fumarate hydratase CRISPR Activation Plasmid (h): sc-401660-ACT

Human FH encodes fumarate hydratase (fumarase), a homotetrameric enzyme of the tricarboxylic acid (TCA) cycle that catalyzes the reversible hydration of fumarate to L-malate, linking mitochondrial energy metabolism to anaplerosis and redox homeostasis. Beyond central carbon flux, fumarate levels influence cellular signaling through protein succination and modulation of epigenetic and hypoxia-associated programs, connecting FH activity to mitochondrial stress responses and metabolic rewiring. Perturbation of FH function has been associated with oncogenic metabolism and altered reactive oxygen species handling, making FH a key node for studying mitochondrial dysfunction, metabolic checkpoints, and genome-to-metabolite relationships in human cells.

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

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

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