Raf-1 CRISPR Activation Plasmid (h): sc-400202-ACT

RAF1 encodes Raf-1, a serine/threonine kinase that functions as a central relay between activated RAS and the MAPK cascade, propagating signals through MEK and ERK to regulate proliferation, differentiation, and stress-responsive transcription. Raf-1 activity is controlled by phosphorylation, 14-3-3 binding, and subcellular localization, integrating cues from receptor tyrosine kinases and other upstream inputs. Beyond canonical MAPK signaling, Raf-1 modulates apoptosis and cytoskeletal dynamics through kinase-dependent and scaffold functions that influence cell survival programs. Dysregulated RAF1 signaling and RAF1 variants are linked to aberrant growth control and developmental disorders, making it a frequent node of interest in pathway mapping and mechanistic disease modeling.

Raf-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RAF1 expression without altering the underlying DNA sequence.

Raf-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RAF1 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 RAF1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Raf-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RAF1 locus and enabling the study of Raf-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Raf-1 pathway restoration in tumor cells with silenced or reduced RAF1 expression.

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