loci.
• Large-scale screening of diverse proteins across multiple cell lines.
• Validation using luminescence-based assays, blotting, and imaging.
• Comparative functional assays between endogenously tagged and plasmid-expressed proteins.

Main Results:

• Successful HiBiT tag integration was achieved in 86% of targeted endogenous proteins.
• Endogenously tagged cell lines accurately reproduced known biological functions, unlike plasmid-based expression.
• HiBiT fusion proteins expressed from endogenous loci provided more representative biological insights.

Conclusions:

• CRISPR-mediated HiBiT tagging is a scalable and efficient strategy for generating reporter cell lines.
• Endogenous HiBiT tagging provides more faithful representation of protein biology compared to overexpression.
• This method enables rapid generation of cellular models for studying protein dynamics and function.