GRAMD1A Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited population of HAP1 cells with targeted disruption of the GRAMD1A gene, creating a loss-of-function model for cholesterol transport and calcium signaling studies. The polyclonal knockout format provides a heterogeneous mixture of edited cells, suitable for pooled functional screens and population-based assays where clonal homogeneity is not required. This approach allows researchers to assess bulk population effects of GRAMD1A loss without isolating single-cell clones.
The HAP1 cell line is a suspension-adapted, near-haploid human cell line derived from the chronic myeloid leukemia cell line KBM-7. It originated from a male patient and retains a stable haploid karyotype except for a single chromosome 8 disomy. HAP1 cells lack wild-type p53, which facilitates high gene-editing efficiencies and robust selection. Their hematopoietic background and suspension growth enable scalable, automated screening workflows widely used in functional genomics and drug discovery.
GRAMD1A (GRAM domain containing 1A) operates as an ER-anchored cholesterol sensor and transporter that mediates non-vesicular cholesterol movement from the plasma membrane to the ER at contact sites. Its GRAM domain recognizes accessible cholesterol, triggering interaction with VAPA and VAPB and promoting cholesterol esterification and SREBP2 activation to link lipid sensing with transcription. Separately, GRAMD1A interacts with STIM1 to hydrolyze PI(4,5)P2, thereby regulating store-operated calcium entry via ORAI1. Upstream regulators include plasma membrane cholesterol, LXR agonists, oxysterols, and STIM1 activation, while downstream targets involve AKT phosphorylation and calcium influx. The protein also binds NPC1 and OSBP, integrating cholesterol trafficking with broader lipid exchange networks.
In HAP1 cells, GRAMD1A knockout exploits the near-haploid genome for straightforward genotype-phenotype correlations, minimizing interference from wild-type alleles. Although p53 deficiency may modulate metabolic stress responses, it enhances editing efficiency, enabling robust population-level analyses. This model is particularly valuable for dissecting how cholesterol sensing intersects with calcium dynamics in hematopoietic contexts, where lipid metabolic reprogramming often supports malignant proliferation. The polyclonal composition captures a range of editing efficiencies, facilitating dose-response or competitive fitness studies.
Researchers can apply these cells in cholesterol efflux and filipin staining assays to assess cellular cholesterol distribution, and calcium imaging with Fura-2 or fluo-4 to monitor store-operated calcium entry. Co-immunoprecipitation and immunofluorescence localize STIM1 and its partners at membrane contact sites. Complementary readouts include lipidomics, gene expression analysis of SREBP2 targets like HMGCR and LDLR, and flow cytometry with perfringolysin O binding. These tools support investigations into atherosclerosis, Alzheimer’s disease, Niemann-Pick type C, metabolic syndrome, and cancer, with drug screening for cholesterol modulators. For further details, please contact Ascent Research.
