The ATG4A Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-mediated polyclonal knockout cell population in which the ATG4A gene has been disrupted in the near-haploid HAP1 cell line. This product provides a heterogeneous pool of cells carrying targeted gene disruption events, enabling loss-of-function analysis of ATG4A without clonal selection. The polyclonal format is suitable for pooled screening experiments and studies where clonal variation is to be averaged, offering a robust model to interrogate autophagy-related processes.
HAP1 is a near-haploid human cell line derived from the chronic myeloid leukemia (CML) line KBM-7. Its near-haploid karyotype, with a single copy of most chromosomes, simplifies genetic manipulation and phenotypic interpretation, making it a preferred host for knockout screening and functional genomics. HAP1 cells retain many signaling pathways relevant to cancer and cell biology, and their adherent growth facilitates a wide range of cell-based assays, including immunofluorescence, live-cell imaging, and biochemical analyses.
ATG4A encodes a cysteine protease essential for autophagy. It cleaves pro-LC3 and GABARAP family proteins, enabling their lipidation and autophagosome conjugation. ATG4A activity is regulated by nutrient deprivation and mTORC1 inhibition upstream, acting downstream of the ULK1 complex. The protease interacts with LC3 and GABARAP family members and functions in concert with the ATG12-ATG5 conjugate. Knockout of ATG4A disrupts LC3-I to LC3-II conversion, blocks autophagy flux, and leads to accumulation of p62/SQSTM1. Representative pathway components include ULK1, ATG13, FIP200, Beclin-1, VPS34, and ATG16L1.
In the HAP1 background, disruption of ATG4A creates a powerful loss-of-function model for autophagy research. The near-haploid nature of HAP1 ensures that a single knockout event per cell leads to complete loss of protein function, enabling clear interpretation of phenotype. This cell model is particularly useful for studying autophagy-dependent processes in cancer, including resistance to chemotherapy and targeted agents, as well as for exploring autophagy??s role in neurodegenerative disorders and host-pathogen interactions. The polyclonal population captures a range of genetic alterations, which can be leveraged in pooled screens to identify synthetic lethal interactions or drug sensitivities.
Typical applications include Western blotting for LC3 and p62 to monitor autophagy, autophagy flux assays using chloroquine, and immunofluorescence for LC3 puncta. Cell viability under nutrient starvation and RT-qPCR for autophagy gene expression further characterize the knockout phenotype. The polyclonal population is amenable to pooled genetic screens and drug sensitivity profiling. For technical inquiries, please contact Ascent Research.
