brachyury CRISPR Activation Plasmid (h): sc-416539-ACT

Human T encodes brachyury, a T-box transcription factor that is essential for mesoderm specification, notochord formation, and posterior axis development. Brachyury regulates lineage programs by binding T-box motifs and coordinating transcriptional networks that interface with EMT-associated processes, cell migration, and developmental patterning pathways. In adult contexts, dysregulated T expression has been linked to altered differentiation states and transcriptional reprogramming observed in multiple tumor types and chordoma, where brachyury is frequently studied as a lineage-associated marker and driver. As a developmental regulator, brachyury provides a tractable node for interrogating gene regulatory circuits controlling epithelial–mesenchymal transitions and mesodermal fate decisions in human cell models.

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

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

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