Calpastatin CRISPR Activation Plasmid (m): sc-419474-ACT

Mouse Cast encodes calpastatin, the endogenous inhibitor of calcium-dependent calpain proteases that constrains limited proteolysis of cytoskeletal and signaling substrates. By tuning calpain activity, calpastatin helps regulate cytoskeletal remodeling, focal adhesion turnover, membrane trafficking, and apoptosis, with downstream effects on inflammatory signaling and stress responses. CAST–calpain balance is broadly relevant to tissue integrity in muscle and nervous system, and its dysregulation has been linked in the literature to mechanisms underlying myopathy, neurodegeneration, and altered immune cell activation. In cell and animal models, modulating Cast expression is used to dissect protease-driven remodeling programs and calcium-dependent signaling networks.

Calpastatin CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Cast expression without altering the underlying DNA sequence.

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

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