Calnexin CRISPR Activation Plasmid (h): sc-400154-ACT

Human CANX encodes calnexin, an endoplasmic reticulum (ER) membrane lectin chaperone that recognizes monoglucosylated N-linked glycans and promotes productive folding and quality control of nascent glycoproteins. Calnexin functions in the calnexin/calreticulin cycle with ERp57 and glycoprotein processing enzymes to coordinate disulfide bond formation, ER retention, and ER-associated degradation (ERAD) of misfolded substrates. Through its central role in proteostasis, CANX links to unfolded protein response signaling, calcium homeostasis at the ER, and regulation of secretory pathway flux. Altered calnexin-dependent folding capacity has been associated with cellular stress phenotypes and disease-relevant mechanisms involving protein misfolding and aberrant trafficking.

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

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

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