NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A_2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A_2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A_2B. However, in cells expressing gene-edited Nluc/A_2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (K_d = 21.4 nM). Additionally, at gene-edited Nluc/A_2B receptors we derived pharmacological parameters of ligand binding; K_d as well as K_on and K_off for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A_2B were used to determine the pK_i of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.