The HADHA Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population with disrupted HADHA, creating a loss-of-function model for the mitochondrial trifunctional protein (MTP) ??-subunit. Derived from the human HAP1 cell line, this heterogeneous knockout pool is ideal for pooled functional screens and metabolic studies, circumventing clonal selection artifacts.
HAP1 is a near-haploid, fibroblast-like human cell line originating from KBM-7 chronic myeloid leukemia cells. Its haploid genome simplifies CRISPR-based knockout generation and ensures complete gene disruption, while retaining key metabolic pathways functional for mitochondrial and lipid metabolism research.
HADHA encodes the ??-subunit of MTP, which with HADHB (??-subunit) catalyzes long-chain fatty acid ??-oxidation??s final steps: enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. Transcriptionally controlled by PPAR?? and PGC-1??, and regulated by AMPK and insulin/glucagon, HADHA disruption abolishes ??-oxidation, causing long-chain acylcarnitine accumulation, reduced ketogenesis, and ATP depletion. SREBP1c is activated as a compensatory lipogenic response. MTP assembly requires HSP60 and operates downstream of carnitine shuttle (CPT1/CPT2) and upstream of electron transfer flavoprotein and respiratory chain.
In HAP1 haploid cells, HADHA knockout yields a clean loss-of-function phenotype, modeling mitochondrial trifunctional protein deficiency disorders such as acute fatty liver of pregnancy, HELLP syndrome, cardiomyopathy, and hypoglycemia. The absence of wild-type alleles ensures unambiguous interpretation of metabolic flux changes. These cells exhibit heightened sensitivity to fatty acid overload and glucose deprivation, enabling dissection of metabolic crisis mechanisms and lipid-induced cellular stress.
Applications include modeling fatty acid oxidation diseases, screening metabolic modulators, studying cancer lipid metabolism, and assessing mitochondrial function. Commonly used assays: HADHA western blot, RT-qPCR, fatty acid oxidation flux with labeled palmitate, acylcarnitine LC-MS profiling, ATP bioluminescence, Seahorse respirometry, lipid droplet staining (Nile Red/BODIPY), and viability under metabolic stress. These cells are also suitable for genetic screens and drug synergy studies. For further information, contact Ascent Research.
