Construction Of Gqi9-Knockin HEK293 Cell Line By CRISPR/Cas9 System

G protein-coupled receptors?GPCRs?are the largest class of receptors in the human genome,there are more than 800 human genes encoding GPCRs.GPCR-mediated signaling pathways are involved in the regulation of many physiological processes in humans.Dysfunction of GPCR can lead to a variety of diseases.Therefore,they are very important drug targets.Upon stimulation,G protein-coupled receptors?GPCRs?can initiate canonical G protein-dependent signaling cascades by coupling to an array of various heterotrimeric G proteins.Activation of G proteins will promote the formation of second-messenger effectors and trigger a series of complex biochemical reactions within the cell.G protein can be grouped into four families,Gs,Gi/Go,Gq/G11,and G12/G13.When Gs and Gq/11subtypes are activated,they cause an increase in intracellular second messenger cAMP/Ca²⁺concentrations.In contrast,activation of Gi protein will inhibit adenylate cyclase activity,resulting in decreases in the intracellular cAMP concentration.This signal can not be directly detected,making the relatively study of Gi protein-coupled receptors cumbersome.Gqi9 is a chimeric G-protein in which the extreme 9 C-terminal residues of G?q were replaced by the corresponding residues of alpha i.Gqi9 can mediate stimulation of phospholipase C by receptors otherwise coupled exclusively to Gi.The emergence of Gqi9 provides an effective tool for the study of Gi protein-coupled receptors.Currently,Gqi9 is always co-transfected with receptors,but this method has the disadvantages of unstable expression and uneven expression.The aim of this study is to generate Gqi9 gene knockin HEK293 cell line by CRISPR/Cas9 system to achieve stable endogenous expression of Gqi9 protein.We designed to knock Gqi9 gene into the AAVS1 locus,which was one of the most widely used genomic safe harbors GSH?GSHs?loci in the human genome.This locus is reported to ensure normal transcription of the foreign gene,and the insertion of the foreign gene at this site has no known side effects on the cell.Finally,the Gqi9 gene insertion in HEK293 cells were successfully obtained.Gqi9 gene expression was detected by PCR and qPCR.And Gqi9 protein function was detected by measurement of intracellular Ca²⁺mobilization upon Gi-coupled receptors activation using Fluo-4 AM and Flexstation3.The results showed that the cell line can express Gqi9gene stablely.An increase in intracellular calcium signal can be detected after exogenous expression of a Gi protein-binding receptor in the cell line.In summary,this study provides an effective tool for the study of Gi protein-coupled receptors,overcoming the shortcomings of previous research methods.This cell line can be used for the structural and functional studies of Gi protein-coupled receptors,and also provides great convenience for drug screening and the study of various mutants.This study also provides a reference for the construction of other useful cell lines.