The CCDC91 Knockout HAP1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout population derived from HAP1 cells, designed for targeted disruption of CCDC91. This heterogeneous pool enables loss-of-function studies without clonal selection, maintaining genetic variability while ensuring robust knockout representation across the population. As a research tool, it allows investigation of CCDC91-dependent processes in a defined genetic background. The cells are produced using CRISPR/Cas9 genome editing to achieve stable gene knockout.
The HAP1 cell line is a near-haploid human line derived from KBM-7 chronic myeloid leukemia cells, exhibiting fibroblast-like morphology and a near-haploid karyotype. It carries the BCR-ABL oncogene, modeling CML. Haploidy simplifies genetic manipulation, as single-allele disruption often yields functional null phenotypes, making HAP1 a preferred system for functional genomics, CRISPR screening, and trafficking studies. Its rapid growth and ease of editing further contribute to its popularity.
CCDC91 functions as a cargo adaptor at the trans-Golgi network (TGN), linking lysosomal enzyme receptors to clathrin/AP-1 coats to drive vesicle formation. It interacts with AP-1 subunits (AP1M1, AP1G1), clathrin heavy chain (CLTC), GGA adaptors, and mannose-6-phosphate receptors (M6PR). This bridging is regulated upstream by ARF GTPases and targets lysosomal hydrolases, cathepsins, and lysosomal membrane proteins downstream. Defects in this process can lead to aberrant lysosomal enzyme secretion and impaired lysosome function. CCDC91 is thus a core sorting adaptor in the Golgi-to-lysosome pathway ensuring lysosomal enzyme delivery.
In the near-haploid HAP1 context, CCDC91 knockout eliminates residual gene activity, enabling clear phenotype assessment. The myeloid/Bcr-Abl background adds relevance to leukemia-related trafficking perturbations and potential lysosomal storage disorder links. Polyclonal populations provide mutation diversity, modeling a range of loss-of-function intensities for robust, reproducible studies. This model is particularly advantageous for examining endolysosomal trafficking in a disease-relevant, genetically simplistic system.
This model supports investigations into vesicle-mediated transport, lysosome biogenesis, and protein sorting. Researchers can perform immunofluorescence co-localization of AP-1 and M6PR, co-immunoprecipitation for CCDC91?CAP-1 complex disruption, and western blotting for cathepsin maturation. Lysosomal enzyme activity assays, electron microscopy of vesicles, and RNAi rescue provide additional phenotypic depth. The polyclonal format suits pooled functional genomics and drug discovery targeting lysosomal storage disorders. It also facilitates high-content screening approaches. For technical support, contact Ascent Research.
