Atg9a CRISPR Activation Plasmid (h): sc-408011-ACT

ATG9A encodes Atg9a, a conserved multispanning membrane protein that supplies membrane to the expanding phagophore during macroautophagy and supports autophagosome biogenesis. Atg9a cycles between the trans-Golgi network, endosomes, and autophagy initiation sites, coordinating membrane trafficking with core autophagy machinery to maintain proteostasis and organelle quality control. Through its roles in autophagic flux, Atg9a influences responses to nutrient stress, ER stress, and selective autophagy pathways that shape inflammation and cellular metabolism. Altered ATG9A regulation has been linked to dysregulated autophagy observed in cancer biology, neurodegeneration, and host–pathogen interactions, making it relevant for mechanistic studies of disease-associated stress adaptation.

Atg9a CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATG9A expression without altering the underlying DNA sequence.

Atg9a CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATG9A 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 ATG9A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Atg9a expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATG9A locus and enabling the study of Atg9a-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Atg9a pathway restoration in tumor cells with silenced or reduced ATG9A expression.

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