ATG4B Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout population engineered to disrupt the ATG4B gene in the near-haploid HAP1 human cell line. This polyclonal pool provides a powerful loss-of-function model for studying ATG4B, a cysteine protease that is indispensable for autophagosome biogenesis and the processing of LC3 family proteins. By avoiding clonal selection, these cells enable robust, unbiased investigation of autophagy-dependent pathways in cancer and neurodegenerative disease contexts.
HAP1 is a near-haploid human cell line originally derived from the peripheral blood of a male patient diagnosed with chronic myeloid leukemia. With a karyotype that is haploid except for a disomic chromosome 8, HAP1 cells greatly simplify genetic analyses by reducing allelic complexity and allowing straightforward interpretation of gene knockout phenotypes. They maintain functional signaling modules central to autophagy, stress adaptation, and oncogenic transformation, making them an ideal host for high-throughput functional genomics and drug screening studies targeting the autophagy-lysosomal system.
The ATG4B gene product is a cysteine protease that serves a dual role in macroautophagy. It cleaves the C-terminal tail of pro-LC3 family members??including MAP1LC3A, MAP1LC3B, GABARAP, GABARAPL1, and GABARAPL2??to generate LC3-I. Following autophagic induction, ATG4B delipidates LC3-II, recycling LC3 for sustained autophagosome growth. This dual processing activity is indispensable for autophagosome maturation and cargo degradation. ATG4B activity is stimulated by nutrient deprivation, mTORC1 inhibition via rapamycin, the transcription factors TFEB and FOXO3, and cellular ROS. It coordinates with ATG7 (E1-like), ATG3 (E2-like), and the ATG12-ATG5-ATG16L1 E3-like complex. Disruption of ATG4B via CRISPR/Cas9 results in failed LC3 lipidation and recycling, leading to pro-LC3 accumulation, defective autophagosome closure, and a complete block in autophagic flux.
In HAP1 cells, ATG4B knockout creates a streamlined model for autophagy research. The haploid background allows precise quantification of LC3 conversion, p62/SQSTM1 degradation, and starvation-induced cell death. The leukemic origin enables study of autophagy’s role in chronic myeloid leukemia, where it can either support tumorigenesis or mediate drug-induced cytotoxicity. This system is also pertinent to neurodegenerative disease models, as autophagy impairment is a common feature. The polyclonal nature avoids clonal bias, ensuring that phenotypes represent the integrated ATG4B loss-of-function.
Typical applications include Western blotting for LC3-I/II conversion and p62 turnover, fluorescence microscopy for GFP-LC3 puncta, and flow-based quantification of autophagic flux following lysosomal inhibition. Researchers can further examine cell survival under starvation or chemotherapeutic stress, measure ATG4B enzymatic activity, and investigate pathways underlying drug resistance. For technical inquiries, please reach out to Ascent Research.
