The ATG101 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population in which the ATG101 gene has been disrupted in the near-haploid HAP1 cell line, creating a loss-of-function model for autophagy research. This polyclonal pool provides a genetically heterogeneous population of knockout cells, enabling robust functional assays without the need for single-cell clonal isolation.
The HAP1 cell line is a human near-haploid chronic myeloid leukemia (CML) cell line of male origin, derived from the KBM-7 parental line and characterized by a haploid karyotype with disomy of chromosome 8. Its near-haploid genome simplifies genetic manipulation, as CRISPR/Cas9-mediated disruption of a single allele often yields a full loss-of-function phenotype, facilitating clean genotype-phenotype correlations in functional studies.
ATG101 is an essential subunit of the ULK1 complex, which also includes ULK1, ATG13, and RB1CC1/FIP200. This complex integrates nutrient signals from mTORC1 and AMPK to control autophagy initiation. Under nutrient-rich conditions, mTORC1 phosphorylates ULK1 and ATG13, inhibiting the complex. Upon starvation, mTORC1 is suppressed and AMPK activated, leading to ULK1 activation. Active ULK1 then phosphorylates components of the class III PI3K complex I, such as ATG14L, BECN1, and PIK3C3/VPS34, to generate PI3P. This lipid recruits WIPI2 and facilitates LC3/GABARAP lipidation, driving autophagosome formation. ATG101 stabilizes the ULK1 complex by directly interacting with ATG13; loss of ATG101 disrupts autophagy initiation and sensitizes cells to metabolic stress.
In HAP1 cells, ATG101 knockout creates an autophagy-deficient model ideal for investigating autophagy??s role in cancer cell biology. The absence of ULK1 complex activity allows study of phenotypes such as survival under nutrient stress, mitochondrial turnover, and chemoresistance. Given the CML origin, this polyclonal pool is relevant for leukemia research. Moreover, the near-haploid genome ensures that observed effects are directly attributable to ATG101 disruption, with minimal interference from duplicated genes.
Research applications include autophagic flux measurements via LC3 and p62 immunoblotting with bafilomycin A1, immunofluorescence of LC3 puncta, and co-immunoprecipitation of ULK1 complex components. This polyclonal knockout is also suitable for RNA-seq profiling, cell viability assays under starvation, and high-throughput screening for autophagy modulators. Key application areas encompass cancer, neurodegeneration, and inflammatory disease research where autophagy plays a critical role. For further information, technical assistance, or custom requests, please contact Ascent Research.
