c-Kit CRISPR Activation Plasmid (h): sc-400106-ACT

Human KIT encodes the receptor tyrosine kinase c-Kit (CD117), a cell-surface receptor for stem cell factor that regulates survival, proliferation, migration, and differentiation in hematopoietic progenitors, melanocytes, germ cells, and mast cells. Ligand-induced dimerization and autophosphorylation engage PI3K–AKT, RAS–MAPK, JAK/STAT, and PLCγ signaling cascades, integrating cues that control lineage commitment and tissue homeostasis. Dysregulated KIT signaling is implicated in oncogenic transformation and aberrant mast cell biology, and altered c-Kit activity is associated with tumors and hematologic disorders where receptor expression or signaling output changes.

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

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

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