SphK1 CRISPR Activation Plasmid (h): sc-401274-ACT

Human SPHK1 encodes sphingosine kinase 1 (SphK1), a cytosolic lipid kinase that phosphorylates sphingosine to generate sphingosine-1-phosphate (S1P), a bioactive sphingolipid that couples metabolic status to signaling outputs. By shifting the sphingolipid rheostat toward S1P, SphK1 influences membrane trafficking, calcium-dependent signaling, and downstream MAPK/ERK, PI3K/AKT, and NF-κB pathway activity through intracellular actions and S1P receptor-dependent signaling. SPHK1 regulation has been linked to inflammatory responses, stress adaptation, angiogenic signaling, and cell motility programs in diverse cellular contexts. Altered SPHK1/S1P signaling is frequently investigated in cancer biology, fibrosis, and immune-mediated disease models where sphingolipid remodeling affects proliferation, survival, and microenvironmental communication.

SphK1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SPHK1 expression without altering the underlying DNA sequence.

SphK1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SPHK1 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 SPHK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SphK1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SPHK1 locus and enabling the study of SphK1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SphK1 pathway restoration in tumor cells with silenced or reduced SPHK1 expression.

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