HFE CRISPR Activation Plasmid (h): sc-403695-ACT

HFE encodes a major histocompatibility complex class I–like protein that associates with β2-microglobulin and modulates transferrin receptor (TFRC) interactions to regulate cellular iron uptake and systemic iron homeostasis. Through its influence on hepcidin (HAMP) signaling and iron-sensing in hepatocytes and macrophages, HFE impacts ferritin storage, transferrin saturation, and oxidative stress responses. Dysregulated HFE function is linked to iron overload phenotypes and altered inflammatory and metabolic signaling in tissues sensitive to redox imbalance. In research settings, HFE provides a tractable node for interrogating iron-dependent pathways affecting mitochondrial function, lipid metabolism, and immune cell behavior.

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

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

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