NCOAT CRISPR Activation Plasmid (h): sc-402329-ACT

MGEA5 encodes NCOAT (also known as O-GlcNAcase, OGA), a bifunctional enzyme that removes O-GlcNAc modifications from serine/threonine residues on nuclear and cytoplasmic proteins, counterbalancing O-GlcNAc transferase to regulate dynamic O-GlcNAc cycling. By tuning O-GlcNAcylation, NCOAT influences transcriptional control, chromatin organization, proteostasis, and signal transduction in nutrient- and stress-responsive pathways. Altered O-GlcNAc homeostasis has been linked to dysregulated metabolic signaling, cell-cycle control, and neurodegenerative processes, making MGEA5 a relevant node for mechanistic studies of cellular adaptation. In human cells, perturbation of NCOAT activity can reshape protein modification networks that intersect with insulin signaling and stress-response programs.

NCOAT CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MGEA5 expression without altering the underlying DNA sequence.

NCOAT CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MGEA5 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 MGEA5 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NCOAT expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MGEA5 locus and enabling the study of NCOAT-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NCOAT pathway restoration in tumor cells with silenced or reduced MGEA5 expression.

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