BCKDE1A CRISPR Activation Plasmid (h): sc-417950-ACT

BCKDHA encodes the E1α subunit of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, a mitochondrial multienzyme system that catalyzes the oxidative decarboxylation of branched-chain α-ketoacids derived from leucine, isoleucine, and valine. This reaction is central to branched-chain amino acid catabolism and links nutrient status to acetyl-CoA/succinyl-CoA production, mitochondrial redox balance, and cellular energy metabolism. BCKDH activity is tightly regulated by phosphorylation and dephosphorylation, integrating signals from BCKDK and PPM1K to modulate metabolic flux. Dysregulation of BCKDHA and impaired BCKDH function are associated with inborn errors of metabolism and are widely studied in the context of mitochondrial dysfunction and altered amino acid homeostasis.

BCKDE1A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BCKDHA expression without altering the underlying DNA sequence.

BCKDE1A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BCKDHA 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 BCKDHA transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous BCKDE1A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BCKDHA locus and enabling the study of BCKDE1A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of BCKDE1A pathway restoration in tumor cells with silenced or reduced BCKDHA expression.

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