CAD CRISPR Activation Plasmid (h): sc-403028-ACT

DNA fragmentation factor subunit beta (DFFB), also known as caspase-activated DNase (CAD), is an endonuclease that executes internucleosomal DNA cleavage during apoptosis after release from its inhibitor ICAD. Activated CAD contributes to chromatin fragmentation, apoptotic body formation, and DNA damage signaling, linking programmed cell death to genome integrity pathways. DFFB activity is therefore relevant to studies of apoptosis regulation, immune homeostasis, and cellular responses to genotoxic stress. Altered control of apoptotic DNA fragmentation has been associated with oncogenic survival, inflammatory phenotypes, and neurodegenerative processes, making DFFB a useful node for mechanistic investigation.

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

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

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