P504S CRISPR Activation Plasmid (h): sc-403603-ACT

Human AMACR encodes alpha-methylacyl-CoA racemase (P504S), a peroxisomal and mitochondrial enzyme that interconverts (R)- and (S)-2-methyl-branched acyl-CoA thioesters to enable subsequent β-oxidation. This activity supports catabolism of branched-chain fatty acids and bile acid intermediates, linking AMACR to lipid metabolic homeostasis and peroxisome–mitochondria crosstalk. Dysregulated AMACR expression and altered fatty-acid oxidation programs have been associated with tumor-associated metabolic remodeling, making AMACR a widely used molecular marker in cancer biology. AMACR is therefore relevant for studying metabolic pathway dependencies, redox balance, and lipid-derived signaling in human cells.

P504S CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AMACR expression without altering the underlying DNA sequence.

P504S CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AMACR 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 AMACR transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous P504S expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AMACR locus and enabling the study of P504S-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of P504S pathway restoration in tumor cells with silenced or reduced AMACR expression.

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