Calpain 8 CRISPR Activation Plasmid (h): sc-402847-ACT

Human CAPN8 encodes calpain 8, a calcium-dependent cysteine protease of the calpain family that contributes to regulated proteolysis and protein turnover in a context-dependent manner. By modulating cleavage of selected substrates, calpain 8 can influence cytoskeletal remodeling, membrane trafficking, and signal transduction events that shape cellular stress responses and differentiation programs. Calpain-mediated processing intersects with calcium signaling networks and proteostasis pathways that coordinate adaptation to environmental cues. Dysregulated calpain activity has been implicated in inflammatory and epithelial pathobiology, making CAPN8 a useful target for mechanistic studies of protease-dependent regulation in human cells.

Calpain 8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CAPN8 expression without altering the underlying DNA sequence.

Calpain 8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CAPN8 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 CAPN8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Calpain 8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CAPN8 locus and enabling the study of Calpain 8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Calpain 8 pathway restoration in tumor cells with silenced or reduced CAPN8 expression.

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