AMACR Knockout HAP1 Polyclonal Cells are a polyclonal knockout cell population produced by CRISPR/Cas9-mediated disruption of the AMACR gene in the near-haploid HAP1 human fibroblast-like cell line. This loss-of-function model enables the study of AMACR-dependent metabolic processes without clonal bias, suitable for pooled screening and functional assays.
The HAP1 cell line is derived from KBM-7 chronic myeloid leukemia cells and maintains a stable near-haploid karyotype. This haploid background facilitates unambiguous gene targeting and CRISPR-based knockout validation, making HAP1 a robust platform for genetic screening and functional genomic studies.
AMACR encodes alpha-methylacyl-CoA racemase, a peroxisomal enzyme essential for the racemization of (R)-alpha-methyl branched-chain fatty acyl-CoA esters to (S)-isomers, a prerequisite for peroxisomal beta-oxidation and primary bile acid synthesis. Its expression is transcriptionally regulated by PPARA, androgen receptor signaling, and SREBF1. Following import into peroxisomes via interaction with PEX5 and PEX7, AMACR converts substrates such as pristanoyl-CoA and di- and trihydroxycholestanoyl-CoA. The resulting (S)-isomers are then catabolized by peroxisomal beta-oxidation enzymes including ACOX1 and DBP, yielding acetyl-CoA and propionyl-CoA, and further processed by CYP27A1 in bile acid synthesis. Disruption of AMACR impairs peroxisomal lipid metabolism and bile acid production, leading to accumulation of branched-chain fatty acid intermediates and altered cellular energy homeostasis.
In the HAP1 cellular context, AMACR knockout provides a genetically clean system to investigate peroxisomal metabolic defects. The near-haploid genome ensures that the knockout allele directly manifests as functional loss without allelic compensation, recapitulating aspects of alpha-methylacyl-CoA racemase deficiency. Given AMACR’s overexpression in prostate and colorectal cancers, this model also enables studies of its role in cancer cell metabolism and proliferation.
This polyclonal knockout population is applicable to prostate cancer biomarker validation, peroxisomal disorder modeling, and metabolic pathway dissection. Typical assays include enzyme activity assays with pristanoyl-CoA, fatty acid oxidation flux analysis, viability assays under lipid-rich conditions, and target gene disruption confirmation by western blotting or RT-qPCR. The heterogeneous population is compatible with pooled CRISPR screens and drug discovery efforts targeting peroxisomal function.
