Endogenous Promotor-Driven Split Nanoluciferase Biosensor for Assessing G Protein Recruitment

Abstract

HEK293 cells are a common immortal cell line used in biological research, and their popularity has led to different distinct lineages across the world. Commonly used for overexpression of proteins, HEK293 cells also natively express biological targets, such as G protein coupled receptors (GPCRs) and their downstream signalling partners, G proteins, although this often confounds rather than compliments research. CRISPR/Cas9 gene editing can be used to harness these native proteins and make use of their presence. Here, a cost- and time-effective, plasmid-based CRISPR/Cas9 approach is used to tag well-characterised GPCRS - the {beta}-adrenoceptors 1 and 2 - with one part of a split Nanoluciferase and replace the Gs coupling partner with the complimentarily tagged minimal Gs protein in HEK293T cells. Compared to untagged proteins, the CRISPR/Cas9 cells allow for better selective-ligand characterisation at the native {beta}-adrenoceptors. Overexpressed tagged systems produce similar results to the CRISPR/Cas9 cells, however subtle changes in the characterisation of partial agonists, such as salbutamol, demonstrate the potential for utilising tagged native receptors in analysing biological effectors. Summary StatementFor the first time, a split-luciferase tagged minimal Gs protein and {beta}1AR is inserted under endogenous promotors in HEK293T cells using CRISPR/Cas9 gene modification, avoiding protein overexpression in the assay.