GS1 CRISPR Activation Plasmid (h): sc-405996-ACT

Human PUDP (pseudouridine 5′-phosphatase), also referred to as GS1, is a cytosolic enzyme in the pyrimidine salvage network that dephosphorylates pseudouridine 5′-monophosphate to pseudouridine, supporting turnover and recycling of modified nucleosides derived from RNA catabolism. By regulating levels of pseudouridine nucleotides, PUDP influences nucleotide homeostasis and intersects with pathways governing RNA modification dynamics, ribonucleotide metabolism, and cellular responses to metabolic stress. Altered handling of modified nucleosides is increasingly linked to dysregulated RNA turnover and metabolic reprogramming observed across diverse disease contexts, making PUDP a useful node for mechanistic studies. Modulation of PUDP/GS1 expression enables interrogation of how pseudouridine metabolism impacts gene expression programs and cellular physiology.

GS1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PUDP expression without altering the underlying DNA sequence.

GS1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PUDP 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 PUDP transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GS1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PUDP locus and enabling the study of GS1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GS1 pathway restoration in tumor cells with silenced or reduced PUDP expression.

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