PC-1 CRISPR Activation Plasmid (h): sc-402336-ACT

ENPP1 encodes the ectoenzyme PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), a type II transmembrane glycoprotein that hydrolyzes extracellular nucleotides to generate AMP and inorganic pyrophosphate. By controlling pyrophosphate availability, PC-1 is a central regulator of extracellular matrix mineralization and calcification biology, influencing osteogenic signaling and tissue phosphate homeostasis. ENPP1 activity also interfaces with purinergic signaling through modulation of nucleotide and nucleoside pools, shaping local inflammatory and metabolic responses. Genetic or functional disruption of ENPP1 is associated with disorders of ectopic calcification and skeletal mineralization, and altered PC-1 expression has been studied in metabolic disease contexts, including insulin signaling dysregulation.

PC-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ENPP1 expression without altering the underlying DNA sequence.

PC-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ENPP1 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 ENPP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PC-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ENPP1 locus and enabling the study of PC-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PC-1 pathway restoration in tumor cells with silenced or reduced ENPP1 expression.

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