DEPTOR CRISPR Activation Plasmid (h): sc-402253-ACT

DEPTOR (DEP domain containing MTOR interacting protein) is an endogenous inhibitor of mTOR signaling that binds and modulates both mTORC1 and mTORC2 complexes. By tuning phosphorylation outputs that control protein synthesis, autophagy, metabolism, and cytoskeletal dynamics, DEPTOR helps coordinate growth and survival programs downstream of nutrient and growth factor cues. Altered DEPTOR expression has been linked to dysregulated PI3K–AKT–mTOR network activity observed across multiple disease-associated contexts, including cancer biology and metabolic stress responses. As a node that couples mTOR complex activity to feedback regulation, DEPTOR is frequently studied for its effects on cell proliferation, apoptosis sensitivity, and adaptive signaling rewiring.

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

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

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