Atg14 CRISPR Activation Plasmid (h): sc-402883-ACT

Human ATG14 encodes Atg14, a core component of the class III phosphatidylinositol 3-kinase (PI3KC3) complex I that promotes phosphatidylinositol 3-phosphate production at nascent autophagosomal membranes. Atg14 helps specify autophagy initiation by directing the Beclin 1–VPS34 complex to the endoplasmic reticulum and phagophore assembly sites, supporting autophagosome nucleation and maturation. Through control of autophagic flux and selective degradation pathways, ATG14 influences proteostasis, organelle quality control, and cellular responses to nutrient stress. Dysregulated autophagy involving ATG14 has been associated with mechanisms relevant to cancer biology, neurodegeneration, and inflammatory signaling, making it a useful target for pathway interrogation.

Atg14 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATG14 expression without altering the underlying DNA sequence.

Atg14 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATG14 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 ATG14 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Atg14 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATG14 locus and enabling the study of Atg14-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Atg14 pathway restoration in tumor cells with silenced or reduced ATG14 expression.

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