TORC2 CRISPR Activation Plasmid (h): sc-402481-ACT

Human CRTC2 (TORC2) is a CREB-regulated transcription coactivator that integrates cAMP/PKA and calcium signaling with metabolic gene expression programs. By coupling CREB to promoter/enhancer activity, TORC2 helps control gluconeogenic and lipid metabolic transcriptional outputs and contributes to adaptive responses such as energy balance and stress signaling. Its activity is modulated by phosphorylation-dependent cytoplasmic sequestration and nuclear translocation, linking upstream kinase pathways to transcriptional control. Dysregulated CRTC2 signaling has been associated with altered metabolic homeostasis and perturbations in transcriptional networks relevant to metabolic and proliferative phenotypes.

TORC2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CRTC2 expression without altering the underlying DNA sequence.

TORC2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CRTC2 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 CRTC2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TORC2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CRTC2 locus and enabling the study of TORC2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TORC2 pathway restoration in tumor cells with silenced or reduced CRTC2 expression.

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