EHHADH CRISPR Activation Plasmid (h): sc-404068-ACT

EHHADH (enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase) encodes a bifunctional peroxisomal enzyme that catalyzes consecutive steps in peroxisomal fatty acid β-oxidation, supporting chain-shortening of very-long-chain and branched-chain fatty acyl-CoAs. Through its role in lipid catabolism and redox balance, EHHADH contributes to peroxisome–mitochondria metabolic crosstalk and regulation of cellular energy homeostasis. Altered EHHADH expression or activity has been associated with peroxisomal dysfunction phenotypes and metabolic disease-relevant processes, including lipid accumulation, oxidative stress, and liver metabolic adaptation. EHHADH is therefore a useful target for studying peroxisome-driven lipid metabolism, organelle homeostasis, and transcriptional programs linked to metabolic stress.

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

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

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