HNF-6 CRISPR Activation Plasmid (h): sc-401082-ACT

ONECUT1 encodes the transcription factor HNF-6, a homeodomain protein that orchestrates tissue-specific gene programs during endodermal development and adult epithelial homeostasis. In human cells, HNF-6 integrates promoter and enhancer inputs to regulate lineage specification and differentiation pathways, with prominent roles in hepatic and pancreatic transcriptional networks and epithelial morphogenesis. By modulating expression of metabolic, secretory, and cell-identity genes, ONECUT1 influences processes such as organogenesis, cell fate maintenance, and stress-responsive transcriptional control. Dysregulated ONECUT1/HNF-6 activity has been associated with altered differentiation states and transcriptional rewiring observed in metabolic and epithelial disease contexts, supporting its utility as a node for mechanistic studies of gene regulatory circuits.

HNF-6 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ONECUT1 expression without altering the underlying DNA sequence.

HNF-6 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ONECUT1 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 ONECUT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HNF-6 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ONECUT1 locus and enabling the study of HNF-6-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HNF-6 pathway restoration in tumor cells with silenced or reduced ONECUT1 expression.

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