Rak CRISPR Activation Plasmid (h): sc-405590-ACT

Human FRK encodes Rak, a non-receptor tyrosine kinase of the BRK family that modulates intracellular signaling by phosphorylating substrates involved in growth factor responses, adhesion, and cytoskeletal organization. Rak has been implicated in the regulation of epithelial differentiation and cell-cycle control through pathways intersecting with receptor tyrosine kinase signaling and downstream MAPK/ERK and PI3K/AKT network components. Altered FRK expression or signaling balance has been associated with changes in proliferation, migration, and stress responses observed across multiple disease-relevant contexts, including cancer biology. As a node in kinase-driven signaling circuits, FRK is frequently studied to define how tyrosine phosphorylation reshapes transcriptional programs and phenotypic state.

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

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

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