PNK CRISPR Activation Plasmid (h): sc-404266-ACT

Human PNKP encodes polynucleotide kinase 3′-phosphatase (PNK), a bifunctional DNA end-processing enzyme that restores 5′-phosphate and 3′-hydroxyl termini required for ligation during DNA single-strand break repair. PNK functions downstream of PARP1 and XRCC1 and interfaces with base excision repair and non-homologous end joining by preparing damaged DNA ends generated by reactive oxygen species, ionizing radiation, and abortive topoisomerase reactions. Through these pathways, PNKP helps preserve genome stability and supports normal replication and transcription in proliferating and post-mitotic cells. Disruption of PNKP-mediated end processing is linked to heightened sensitivity to genotoxic stress and has been associated with neurodevelopmental and neurodegenerative phenotypes arising from defective DNA repair capacity.

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

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

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