CRISPR/Cas9-mediated disruption of the ITPA gene in HAP1 cells yields a polyclonal knockout population designed for loss-of-function studies in purine metabolism and genomic stability. This product provides a heterogeneous pool of gene-edited near-haploid human cells in which ITPA function is abrogated, enabling researchers to investigate the consequences of inosine triphosphate pyrophosphatase deficiency without the confounding effects of clonal selection or defined editing outcomes. The polyclonal format preserves the genetic diversity inherent to population-level responses, making it particularly suitable for drug sensitivity screens and assays that benefit from a range of knockout efficiencies across the cell pool.
The host HAP1 cell line is a near-haploid chronic myeloid leukemia-derived model with a karyotype of approximately one copy per chromosome, originally derived from the KBM-7 line. It carries the BCR-ABL1 fusion oncogene, which drives constitutive tyrosine kinase activity and proliferation. The near-haploid genomic architecture simplifies genetic manipulation and reduces functional redundancy, facilitating straightforward genotype-phenotype association in knockout screens. This background has been widely adopted for haploid genetic screening, CRISPR-based hit identification, and investigation of signaling pathways in a leukemia-relevant context.
ITPA encodes inosine triphosphate pyrophosphatase, which functions as a homodimer to hydrolyze the non-canonical nucleotides inosine triphosphate (ITP) and deoxyinosine triphosphate (dITP) into inosine monophosphate (IMP) and deoxyinosine monophosphate (dIMP). This enzymatic activity is essential for preventing the accumulation and misincorporation of these aberrant nucleotides into RNA and DNA, thereby maintaining nucleotide pool fidelity and preventing mutagenesis. ITPA expression is constitutively regulated, with the transcription factor NRF2 (NFE2L2) acting as a key upstream regulator. Downstream, the generated IMP integrates into the purine salvage pathway through enzymes such as IMP dehydrogenase (IMPDH), adenylosuccinate lyase, and hypoxanthine-guanine phosphoribosyltransferase (HPRT1), supporting balanced nucleotide homeostasis. Disruption of ITPA leads to elevated ITP and dITP levels, triggering DNA damage signaling, increased mutagenic potential, and hypersensitivity to thiopurine drugs like 6-mercaptopurine.
In the HAP1 leukemia background, loss of ITPA creates a relevant model for studying the intersection of purine metabolism defects with BCR-ABL1-driven signaling. The near-haploid state amplifies the phenotypic consequences of single-gene knockout, making it easier to detect subtle effects on nucleotide pools, energy metabolism, and drug sensitivity. This system allows for precise dissection of how ITPA deficiency modulates DNA damage responses and oxidative stress, particularly in the context of thiopurine-based therapies that are commonly used in hematological malignancies. The polyclonal nature captures a spectrum of editing outcomes, reflecting the heterogeneous behavior often observed in patient samples.
Researchers can employ this knockout model for a variety of applications, including quantitative nucleotide pool analysis by HPLC, enzymatic activity assays to measure residual ITPase function, and comet assays to assess DNA damage accumulation. Drug sensitivity studies with 6-mercaptopurine in combination with cell viability and ROS detection assays can elucidate mechanisms of thiopurine toxicity and resistance. Additionally, the cells serve as a platform for investigating ITPA deficiency syndrome, developmental and epileptic encephalopathy, and azathioprine sensitivity. This product provides a robust and versatile tool for advancing understanding of purine metabolism and its implications in disease and therapy. For additional information, please contact Ascent Research.
