DR3 CRISPR Activation Plasmid (h): sc-403461-ACT

TNFRSF25 encodes death receptor 3 (DR3), a TNF receptor superfamily member expressed predominantly in immune cell subsets where it modulates lymphocyte activation, survival, and cytokine production. DR3 is engaged by TNFSF15 (TL1A) to initiate receptor-proximal signaling that can converge on NF-κB and MAPK pathways, shaping inflammatory gene programs and T cell effector functions. Through its death domain, DR3 can also interface with apoptosis- and necroptosis-associated signaling complexes in a context-dependent manner, linking immune activation to regulated cell death. Dysregulated TL1A–DR3 signaling has been implicated in immune-mediated inflammation and tissue remodeling, making TNFRSF25 a useful node for studying mechanisms of chronic inflammatory pathology and immune homeostasis.

DR3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFRSF25 expression without altering the underlying DNA sequence.

DR3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNFRSF25 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 TNFRSF25 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous DR3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNFRSF25 locus and enabling the study of DR3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of DR3 pathway restoration in tumor cells with silenced or reduced TNFRSF25 expression.

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