Ref-1 CRISPR Activation Plasmid (h): sc-400932-ACT

APEX1 encodes Ref-1 (APE1), an essential apurinic/apyrimidinic endonuclease that coordinates base excision repair by cleaving abasic DNA sites generated after oxidative damage, alkylation, or spontaneous base loss. Beyond DNA repair, Ref-1 also functions as a redox regulator that modulates the DNA-binding activity of transcription factors involved in stress and inflammatory signaling, including AP-1, NF-κB, and HIF-1. Through these roles, APEX1 supports genome stability, replication-associated damage tolerance, and cellular responses to reactive oxygen species. Dysregulated APEX1 activity and altered redox/repair balance have been associated with tumor biology, neurodegeneration, and inflammatory pathologies, making it a key node for mechanistic studies of DNA damage responses.

Ref-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous APEX1 expression without altering the underlying DNA sequence.

Ref-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the APEX1 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 APEX1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ref-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native APEX1 locus and enabling the study of Ref-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ref-1 pathway restoration in tumor cells with silenced or reduced APEX1 expression.

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