Rheb CRISPR Activation Plasmid (h): sc-400539-ACT

Human RHEB encodes Rheb, a small Ras-related GTPase that serves as a key upstream activator of mTORC1, integrating growth factor and nutrient cues to coordinate protein synthesis, autophagy suppression, and cellular metabolism. Rheb activity is controlled by the TSC1–TSC2 complex, linking PI3K–AKT signaling and energy stress responses to mTOR pathway output. Dysregulated RHEB–mTORC1 signaling is implicated in aberrant cell growth and metabolic reprogramming and is frequently studied in the context of cancer biology and neurodevelopmental disorders. As a proximal mTORC1 regulator, Rheb is widely used to interrogate translational control, lysosomal signaling, and stress-adaptive homeostasis.

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

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

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