Atg4b CRISPR Activation Plasmid (h): sc-404173-ACT

Human ATG4B encodes Atg4b, a cysteine protease that catalyzes processing and delipidation of ATG8 family proteins such as LC3 and GABARAP, enabling their conjugation to phosphatidylethanolamine and recycling from autophagosomal membranes. Through these activities, Atg4b regulates autophagosome biogenesis, maturation, and cargo flux across macroautophagy and selective autophagy pathways. ATG4B function influences proteostasis, mitochondrial and organelle quality control, and cellular responses to nutrient stress and hypoxia. Dysregulated ATG4B-dependent autophagy has been implicated in contexts relevant to cancer biology, neurodegeneration, and infection, where altered turnover of protein aggregates and damaged organelles can reshape cell survival and inflammatory signaling.

Atg4b CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATG4B expression without altering the underlying DNA sequence.

Atg4b CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATG4B 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 ATG4B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Atg4b expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATG4B locus and enabling the study of Atg4b-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Atg4b pathway restoration in tumor cells with silenced or reduced ATG4B expression.

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