HXK III CRISPR Activation Plasmid (h): sc-403846-ACT

Human HK3 encodes hexokinase III (HXK III), a glucose-phosphorylating enzyme that catalyzes the ATP-dependent conversion of glucose to glucose-6-phosphate, committing hexose substrates to intracellular metabolism. By generating glucose-6-phosphate, HXK III helps regulate glycolytic flux and supports branching into the pentose phosphate pathway for NADPH production and redox homeostasis. HK3 expression is frequently studied in immune and hematopoietic contexts where metabolic reprogramming shapes inflammatory signaling, differentiation, and stress responses. Dysregulated hexokinase activity and altered HK3 transcription have been linked to disease-relevant metabolic phenotypes, including aberrant glucose utilization and oxidative stress pathways.

HXK III CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous HK3 expression without altering the underlying DNA sequence.

HXK III CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HK3 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 HK3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HXK III expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HK3 locus and enabling the study of HXK III-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HXK III pathway restoration in tumor cells with silenced or reduced HK3 expression.

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