The utility of CRISPR/Cas technology to uncover the signalling of orphan GPCRs

J. Iyinikkel, J. Hislop, D. Thompson, F. Murray. Metabolic and Cardiovascular Health, University of Aberdeen, Aberdeen, United Kingdom.

Introduction: G protein-coupled receptors (GPCRs) are successful therapeutic targets due to their accessibility, distribution and role in amplifying signalling (Insel et al., 2012). Roughly 800 GPCRs are expressed in humans, 120 of which are orphan GPCRs whose endogenous ligand is unknown. Recent strategies profiling the expression of GPCRs in cells have identified several orphan GPCRs that could serve as novel targets, however limited reagents are available to assess their physiological roles. Molecular approaches, such as overexpression in heterologous cells and RNA interference have been useful in determining signalling. Our lab focuses on GPR75, an orphan receptor also termed retinal-GPCR. We aimed to use CRISPR/Cas9, a precise gene editing tool, to characterise the signalling of GPR75.

Methods: GPR75 cDNA (Origene) was used to generate a stable GPR75-HEK293 cell line. Guide oligos (∼20 bp) targeting GPR75 were designed (Addgene- Zhang lab’s Target Finder) and cloned into pX330 vector (S. pyogenes Cas9, Addgene). sgRNA was in-vitro transcribed from the plasmid template (Yang et al., 2014) using the MEGAshortscript T7 kit (Ambion). sgRNA guides and Cas9 mRNA were electroporated into GPR75-HEK293 cells. GPR75 expression was confirmed using real-time PCR and western blots. Gprotein-coupling was investigated by measuring cAMP accumulation (ELISA, Enzo Life Science) in cells pre-incubated with IBMX (200 μM) then stimulated with forskolin (1 μM). Data expressed as mean ±SEM. Statistical comparisons were performed using Student's t-tests or one-way ANOVA, P<0.05 considered significant.

Results: GPR75 mRNA [35.1 ±0.3 fold, n=3, p<0.05] and protein [5.9 ±2.3 fold, n=3, p<0.05] expression increased in GPR75-HEK293 compared to control. Forskolin-induced cAMP accumulation was significantly reduced in GPR75-HEK293 compared to control [42.1 ±1.7 vs. 58.7 ±2.1 pmol/million cells respectively, n=3, p<0.05]. Pre-treatment with pertussis toxin (100 ng/ml) blocked the reduction in cAMP accumulation in GPR75-HEK293 [51.2 ±1.2 vs. 42.1 ±1.7 pmol/million cells respectively, n=3, p<0.05]. GPR75 gene expression was significantly reduced in the CRISPR-KO cells [Guide 2, -2.1 ±0.2 and Guide 7, -3.04 ±0.36 fold, n=3, p<0.001], which correlated with a decrease in protein expression. The reduction in forskolin-induced cAMP accumulation in GPR75-HEK293 cells was reversed with CRISPR-KO of GPR75.

Conclusion: We conclude GPR75, which is constitutively active, inhibits cAMP accumulation thereby indicating Gαi coupling. These data suggest CRISPR/Cas9 can help determine signalling of orphan GPCRs, which could also have utility in-vivo to define their physiological role.

References:

1. Insel P et al. (2012). Br J Pharmacol 149, 490-497.

2. Yang H et al. (2014). Nature Protocols 9, 1956-1968.