cystatin A CRISPR Activation Plasmid (h): sc-416374-ACT

Human CSTA encodes cystatin A, an intracellular type I cystatin that inhibits papain-like cysteine proteases such as cathepsins B, H, and L, thereby modulating proteolysis, antigen processing, and barrier-associated cellular homeostasis. By restraining lysosomal and cytosolic protease activity, cystatin A contributes to regulation of keratinocyte differentiation, epithelial integrity, and stress responses linked to protease–antiprotease balance. Dysregulated CSTA expression has been associated with inflammatory skin conditions and epithelial remodeling, and altered cystatin–cathepsin dynamics is frequently explored in studies of tumor invasion, immune signaling, and extracellular matrix turnover. As a result, CSTA serves as a useful node for dissecting protease-controlled pathways that influence cell adhesion, migration, and programmed cell death.

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

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

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