XAF1 CRISPR Activation Plasmid (h): sc-402427-ACT

XAF1 (XIAP-associated factor 1) is a pro-apoptotic tumor suppressor that antagonizes inhibitor of apoptosis proteins, including XIAP, to facilitate caspase activation and mitochondrial apoptosis. Its expression is frequently induced by interferon signaling and cellular stress, linking XAF1 to innate immune responses and transcriptional programs that constrain survival under damaging conditions. Reduced XAF1 expression through promoter hypermethylation or transcriptional repression has been reported across multiple malignancies and is often associated with apoptosis resistance and altered sensitivity to stress pathways. As a regulator of cell fate decisions, XAF1 is widely studied in contexts of cell-cycle control, DNA damage responses, and inflammatory signaling cross-talk.

XAF1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous XAF1 expression without altering the underlying DNA sequence.

XAF1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the XAF1 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 XAF1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous XAF1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native XAF1 locus and enabling the study of XAF1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of XAF1 pathway restoration in tumor cells with silenced or reduced XAF1 expression.

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