Cdc25A CRISPR Activation Plasmid (h): sc-400345-ACT

CDC25A encodes Cdc25A, a dual-specificity phosphatase that activates cyclin-dependent kinases by removing inhibitory phosphates, thereby promoting G1/S progression and coordinating DNA replication with checkpoint control. Cdc25A integrates signaling from DNA damage response pathways, including ATM/ATR–CHK1/CHK2, which regulate its stability and activity to restrain cell cycle entry under genotoxic stress. Dysregulated CDC25A expression or turnover can perturb checkpoint fidelity, increase replicative stress, and contribute to genomic instability observed in proliferative disorders. As a result, CDC25A is widely studied in cell cycle regulation, replication timing, and stress-response network remodeling in human cells.

Cdc25A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CDC25A expression without altering the underlying DNA sequence.

Cdc25A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CDC25A 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 CDC25A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdc25A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CDC25A locus and enabling the study of Cdc25A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdc25A pathway restoration in tumor cells with silenced or reduced CDC25A expression.

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