A High‐throughput CRISPR Interference Screen to Dissect Functional Regulators of GPCR/cAMP Signaling

Abstract

The beta‐adrenergic (β2‐AR) receptor is a prototypical GPCR that controls important functions in the nervous, pulmonary, and cardiovascular systems. β2‐AR signaling involves multiple factors in both G‐protein dependent and independent signaling, resulting in diverse signaling outcomes. To understand the complexity of this pathway, we have performed the first unbiased, high‐throughput functional genomics screen of β2‐AR signaling using cAMP reporter and CRISPR interference (CRISPRi) silencing. We generated a reporter cell line using a construct encoding cAMP‐response element (CREB) fused to a ProteoTuner destabilizing domain and green fluorescent protein and another construct encoding catalytically‐dead Cas9 (dCas9) fused to the transcriptional repressor KRAB. Reporter cells were transduced with genome‐wide lentiviral sgRNA library to silence individual genes. After induction of β2‐AR signaling, we sorted the edited cells based on reporter expression and subjected them to deep sequencing and statistical analysis of sgRNA enrichment. We obtained 32 positive and 14 negative β2‐AR/cAMP signaling modulators enriched in factors involved in transcription initiation, RNA splicing, and RNA metabolic processes. We validated TP53 and NUP93 as novel positive modulators of β2‐AR signaling using dual luciferase cAMP assay and RT‐qPCR of cAMP‐dependent target gene, consistent with our cAMP reporter result. We found that the TP53‐dependent modulation of this pathway can partially be explained by lower transcription and surface expression of the β2‐AR receptors. These findings demonstrate a successful application of our reporter in a high‐throughput approach to identify previously uncharacterized modulators of the β2‐AR/cAMP pathway. This study reveals broader potential applications of our cAMP reporter as a platform to understand other aspects of GPCR signal transduction such as biased signaling, receptor‐specific molecular mechanisms, and drug discovery. Support or Funding Information This work is supported by the National Institute of Mental Health R00 award (MH109633) awarded to Nikoleta Tsvetanova.