GLI-1/GLI1 CRISPR Activation Plasmid (h): sc-400266-ACT

GLI1 encodes a zinc finger transcription factor that functions as a central terminal effector of Hedgehog signaling, integrating upstream cues from PTCH1 and SMO to regulate gene programs controlling proliferation, differentiation, and tissue patterning. In the nucleus, GLI-1 binds GLI consensus elements to modulate expression of targets involved in cell-cycle progression, stem-like states, and epithelial–mesenchymal transition. Crosstalk with pathways such as TGF-β, MAPK, and PI3K/AKT can tune GLI1 transcriptional output and context-specific phenotypes. Dysregulated GLI1 activity is frequently used as a molecular readout of aberrant Hedgehog pathway signaling in cancers and other disorders of development.

GLI-1/GLI1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GLI1 expression without altering the underlying DNA sequence.

GLI-1/GLI1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GLI1 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 GLI1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GLI-1/GLI1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GLI1 locus and enabling the study of GLI-1/GLI1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GLI-1/GLI1 pathway restoration in tumor cells with silenced or reduced GLI1 expression.

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