PREP-1 CRISPR Activation Plasmid (h): sc-402314-ACT

PKNOX1 encodes the human TALE homeobox transcription factor PREP-1, a nuclear DNA-binding protein that partners with PBX and MEIS family cofactors to regulate enhancer and promoter activity during development and cell fate decisions. PREP-1 contributes to transcriptional programs controlling differentiation, proliferation, and metabolic homeostasis, and its activity integrates with broader homeobox-regulated gene networks. Dysregulated PKNOX1/PREP-1 expression has been associated with altered lineage specification and oncogenic transcriptional states in multiple contexts, supporting its use as a node for studying transcriptional control in disease-relevant models. As a chromatin-associated regulator, PREP-1 is also pertinent to investigations of gene regulatory architecture and context-dependent enhancer function.

PREP-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PKNOX1 expression without altering the underlying DNA sequence.

PREP-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PKNOX1 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 PKNOX1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PREP-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PKNOX1 locus and enabling the study of PREP-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PREP-1 pathway restoration in tumor cells with silenced or reduced PKNOX1 expression.

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