ENX-1 CRISPR Activation Plasmid (h): sc-417028-ACT

EZH2 encodes the human ENX-1 protein, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) that trimethylates histone H3 at lysine 27 (H3K27me3) to establish transcriptionally repressive chromatin. Through Polycomb-mediated silencing, ENX-1 regulates lineage specification, cell-cycle control, and maintenance of cellular identity by modulating chromatin accessibility and long-range epigenetic programs. EZH2-dependent repression intersects with key pathways controlling proliferation and differentiation, including chromatin remodeling and developmental transcription networks. Dysregulated EZH2 activity and altered H3K27 methylation landscapes are frequently associated with oncogenic transcriptional states and disease-relevant epigenetic remodeling, supporting its use as a model gene in cancer and chromatin biology research.

ENX-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EZH2 expression without altering the underlying DNA sequence.

ENX-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EZH2 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 EZH2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ENX-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EZH2 locus and enabling the study of ENX-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ENX-1 pathway restoration in tumor cells with silenced or reduced EZH2 expression.

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