GlcNAc kinase CRISPR Activation Plasmid (h): sc-407809-ACT

NAGK encodes human GlcNAc kinase, a cytosolic enzyme that phosphorylates N-acetylglucosamine to GlcNAc-6-phosphate, linking GlcNAc salvage to hexosamine metabolism. This activity contributes to the generation of UDP-GlcNAc, a central donor substrate for N- and O-linked glycosylation and other glycan-dependent processes that regulate protein folding, trafficking, and signal transduction. By influencing glycosylation capacity, NAGK can impact cellular responses to nutrient availability and stress, with downstream effects on cell growth and differentiation programs. Dysregulated hexosamine flux and glycosylation are recurrent features in metabolic and proliferative disease contexts, making NAGK a useful node for mechanistic pathway interrogation.

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

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

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