Smad6 CRISPR Activation Plasmid (h): sc-401411-ACT

Human SMAD6 encodes Smad6, an inhibitory SMAD that dampens signaling downstream of the TGF-β superfamily, particularly the BMP branch. Smad6 acts as a negative feedback regulator by interfering with receptor-regulated SMAD phosphorylation and by modulating SMAD complex formation and transcriptional output. Through these mechanisms it influences developmental patterning, osteogenic differentiation, endothelial and vascular homeostasis, and inflammatory signaling crosstalk. Altered SMAD6 activity or expression has been associated with congenital cardiovascular and craniofacial phenotypes and has been studied in contexts where BMP/TGF-β pathway imbalance contributes to disease-relevant remodeling and calcification processes.

Smad6 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SMAD6 expression without altering the underlying DNA sequence.

Smad6 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SMAD6 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 SMAD6 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Smad6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SMAD6 locus and enabling the study of Smad6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Smad6 pathway restoration in tumor cells with silenced or reduced SMAD6 expression.

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