citrate synthase CRISPR Activation Plasmid (h): sc-402227-ACT

Human CS encodes citrate synthase, the mitochondrial matrix enzyme that catalyzes condensation of oxaloacetate and acetyl‑CoA to form citrate, initiating the tricarboxylic acid (TCA) cycle. By controlling entry into oxidative metabolism, citrate synthase supports ATP production, anaplerosis, and provision of citrate for lipid and cholesterol biosynthesis via mitochondrial–cytosolic carbon flux. CS activity is closely linked to mitochondrial biogenesis, redox homeostasis, and metabolic adaptation under nutrient stress. Altered CS expression or mitochondrial TCA cycle function is frequently studied in contexts of metabolic dysfunction and bioenergetic remodeling associated with neurodegeneration, cardiometabolic phenotypes, and tumor metabolism.

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

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

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