Waf1/Cip1/CDKN1A p21 CRISPR Activation Plasmid (m): sc-419607-ACT

Cdkn1a encodes the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/CDKN1A), a central effector of p53-dependent cell-cycle control that restrains CDK2/CDK4 activity and enforces G1/S checkpoint arrest. By modulating cyclin–CDK complexes and proliferating cell nuclear antigen (PCNA)-associated DNA replication and repair, p21 integrates DNA damage signaling with senescence, differentiation, and stress responses. Altered Cdkn1a regulation is frequently linked to dysregulated proliferation, impaired genome maintenance, and context-dependent changes in apoptosis and senescence programs in models of tumorigenesis and tissue injury. As a node in p53, MAPK, and TGF-β-responsive networks, p21 is widely used to interrogate checkpoint integrity, replicative stress, and cellular aging phenotypes in mouse systems.

Waf1/Cip1/CDKN1A p21 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Cdkn1a expression without altering the underlying DNA sequence.

Waf1/Cip1/CDKN1A p21 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Cdkn1a 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 Cdkn1a transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Waf1/Cip1/CDKN1A p21 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Cdkn1a locus and enabling the study of Waf1/Cip1/CDKN1A p21-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Waf1/Cip1/CDKN1A p21 pathway restoration in tumor cells with silenced or reduced Cdkn1a expression.

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