ATP9A Knockout HAP1 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal knockout cell population designed for functional dissection of the ATP9A gene in a near-haploid human cell background. This product provides a heterogeneous pool of HAP1 cells carrying diverse CRISPR-mediated disruptions at the ATP9A locus, enabling pool-level loss-of-function studies without clonal isolation. The polyclonal format preserves population-level heterogeneity and is well-suited for phenotypic screens, biochemical assays, and imaging-based applications where a purely monoclonal population is not required.
HAP1 is a human haploid chronic myeloid leukemia cell line derived from the KBM-7 parental line. It retains a near-haploid karyotype except for a disomic segment of chromosome 15 and is male in origin. The haploid nature of HAP1 simplifies CRISPR-based gene disruption, as a single allele can be targeted to achieve functional knockout, yielding unambiguous genotype-phenotype relationships. This feature makes HAP1 a powerful platform for functional genomics, drug target validation, and membrane trafficking studies, where the consequences of complete gene inactivation can be assessed without allele redundancy.
ATP9A encodes a P4-ATPase phospholipid flippase that actively translocates aminophospholipids from the luminal to the cytoplasmic leaflet of endosomal membranes, thereby generating and maintaining lipid asymmetry. ATP9A couples ATP hydrolysis to phospholipid flipping, and its activity is critically dependent on heterodimerization with CDC50A (TMEM30A) or CDC50B chaperone subunits that are required for proper folding and trafficking of the flippase complex. Within the endocytic recycling pathway, ATP9A promotes membrane curvature and tubulation, facilitating the formation of recycling carriers together with RAB11, EHD1, and the cargo adaptor SNX17. This mechanism is essential for the efficient retrograde transport of internalized receptors from endosomes to the trans-Golgi network and for the sorting of cargoes back to the plasma membrane.
In the HAP1 haploid background, disruption of ATP9A leads to a complete loss of flippase function, resulting in impaired endosomal phospholipid asymmetry and defective endocytic recycling. This cellular context provides a clean genetic model to dissect ATP9A-dependent membrane remodeling processes and to explore its role in maintaining organelle identity and cargo retrieval. Because membrane lipid asymmetry is also linked to neurodevelopmental disorders, the ATP9A knockout in HAP1 serves as a tractable system for investigating disease-relevant molecular mechanisms and for identifying genetic interactions that modulate retrograde trafficking.
Researchers can employ this polyclonal knockout population in a variety of assays, including transferrin recycling assays to quantify endocytic retrieval kinetics, immunofluorescence microscopy for endosomal markers such as EEA1 and RAB11 to assess organelle morphology, and annexin V?Cbased flippase activity measurements. Western blotting for CDC50A and key cargo proteins, SNAP-tag pulse-chase labeling, and proliferation assays further enable in-depth mechanistic studies. CRISPR-based complementation experiments with wild-type or mutant ATP9A allow confirmation of phenotypic specificity. For further details, please contact Ascent Research.
