Aminopeptidase A CRISPR Activation Plasmid (h): sc-403989-ACT

ENPEP encodes aminopeptidase A (APA), a zinc-dependent membrane aminopeptidase that preferentially removes N-terminal acidic residues from bioactive peptides. In the renin–angiotensin system, APA contributes to angiotensin II processing and influences downstream signaling linked to vascular tone and neuroendocrine regulation, with additional roles in peptide metabolism at the cell surface. ENPEP expression is prominent in kidney and other epithelial compartments, where it intersects with proteolytic processing, membrane trafficking, and local peptide hormone homeostasis. Dysregulated APA activity or ENPEP expression has been associated with altered blood pressure regulation and renal pathophysiology, motivating mechanistic studies in cardiometabolic and kidney-relevant cell models.

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

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

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