Perforin 1 CRISPR Activation Plasmid (h): sc-400976-ACT

PRF1 encodes perforin 1, a pore-forming cytolytic effector that is essential for granule-mediated killing by cytotoxic T lymphocytes and natural killer cells. Upon immune synapse formation, perforin facilitates delivery of granzymes into target cells to trigger apoptotic programs, integrating with pathways controlling immune surveillance, antiviral defense, and tumor immunoediting. PRF1 activity is closely linked to lymphocyte activation state and degranulation dynamics, and altered expression or function is associated with immune dysregulation phenotypes including impaired cytotoxicity and hyperinflammatory syndromes. As a core component of cell-mediated cytotoxicity, PRF1 is widely studied in contexts such as infection biology, immune evasion, and inflammatory tissue damage.

Perforin 1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PRF1 expression without altering the underlying DNA sequence.

Perforin 1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PRF1 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 PRF1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Perforin 1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PRF1 locus and enabling the study of Perforin 1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Perforin 1 pathway restoration in tumor cells with silenced or reduced PRF1 expression.

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