HADHB CRISPR Activation Plasmid (h): sc-403234-ACT

HADHB encodes the mitochondrial trifunctional protein beta subunit, a core component of long-chain fatty acid β-oxidation that catalyzes the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase steps in concert with HADHA. This enzyme complex supports energy homeostasis by channeling long-chain acyl-CoAs into acetyl-CoA production, coupling lipid catabolism to mitochondrial respiration and metabolic flexibility. Disruption of HADHB function is linked to mitochondrial fatty acid oxidation disorders and metabolic stress phenotypes, including impaired energy production during fasting or high-demand conditions. In experimental systems, HADHB expression and activity are commonly studied in the context of mitochondrial metabolism, redox balance, and lipid-driven signaling responses.

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

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

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