HADHA CRISPR Activation Plasmid (h): sc-402803-ACT

HADHA encodes the alpha subunit of the mitochondrial trifunctional protein, a multi-enzyme complex required for long-chain fatty acid β-oxidation. HADHA contributes enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities that support mitochondrial energy production and lipid catabolism, linking fatty acid flux to redox balance and metabolic homeostasis. Disrupted HADHA function is associated with mitochondrial fatty acid oxidation defects and lipid-associated cellular stress, making it relevant to studies of metabolic remodeling and mitochondrial dysfunction. Because fatty acid oxidation intersects with oxidative stress responses and organellar quality control, HADHA is frequently examined in pathways governing mitochondrial fitness and lipid-driven signaling.

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

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

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