RIP3 CRISPR Activation Plasmid (h): sc-401008-ACT

Human RIPK3 encodes receptor-interacting serine/threonine-protein kinase 3 (RIP3), a central mediator of programmed necrosis (necroptosis) and inflammatory cell death. RIP3 integrates upstream signals from death receptors and innate immune pathways to activate MLKL-driven membrane disruption, while also intersecting with NF-κB signaling, inflammasome crosstalk, and metabolic stress responses. Dysregulated RIPK3 activity is linked to aberrant inflammation and tissue injury mechanisms implicated in neurodegeneration, ischemia-reperfusion damage, infection-associated immunopathology, and cancer biology. As a pathway node, RIP3 is frequently studied to dissect context-dependent switches between apoptosis, necroptosis, and pro-survival signaling.

RIP3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RIPK3 expression without altering the underlying DNA sequence.

RIP3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RIPK3 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 RIPK3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RIP3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RIPK3 locus and enabling the study of RIP3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RIP3 pathway restoration in tumor cells with silenced or reduced RIPK3 expression.

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