Raptor CRISPR Activation Plasmid (h): sc-400960-ACT

Human RPTOR encodes Raptor, an essential scaffolding component of mTOR complex 1 (mTORC1) that recruits substrates such as S6K and 4E-BP1 to coordinate nutrient, energy, and growth factor inputs with protein synthesis, autophagy suppression, and metabolic reprogramming. Through regulation of mTORC1 substrate specificity and localization, Raptor influences cell growth, cell cycle progression, ribosome biogenesis, and lysosome-associated signaling. Dysregulated RPTOR/mTORC1 activity has been associated with altered anabolic signaling observed across cancer biology, metabolic disease, and neurodevelopmental and neurodegenerative contexts, making it a widely used node for pathway dissection. Modulating RPTOR expression provides a direct approach to study feedback between PI3K–AKT signaling, AMPK, and nutrient-sensing pathways that shape cellular homeostasis.

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

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

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