GCKR CRISPR Activation Plasmid (h): sc-401623-ACT

Human GCKR encodes glucokinase regulatory protein, a hepatocyte-enriched modulator of glucose utilization that binds and sequesters glucokinase to tune glycolytic flux in response to nutrient and phosphate status. By regulating glucokinase availability, GCKR influences hepatic glucose sensing, glycogen synthesis, and the balance between glycolysis and de novo lipogenesis, integrating carbohydrate metabolism with triglyceride production. Genetic variation or altered expression of GCKR has been associated with shifts in fasting glucose, insulin sensitivity, and circulating lipid traits, making it relevant to metabolic disease biology. In cell-based models, perturbing GCKR provides a tractable way to interrogate liver metabolic pathway wiring and nutrient-responsive transcriptional programs.

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

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

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