PFKL CRISPR Activation Plasmid (h): sc-402662-ACT

PFKL encodes the liver isoform of phosphofructokinase-1, a rate-limiting enzyme that catalyzes conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, committing glucose to glycolysis. As a key metabolic control point, PFKL integrates allosteric regulation by ATP, AMP, citrate, and fructose-2,6-bisphosphate to tune glycolytic flux and downstream carbon flow into the TCA cycle and biosynthetic pathways. Altered PFKL expression or activity contributes to metabolic reprogramming, influencing cellular proliferation, redox balance, and nutrient-sensing responses. Dysregulated glycolysis involving PFKL has been linked to tumor metabolism and other disorders characterized by perturbed energy homeostasis, supporting its use in mechanistic studies of metabolism-driven phenotypes.

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

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

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