TFEB CRISPR Activation Plasmid (h): sc-401388-ACT

Human TFEB (transcription factor EB) is a master regulator of lysosomal biogenesis and autophagy, coordinating expression of CLEAR network genes that control endolysosomal function, membrane trafficking, and cellular clearance. TFEB activity is tightly linked to nutrient sensing through mTORC1-dependent phosphorylation and nuclear translocation, integrating metabolic stress signals with catabolic programs. Dysregulated TFEB signaling has been associated with impaired proteostasis and lysosome-related dysfunction observed across neurodegeneration, lysosomal storage disorders, and cancer biology. As a transcriptional node, TFEB provides a tractable entry point to study autophagic flux, lysosome remodeling, and stress-adaptive transcriptional networks.

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

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

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