Ski CRISPR Activation Plasmid (h): sc-401509-ACT

Human SKI encodes the Ski proto-oncogene, a nuclear transcriptional coregulator that modulates gene expression programs controlling cell proliferation, differentiation, and lineage commitment. Ski is best known as a negative regulator of TGF-β/SMAD signaling, where it interacts with SMAD2/3/4 complexes and corepressors to attenuate transcription of TGF-β-responsive genes, thereby influencing epithelial–mesenchymal transition and extracellular matrix remodeling. Through crosstalk with pathways such as MAPK and retinoic acid–dependent transcription, SKI contributes to developmental patterning and tissue homeostasis. Dysregulated SKI activity has been linked to altered growth control and differentiation states in cancer biology, while germline perturbations in SKI are associated with connective tissue and craniofacial phenotypes relevant to developmental disease research.

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

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

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