Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein.

Automated summary

E-selectin is a protein expressed on endothelial cells in response to inflammatory cytokines, and it plays a role in leukocyte rolling and extravasation. This study used CRISPR-Cas9 genome editing and NanoLuc Binary Technology (NanoBiT) to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) and monitor its expression in real time. The combination of NanoBiT and CRISPR-Cas9 gene editing provides a powerful tool for monitoring dynamic changes in E-selectin expression on cell surfaces, which can contribute to the discovery of drugs targeting this important inflammatory protein.