mTOR CRISPR Activation Plasmid (h): sc-400140-ACT

MTOR encodes mTOR, a serine/threonine kinase that integrates nutrient availability, growth factor signaling, cellular energy status, and stress cues to coordinate anabolic and catabolic programs. As the catalytic core of mTORC1 and mTORC2, mTOR regulates protein synthesis via S6K and 4E-BP1, lipid and nucleotide biosynthesis, autophagy, lysosomal function, cytoskeletal organization, and cell survival through pathways that intersect with PI3K–AKT signaling. Tight control of mTOR activity is essential for cell growth, metabolism, and immune homeostasis, while dysregulation is frequently associated with altered proliferation, metabolic rewiring, and aberrant autophagy. Perturbed MTOR signaling has been linked to diverse disease-relevant contexts including cancer biology, neurodevelopmental and neurodegenerative processes, and metabolic and inflammatory phenotypes.

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

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

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