ITM1 CRISPR Activation Plasmid (h2): sc-405155-ACT-2

Human STT3A encodes the catalytic subunit of the oligosaccharyltransferase (OST) complex that mediates co-translational N-linked glycosylation of nascent polypeptides in the endoplasmic reticulum, shaping proteostasis, membrane/secreted protein maturation, and ER quality control. Through its role in glycoprotein biogenesis, STT3A influences pathways including ER-associated degradation (ERAD), the unfolded protein response (UPR), and trafficking of receptors and transporters that depend on proper N-glycan installation. Dysregulation of STT3A-dependent glycosylation has been linked to congenital disorders of glycosylation and to stress-adaptation phenotypes observed in proliferative diseases, where altered glycan processing can affect signaling and cell–cell interactions. Gene editing or perturbation of STT3A in human cell models is useful for dissecting OST complex function, mapping glycosylation sites and client dependencies, and interrogating how ER stress and glycoprotein maturation impact cellular signaling networks.

ITM1 CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous STT3A expression without altering the underlying DNA sequence.

ITM1 CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the STT3A 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 STT3A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ITM1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native STT3A locus and enabling the study of ITM1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ITM1 pathway restoration in tumor cells with silenced or reduced STT3A expression.

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