Atg16 CRISPR Activation Plasmid (h): sc-404024-ACT

Human ATG16L1 encodes Atg16, a core component of the ATG12–ATG5–ATG16L1 complex that specifies autophagosome formation by directing LC3 lipidation at nascent phagophores. Through its role in macroautophagy, Atg16 supports cytoplasmic quality control, organelle turnover, and cellular adaptation to metabolic stress, while also intersecting with innate immune signaling and antimicrobial responses. Altered ATG16L1 activity has been linked to dysregulated inflammation and epithelial barrier homeostasis, and genetic variation in ATG16L1 is associated with susceptibility to inflammatory bowel disease. Modulating ATG16L1 expression is therefore useful for investigating autophagy flux, immunometabolic crosstalk, and stress-response phenotypes in relevant human cell models.

Atg16 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ATG16L1 expression without altering the underlying DNA sequence.

Atg16 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ATG16L1 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 ATG16L1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Atg16 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ATG16L1 locus and enabling the study of Atg16-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Atg16 pathway restoration in tumor cells with silenced or reduced ATG16L1 expression.

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