DHODH CRISPR Activation Plasmid (h): sc-402983-ACT

Human DHODH encodes dihydroorotate dehydrogenase, a flavin-dependent mitochondrial inner membrane enzyme that catalyzes the fourth step of de novo pyrimidine biosynthesis by oxidizing dihydroorotate to orotate while coupling electron transfer to the respiratory chain via ubiquinone. By controlling intracellular pyrimidine nucleotide availability, DHODH supports DNA/RNA synthesis, cell-cycle progression, and metabolic adaptation during proliferative and stress conditions. DHODH activity interfaces with mitochondrial redox balance and nucleotide homeostasis pathways that influence replication stress responses. Dysregulated pyrimidine metabolism and DHODH-dependent bioenergetic coupling have been implicated in proliferative disease biology and immune cell activation, making DHODH a common mechanistic node in studies of metabolic vulnerability.

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

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

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