EXT1 CRISPR Activation Plasmid (h): sc-404635-ACT

Human EXT1 encodes exostosin-1, a Golgi-resident glycosyltransferase that forms a functional complex with EXT2 to catalyze heparan sulfate chain elongation on proteoglycans. By controlling heparan sulfate biosynthesis, EXT1 shapes extracellular matrix organization and modulates ligand availability for key signaling pathways including FGF, WNT, Hedgehog, and BMP, influencing cell adhesion, migration, and developmental patterning. Disruption or dysregulation of EXT1 alters proteoglycan composition and signaling gradients, and is strongly linked to skeletal dysplasias such as multiple osteochondromas and broader defects in tissue morphogenesis. EXT1 activity is also relevant to studies of tumor microenvironment remodeling and receptor–ligand interactions that depend on heparan sulfate presentation.

EXT1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EXT1 expression without altering the underlying DNA sequence.

EXT1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EXT1 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 EXT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EXT1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EXT1 locus and enabling the study of EXT1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EXT1 pathway restoration in tumor cells with silenced or reduced EXT1 expression.

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