Six2 CRISPR Activation Plasmid (h): sc-402975-ACT

SIX2 encodes the homeobox transcription factor Six2, a developmental regulator that maintains progenitor cell identity and controls lineage specification programs. In human tissues, Six2-driven transcriptional networks influence mesenchymal differentiation, epithelial–mesenchymal interactions, and organogenesis-associated pathways, with downstream effects on cell-cycle timing and chromatin-state maintenance. Dysregulated SIX2 expression has been linked to altered developmental gene programs and has been reported in contexts of aberrant proliferation and differentiation, making it relevant for mechanistic studies of developmental disorders and oncogenic transcriptional rewiring. As a DNA-binding regulator, Six2 is frequently used as a node to interrogate transcriptional control circuits and fate decisions in stem and progenitor models.

Six2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIX2 expression without altering the underlying DNA sequence.

Six2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIX2 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 SIX2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Six2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIX2 locus and enabling the study of Six2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Six2 pathway restoration in tumor cells with silenced or reduced SIX2 expression.

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