Fumarylacetoacetase CRISPR Activation Plasmid (h): sc-402478-ACT

FAH encodes human fumarylacetoacetase, a cytosolic enzyme that catalyzes the terminal step of tyrosine catabolism by hydrolyzing fumarylacetoacetate to fumarate and acetoacetate. This reaction links aromatic amino acid degradation to central carbon metabolism through production of TCA-cycle and ketone body precursors, and helps prevent accumulation of reactive intermediates that can modify cellular macromolecules. Disrupted FAH activity is associated with hereditary tyrosinemia type I and is widely used as a model for hepatocyte metabolic stress, DNA damage responses, and liver-selective selection systems in experimental settings. As such, FAH serves as a key node for studying metabolic pathway flux, detoxification capacity, and hepatocyte homeostasis.

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

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

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