DLD CRISPR Activation Plasmid (h): sc-403207-ACT

Human DLD encodes dihydrolipoamide dehydrogenase, a FAD-dependent oxidoreductase that functions as the E3 subunit shared by the pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain α-ketoacid dehydrogenase complexes in mitochondria. By reoxidizing dihydrolipoamide and transferring electrons to NAD+, DLD helps sustain acetyl-CoA production, TCA cycle flux, and mitochondrial redox balance. This activity links carbohydrate and amino acid catabolism to oxidative phosphorylation and reactive oxygen species control. Altered DLD function is associated with mitochondrial metabolic dysfunction and neurometabolic disease phenotypes, making it a relevant node for studies of energy homeostasis and redox-dependent signaling.

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

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

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