MCAT CRISPR Activation Plasmid (h): sc-404338-ACT

Human MCAT encodes mitochondrial malonyl-CoA:ACP transferase, an essential component of the mitochondrial fatty acid synthesis (mtFAS) pathway that transfers malonyl groups to acyl carrier protein to support de novo acyl chain elongation. This activity is required for production of octanoyl-ACP, a key precursor for protein lipoylation, thereby linking MCAT function to pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complex regulation and broader mitochondrial energy metabolism. Through its role in maintaining oxidative metabolism and mitochondrial homeostasis, altered MCAT expression or activity has been associated with mitochondrial dysfunction phenotypes and is studied in the context of metabolic stress, bioenergetic remodeling, and neuro-metabolic disease mechanisms.

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

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

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