EFP CRISPR Activation Plasmid (h): sc-402678-ACT

Human TRIM25 encodes the E3 ubiquitin ligase EFP, a RING finger protein that regulates innate immune signaling and RNA metabolism through ubiquitination of key substrates. EFP promotes antiviral responses by modulating RIG-I-like receptor pathways and downstream type I interferon signaling, and it can influence NF-κB activation and stress-responsive transcriptional programs. Beyond immunity, TRIM25 participates in post-transcriptional control via interactions with RNA-binding proteins, impacting mRNA stability and translation. Dysregulated TRIM25/EFP activity has been associated with altered inflammatory signaling and tumor-associated phenotypes, making it relevant for mechanistic studies in infection biology, immune regulation, and cancer cell signaling.

EFP CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TRIM25 expression without altering the underlying DNA sequence.

EFP CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TRIM25 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 TRIM25 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EFP expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TRIM25 locus and enabling the study of EFP-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EFP pathway restoration in tumor cells with silenced or reduced TRIM25 expression.

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