p22-phox CRISPR Activation Plasmid (h): sc-400325-ACT

CYBA encodes p22-phox, an essential membrane-bound subunit of the phagocyte NADPH oxidase complex that stabilizes NOX catalytic subunits and supports assembly of the electron transfer machinery required for reactive oxygen species (ROS) generation. Through NOX-dependent redox signaling, p22-phox contributes to innate immune defense, regulation of inflammatory signaling cascades, and modulation of vascular and metabolic processes. Altered CYBA function or expression has been linked to dysregulated oxidative stress and inflammatory phenotypes, with relevance to immunodeficiency presentations and cardiometabolic and vascular pathobiology. In human cells, CYBA is frequently studied to dissect NOX complex regulation, compartmentalized ROS production, and downstream signaling events such as MAPK and NF-κB pathway activation.

p22-phox CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CYBA expression without altering the underlying DNA sequence.

p22-phox CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CYBA 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 CYBA transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous p22-phox expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CYBA locus and enabling the study of p22-phox-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of p22-phox pathway restoration in tumor cells with silenced or reduced CYBA expression.

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