UPase CRISPR Activation Plasmid (h): sc-411050-ACT

Human UPP1 encodes uridine phosphorylase (UPase), a key enzyme in pyrimidine nucleoside metabolism that catalyzes the reversible phosphorolysis of uridine and deoxyuridine to generate uracil and ribose-1-phosphate or deoxyribose-1-phosphate. By regulating nucleoside salvage and the balance of intracellular pyrimidine pools, UPase influences nucleotide homeostasis, RNA/DNA precursor availability, and metabolic adaptation under nutrient stress. UPP1 activity intersects with pathways supporting mitochondrial function and redox balance through ribose-phosphate flux into central carbon metabolism. Altered UPP1 expression has been associated with tumor metabolism, proliferation phenotypes, and sensitivity to fluoropyrimidine metabolism in experimental models, making it a useful node for studying nucleotide turnover in disease-relevant contexts.

UPase CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous UPP1 expression without altering the underlying DNA sequence.

UPase CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the UPP1 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 UPP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous UPase expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native UPP1 locus and enabling the study of UPase-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of UPase pathway restoration in tumor cells with silenced or reduced UPP1 expression.

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