The ATG16L1 Knockout HAP1 Polyclonal Cells from Ascent Research constitute a CRISPR/Cas9-edited polyclonal knockout population targeting the human ATG16L1 gene within the near-haploid HAP1 cell line. This product provides a heterogeneous pool of HAP1 cells harboring diverse gene disruptions at the ATG16L1 locus, enabling robust loss-of-function studies without the need for clonal isolation. The polyclonal format preserves population-level genetic diversity while maintaining a consistent knockout background, making it well-suited for high-throughput functional assays and pathway analyses in autophagy and innate immunity research.
HAP1 cells are a widely used near-haploid human cell line originally derived from the KBM-7 chronic myeloid leukemia cell line isolated from a male patient. Their near-haploid karyotype, with a single copy of most chromosomes, greatly simplifies CRISPR/Cas9-mediated gene targeting because only one allele must be disrupted to achieve a functional knockout. This genetic characteristic dramatically increases knockout efficiencies and reduces the confounding effects of heterozygous mutations. HAP1 cells retain leukemia-cell features while remaining amenable to standard cell culture techniques, and they have become a standard platform for genome-wide screens, gene essentiality studies, and mechanistic cell biology experiments.
ATG16L1 functions as a critical scaffold protein in the autophagy machinery, forming the ATG12?CATG5?CATG16L1 complex that directs the lipidation of LC3 and drives phagophore elongation during autophagosome formation. Its role extends into innate immunity through a well-characterized interaction with NOD2, an intracellular sensor for bacterial muramyl dipeptide. This interaction couples pathogen recognition to the induction of xenophagy, a selective autophagic process that clears intracellular bacteria. Furthermore, ATG16L1 modulates NF-??B signaling downstream of NOD2, influencing the secretion of proinflammatory cytokines such as IL-1?? and IL-18. Upstream signals, including mTORC1 inhibition and AMPK activation during amino acid starvation or ER stress, initiate autophagy through the ULK1 kinase complex, which in turn regulates the Beclin-1?CVPS34 lipid-kinase module. ATG16L1 operates in concert with ATG5, ATG12, ATG3, WIPI2, TMEM59, and Rab33B, orchestrating the expansion of the isolation membrane and the conjugation of LC3 to phosphatidylethanolamine. The pathway additionally engages ATG7 as an E1-like enzyme, ATG3 as an E2-like enzyme, and p62/SQSTM1 as a cargo receptor linking autophagic substrates to LC3. This molecular network places ATG16L1 at a key intersection of catabolic homeostasis and immune defense.
Disruption of ATG16L1 in the HAP1 background creates a simplified cellular model for dissecting autophagy-dependent processes. The loss of ATG16L1 abrogates autophagosome biogenesis, leading to impaired clearance of ubiquitinated protein aggregates and defective bacterial clearance, which recapitulates phenotypes observed in Crohn??s disease-associated ATG16L1 variants. In haploid cells, phenotypic readouts are typically more direct and interpretable without the masking effects of a second functional allele. This allows quantitative assessment of autophagic activity, cytokine secretion, and pathogen resistance. The model is particularly valuable for examining the interplay between the autophagy pathway and NOD2-mediated signaling, offering insights into the mechanisms linking autophagy defects to chronic inflammatory disorders and cancer.
Researchers can apply these polyclonal knockout cells in a variety of experimental workflows: monitoring autophagic flux via Western blotting for LC3 lipidation and p62 degradation, imaging GFP-LC3 puncta formation, and flow cytometric analysis of autophagic vesicle accumulation. The cells are compatible with co-immunoprecipitation to study residual ATG16L1 complex components and with bacterial invasion assays (e.g., Salmonella, Shigella) to quantify xenophagy. ELISA-based measurement of IL-1?? and IL-18 secretion following NOD2 stimulation provides a direct readout of inflammatory signaling. In drug discovery, the knockout line serves as a negative-control background for high-throughput screens identifying autophagy inducers that bypass ATG16L1. For further information, customization, or bulk ordering, please contact Ascent Research.
