ACSF3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-mediated gene-disrupted polyclonal cell population designed for loss-of-function studies of ACSF3 in a near-haploid background. This pool comprises HAP1 cells harboring heterogeneous ACSF3 disruptions, providing a robust model for investigating mitochondrial malonate metabolism without clonal selection bias.
The HAP1 cell line is a male, fibroblast-like, near-haploid derivative of the KBM-7 chronic myeloid leukemia cell line, exhibiting a mosaic karyotype including disomy for chromosome 8 and a 15q;11q translocation. Its near-haploid genome simplifies knockout generation, as a single disruption can abrogate gene function, making HAP1 a versatile platform for CRISPR-based functional genomics in cancer and metabolic research.
ACSF3 encodes a mitochondrial acyl-CoA synthetase that activates malonate and methylmalonate to malonyl-CoA and methylmalonyl-CoA, respectively. These products are essential substrates for mitochondrial fatty acid synthesis (mtFAS), which generates octanoyl-ACP??the precursor for lipoic acid biosynthesis. Transcription of ACSF3 is regulated by the PPARGC1A?CNRF1?CTFAM cascade. Within mtFAS, ACSF3 products are utilized by MCAT, a component of a complex that includes OXSM, MECR, and the acyl carrier protein NDUFAB1. Lipoic acid attachment by LIAS to the pyruvate dehydrogenase complex and other dehydrogenases critically depends on ACSF3 activity. Therefore, ACSF3 disruption blocks mtFAS and downstream lipoylation.
In the HAP1 context, ACSF3 knockout accumulation of malonate and methylmalonate recapitulates hallmark metabolic imbalances of combined malonic and methylmalonic aciduria (CMAMMA). This model permits direct interrogation of mtFAS defects and their impact on lipoic acid-dependent enzyme function, particularly pyruvate and ??-ketoglutarate dehydrogenase complexes. The polyclonal population supports population-level metabolic flux analyses and genetic interaction screens.
Applications include functional dissection of mtFAS, CMAMMA disease modeling, metabolic tracing with stable isotopes, and drug screening for metabolic disorders. Typical assays encompass Western blotting for ACSF3 and lipoylation status, RT-qPCR, LC-MS metabolomics of malonate and methylmalonate, enzyme activity assays, Seahorse respirometry, mitochondrial immunofluorescence, and complementation with wild-type ACSF3. For custom inquiries, contact Ascent Research.
