SphK2 CRISPR Activation Plasmid (h): sc-417921-ACT

Human SPHK2 encodes sphingosine kinase 2 (SphK2), a lipid kinase that phosphorylates sphingosine to generate sphingosine-1-phosphate (S1P), a pleiotropic signaling mediator that shapes cell survival, stress responses, migration, and inflammatory signaling. SphK2 activity influences sphingolipid rheostat balance between pro-apoptotic ceramides/sphingosine and pro-survival S1P, linking it to mitochondrial function, nuclear signaling, and chromatin-associated processes. Through regulation of intracellular S1P pools and downstream pathways, including MAPK/ERK, PI3K-AKT, and NF-κB-related programs, SPHK2 contributes to context-dependent control of proliferation and immune modulation. Dysregulated sphingolipid metabolism involving SPHK2 has been associated with cancer biology, neuroinflammation and neurodegeneration-related mechanisms, and cardiometabolic stress pathways, supporting its use in mechanistic studies of signaling and lipid homeostasis.

SphK2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SPHK2 expression without altering the underlying DNA sequence.

SphK2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SPHK2 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 SPHK2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SphK2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SPHK2 locus and enabling the study of SphK2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SphK2 pathway restoration in tumor cells with silenced or reduced SPHK2 expression.

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