PRPK CRISPR Activation Plasmid (h2): sc-405589-ACT-2

Human TP53RK encodes PRPK (p53-related protein kinase), a catalytic component of the KEOPS complex that supports essential tRNA N6-threonylcarbamoyladenosine (t6A) modification, thereby influencing translational fidelity and global proteostasis. PRPK has been linked to regulation of cell growth and stress-adaptive programs, with reported connections to p53-associated signaling contexts and broader control of proliferation-related processes. Genetic disruption of TP53RK/PRPK is associated with developmental and neurodevelopmental phenotypes, including Galloway–Mowat syndrome, highlighting the importance of KEOPS-mediated RNA metabolism in organismal homeostasis. Gene editing of TP53RK enables mechanistic studies of tRNA modification-dependent translation, KEOPS complex assembly, and downstream effects on cell cycle, genome maintenance, and stress response pathways in human cellular models.

PRPK CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous TP53RK expression without altering the underlying DNA sequence.

PRPK CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TP53RK 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 TP53RK transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PRPK expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TP53RK locus and enabling the study of PRPK-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PRPK pathway restoration in tumor cells with silenced or reduced TP53RK expression.

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