Wip1 CRISPR Activation Plasmid (h): sc-400980-ACT

PPM1D encodes Wip1 (PPM1D), a Mg2+/Mn2+-dependent serine/threonine phosphatase induced by p53 that functions as a key negative feedback regulator of the DNA damage response. Wip1 dephosphorylates multiple checkpoint and stress signaling proteins, including p53, ATM/ATR pathway components, and p38 MAPK signaling nodes, thereby shaping cell-cycle checkpoint recovery, genome stability, and apoptosis thresholds. Through modulation of these pathways, Wip1 influences responses to genotoxic stress and replication stress, with altered PPM1D activity and copy number reported across diverse tumor contexts. Experimental perturbation of Wip1 is widely used to dissect checkpoint dynamics, stress-adaptive signaling, and pathway crosstalk that control proliferation and senescence.

Wip1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PPM1D expression without altering the underlying DNA sequence.

Wip1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PPM1D 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 PPM1D transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Wip1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PPM1D locus and enabling the study of Wip1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Wip1 pathway restoration in tumor cells with silenced or reduced PPM1D expression.

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