A20/TNFAIP3 CRISPR Activation Plasmid (h): sc-400447-ACT

TNFAIP3 encodes A20, a zinc finger ubiquitin-editing enzyme that acts as a central negative regulator of inflammatory signaling. A20 terminates NF-κB and MAPK pathway activation downstream of receptors such as TNFR, IL-1R, and TLRs by modulating ubiquitin chains on key signaling intermediates, thereby limiting cytokine production and preventing prolonged immune activation. Through control of cell death and stress responses, A20 helps maintain immune homeostasis in many cell types, including myeloid and lymphoid lineages. Dysregulated TNFAIP3 expression or function has been linked to immune-mediated pathology and inflammatory disease mechanisms, making it a widely used target for studying signaling feedback, ubiquitin biology, and innate/adaptive immune crosstalk.

A20/TNFAIP3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNFAIP3 expression without altering the underlying DNA sequence.

A20/TNFAIP3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNFAIP3 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 TNFAIP3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous A20/TNFAIP3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNFAIP3 locus and enabling the study of A20/TNFAIP3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of A20/TNFAIP3 pathway restoration in tumor cells with silenced or reduced TNFAIP3 expression.

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