The EHHADH Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population in which the EHHADH gene has been disrupted to abolish expression of the L-bifunctional protein. This pooled format provides a genetically heterogeneous knockout background, enabling robust loss-of-function studies without clonal selection artifacts. The polyclonal nature supports population-level analyses of peroxisomal fatty acid beta-oxidation pathway defects, making it a versatile tool for investigating metabolic dysfunction in a near-haploid human cellular context.
The parental HAP1 cell line is a near-haploid human fibroblast-like cell line originally derived from KBM-7 chronic myeloid leukemia cells. Its adherent morphology and stable near-haploid karyotype simplify gene editing and phenotype?Cgenotype correlations, as the presence of a single gene copy reduces genetic redundancy. This background is widely adopted in functional genomics screens, protein interaction studies, and signaling research, providing a clean genetic backdrop for dissecting the roles of individual genes such as EHHADH in lipid metabolism and peroxisomal biology.
EHHADH encodes the L-bifunctional protein, a peroxisomal enzyme that catalyzes the second and third steps of peroxisomal fatty acid beta-oxidation: enoyl-CoA hydration and 3-hydroxyacyl-CoA dehydrogenation. It operates within a multi-enzyme complex containing ACOX1, HSD17B4, and PEX5/PEX7 import receptors. Upstream, its expression is transcriptionally regulated by PPAR?? in response to fatty acids and reactive oxygen species. Downstream, its activity generates shortened acyl-CoA species, Acetyl-CoA, NADH, and bile acid intermediates, which feed into mitochondrial oxidation and other metabolic pathways. Disruption of EHHADH therefore impairs peroxisomal degradation of very long-chain fatty acids, branched-chain fatty acids, and bile acid precursors, leading to substrate accumulation and metabolic imbalance.
In HAP1 cells, loss of EHHADH effectively recapitulates key biochemical hallmarks of peroxisomal disorders such as Zellweger spectrum disorder and renal Fanconi syndrome. The model permits direct interrogation of how very long-chain fatty acid accumulation impacts lipid homeostasis, organelle crosstalk, and cell viability under lipotoxic stress. Because HAP1 cells retain functional peroxisomes and mitochondrial fatty acid oxidation, this knockout enables dissection of the compartment-specific contributions of peroxisomal beta-oxidation versus mitochondrial oxidation, offering insights into metabolic rewiring caused by EHHADH deficiency.
Researchers can deploy this polyclonal knockout population in targeted assays: very long-chain fatty acid accumulation by LC-MS, fatty acid oxidation flux assays, western blotting for EHHADH and interacting partners, immunofluorescence for peroxisomal proteins, and metabolic flux analysis. The model is suited for drug screening to restore peroxisomal function or bypass metabolic blocks, and for CRISPR modifier screens to identify synthetic lethal interactions or compensatory pathways. For further details and technical support, please contact Ascent Research.
