PNPase CRISPR Activation Plasmid (h): sc-404406-ACT

PNPT1 encodes polyribonucleotide nucleotidyltransferase 1 (PNPase), a conserved 3′–5′ exoribonuclease and poly(A) polymerase–like enzyme that localizes predominantly to mitochondria and regulates RNA turnover and surveillance. PNPase participates in mitochondrial RNA processing, maintenance of mitochondrial gene expression, and control of cytosolic and mitochondrial RNA homeostasis, impacting oxidative phosphorylation and cellular energy metabolism. Disruption of PNPT1 function has been linked to mitochondrial dysfunction phenotypes and is studied in the context of neurodevelopmental and neuromuscular disorders as well as broader pathways involving mitochondrial stress responses and innate immune activation triggered by aberrant RNA species.

PNPase CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PNPT1 expression without altering the underlying DNA sequence.

PNPase CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PNPT1 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 PNPT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PNPase expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PNPT1 locus and enabling the study of PNPase-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PNPase pathway restoration in tumor cells with silenced or reduced PNPT1 expression.

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