PCCA CRISPR Activation Plasmid (h): sc-404791-ACT

PCCA encodes the alpha subunit of propionyl‑CoA carboxylase, a biotin-dependent mitochondrial enzyme that catalyzes the ATP-dependent conversion of propionyl‑CoA to D‑methylmalonyl‑CoA. This reaction is essential for catabolism of branched-chain amino acids, odd-chain fatty acids, and cholesterol side chains, linking amino acid and lipid utilization to mitochondrial anaplerosis and energy metabolism. PCCA function intersects with biotin metabolism, mitochondrial protein homeostasis, and metabolic stress responses that influence redox balance and organic acid handling. Pathogenic disruption of PCCA activity is associated with propionic acidemia, making it a useful target for studying metabolic dysfunction, mitochondrial stress signaling, and genotype–phenotype relationships in human cell models.

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

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

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