Glucosidase IIα CRISPR Activation Plasmid (m): sc-420442-ACT

Ganab encodes the alpha subunit of glucosidase II, a key endoplasmic reticulum (ER) enzyme in N-linked glycan processing that sequentially trims glucose residues from nascent glycoproteins. This activity is integral to ER quality control, supporting the calnexin/calreticulin folding cycle, glycoprotein maturation, and ER-associated degradation (ERAD) of misfolded clients. By shaping proteostasis, GANAB influences secretory pathway output and stress-adaptive programs such as the unfolded protein response (UPR). Disruption or dysregulation of glycoprotein processing pathways linked to GANAB has been associated with protein misfolding phenotypes and disease-relevant biology in tissues with high secretory demand.

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

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

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