Six6 CRISPR Activation Plasmid (h): sc-405096-ACT

SIX6 encodes the homeobox transcription factor Six6, a nuclear DNA-binding regulator essential for embryonic eye and forebrain development and for maintaining neuroretinal cell identity. Six6 modulates gene expression programs controlling progenitor proliferation, cell-cycle timing, and lineage specification, interfacing with developmental signaling networks that include WNT, SHH, and BMP cues. Altered SIX6 dosage or regulatory variation has been associated with ocular phenotypes such as optic nerve head and retinal developmental abnormalities and with genetic risk for glaucoma-related traits, supporting its relevance to neurodevelopmental and ocular disease biology. As a transcriptional regulator, Six6 is frequently studied in models of retinal differentiation, neuronal fate decisions, and gene regulatory network architecture.

Six6 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIX6 expression without altering the underlying DNA sequence.

Six6 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIX6 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 SIX6 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Six6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIX6 locus and enabling the study of Six6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Six6 pathway restoration in tumor cells with silenced or reduced SIX6 expression.

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