FADD CRISPR Activation Plasmid (h): sc-400580-ACT

Human FADD (Fas-associated protein with death domain) is an adaptor molecule that transduces signals from death receptors including FAS and TNFRSF members to initiate extrinsic apoptosis. By bridging receptor complexes to initiator caspases such as CASP8 through homotypic death domain interactions, FADD regulates caspase cascade activation, programmed cell death, and immune homeostasis. Beyond apoptosis, FADD influences NF-κB signaling, inflammatory responses, and cell cycle control in a context-dependent manner. Dysregulated FADD signaling has been implicated in altered apoptotic competence and immune dysregulation observed across diverse cancer and inflammatory disease models.

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

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

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