PAR-4 CRISPR Activation Plasmid (h): sc-401719-ACT

F2RL3 encodes protease-activated receptor 4 (PAR-4), a G protein–coupled receptor activated by thrombin and other serine proteases through proteolytic unmasking of a tethered ligand. PAR-4 signaling engages Gq and G12/13 pathways to stimulate phospholipase C activity, intracellular Ca2+ mobilization, RhoA/ROCK signaling, and downstream MAPK and NF-κB transcriptional programs. In platelets and vascular-associated cells, these cascades regulate activation, secretion, cytoskeletal remodeling, and inflammatory crosstalk. Dysregulated PAR-4 activity and F2RL3 expression patterns have been associated with thrombosis-relevant biology, vascular inflammation, and cancer-related microenvironment signaling, supporting mechanistic studies across hemostasis and tumor–stroma interactions.

PAR-4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous F2RL3 expression without altering the underlying DNA sequence.

PAR-4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the F2RL3 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 F2RL3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PAR-4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native F2RL3 locus and enabling the study of PAR-4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PAR-4 pathway restoration in tumor cells with silenced or reduced F2RL3 expression.

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