NAGAT CRISPR Activation Plasmid (h): sc-401118-ACT

ABO encodes an A/B glycosyltransferase activity commonly referred to as N-acetylgalactosaminyltransferase (NAGAT), a Golgi-localized enzyme that catalyzes terminal carbohydrate modifications on the H antigen to generate A and B blood group determinants. Through regulation of glycoconjugate biosynthesis, ABO influences cell-surface glycosylation patterns that affect membrane protein trafficking, cell–cell recognition, and interactions with lectins and immune mediators. Variation in ABO-dependent glycan structures has been linked to altered hemostatic and inflammatory phenotypes via effects on glycoproteins such as von Willebrand factor and other plasma proteins. These properties make ABO/NAGAT a useful model for studying glycosylation-dependent mechanisms relevant to vascular biology, host–pathogen interactions, and immune regulation in human cells.

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

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

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