GnT-I CRISPR Activation Plasmid (h): sc-409648-ACT

MGAT1 encodes N-acetylglucosaminyltransferase I (GnT-I), a medial-Golgi enzyme that catalyzes the first committed step in the conversion of high-mannose to complex and hybrid N-glycans. By initiating branching in N-linked glycosylation, GnT-I influences protein folding, stability, receptor trafficking, and cell–cell and cell–matrix interactions across secretory and membrane proteins. MGAT1-dependent glycan remodeling integrates with Golgi processing pathways and modulates signaling outputs from glycoproteins involved in adhesion and growth factor responsiveness. Dysregulated MGAT1 activity and altered N-glycan structures are frequently examined in the context of cancer biology, immune regulation, and congenital disorders of glycosylation as mechanistic contributors to aberrant cellular phenotypes.

GnT-I CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MGAT1 expression without altering the underlying DNA sequence.

GnT-I CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MGAT1 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 MGAT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GnT-I expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MGAT1 locus and enabling the study of GnT-I-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GnT-I pathway restoration in tumor cells with silenced or reduced MGAT1 expression.

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