A-Raf CRISPR Activation Plasmid (h): sc-401799-ACT

Human ARAF encodes A-Raf, a RAF family serine/threonine kinase that relays RAS-dependent signals to the MAPK cascade by regulating MEK and downstream ERK activity. Through this pathway, A-Raf contributes to control of cell proliferation, differentiation, and survival, and it can modulate signal strength and duration in a context-dependent manner across tissues. Aberrant RAS–RAF–MEK–ERK signaling is a common feature of oncogenic and developmental dysregulation, making ARAF a useful node for dissecting MAPK pathway wiring, feedback, and crosstalk with PI3K/AKT and stress-response programs. Experimental perturbation of ARAF supports mechanistic studies of kinase network compensation among RAF paralogs and pathway-dependent transcriptional responses.

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

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

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