RBKS CRISPR Activation Plasmid (h): sc-404754-ACT

RBKS (ribokinase) encodes a cytosolic carbohydrate kinase that phosphorylates D-ribose to ribose-5-phosphate, linking ribose salvage to the pentose phosphate pathway and nucleotide biosynthesis. By regulating intracellular pools of ribose-5-phosphate, RBKS influences purine and pyrimidine metabolism, redox buffering through NADPH-generating reactions, and proliferative capacity in metabolically active cells. RBKS activity interfaces with broader carbon flux control that supports DNA/RNA synthesis and cellular stress responses. Dysregulated ribose metabolism and pentose phosphate pathway remodeling are frequently studied in the context of cancer metabolism, immune cell activation, and inborn errors affecting nucleotide homeostasis.

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

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

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