ACOT8 CRISPR Activation Plasmid (h): sc-407262-ACT

Human ACOT8 encodes a peroxisomal acyl-coenzyme A thioesterase that hydrolyzes acyl‑CoA esters to free fatty acids and CoA, helping balance acyl‑CoA pools and maintain CoA availability. By regulating peroxisomal lipid handling, ACOT8 influences fatty acid β‑oxidation, lipid remodeling, and redox homeostasis linked to hydrogen peroxide–producing peroxisomal metabolism. ACOT8 activity intersects with broader metabolic programs coordinating peroxisome–mitochondria crosstalk and cellular responses to nutrient and oxidative stress. Altered peroxisomal lipid metabolism and CoA homeostasis are relevant to metabolic dysfunction and peroxisome-associated disease biology, supporting use of ACOT8 as a target in studies of lipid-driven cellular phenotypes.

ACOT8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ACOT8 expression without altering the underlying DNA sequence.

ACOT8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ACOT8 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 ACOT8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ACOT8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ACOT8 locus and enabling the study of ACOT8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ACOT8 pathway restoration in tumor cells with silenced or reduced ACOT8 expression.

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