cystatin C CRISPR Activation Plasmid (h): sc-402420-ACT

CST3 encodes cystatin C, a secreted cysteine protease inhibitor that restrains cathepsins and other lysosomal/endosomal proteases to help maintain extracellular matrix turnover, antigen processing, and proteostasis. By modulating protease activity in the endolysosomal system and pericellular space, cystatin C influences inflammatory signaling, cell migration, and tissue remodeling. Altered CST3 expression or cystatin C abundance has been linked to neurodegenerative processes, cerebrovascular pathology, and renal and cardiovascular biology, making it a useful node for studying protease–inhibitor balance across tissues. In cell models, CST3 perturbation can be used to probe pathways connecting lysosomal function, oxidative stress responses, and innate immune activation.

cystatin C CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CST3 expression without altering the underlying DNA sequence.

cystatin C CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CST3 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 CST3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous cystatin C expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CST3 locus and enabling the study of cystatin C-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of cystatin C pathway restoration in tumor cells with silenced or reduced CST3 expression.

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