GPR54 CRISPR Activation Plasmid (h): sc-403766-ACT

KISS1R (GPR54) is a class A GPCR that binds kisspeptins encoded by KISS1 to regulate neuroendocrine control of puberty onset and reproductive axis function. Upon ligand engagement, GPR54 primarily couples to Gαq/11 to stimulate phospholipase C signaling, IP3-dependent calcium mobilization, and downstream MAPK/ERK and PKC pathways that influence gene expression and cellular excitability. This signaling node is central to hypothalamic GnRH neuron activation and integrates developmental, metabolic, and environmental inputs that tune gonadotropin release. Altered KISS1R activity or expression has been linked to disorders of pubertal timing and hypogonadotropic hypogonadism, and GPR54 pathway perturbations are also studied in contexts of hormone-dependent tumor biology and cell migration programs.

GPR54 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KISS1R expression without altering the underlying DNA sequence.

GPR54 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KISS1R locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the KISS1R transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GPR54 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KISS1R locus and enabling the study of GPR54-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GPR54 pathway restoration in tumor cells with silenced or reduced KISS1R expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.