CA5B Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the CA5B gene in the HAP1 haploid human cell line. This pool of genetically heterogeneous knockout cells enables loss-of-function studies of mitochondrial carbonic anhydrase 5B, an enzyme critical for CO2 hydration and bicarbonate production. The polyclonal format provides a robust system for studying pH regulation, metabolism, and disease states such as metabolic acidosis and urea cycle disorders, without clonal selection bias.
HAP1 cells, derived from the KBM-7 chronic myeloid leukemia line, are a near-haploid human cell line extensively used in genetic research. The haploid karyotype simplifies genotype-phenotype associations, enabling clear functional readouts in knockout and screening experiments. HAP1 retains features of the parental leukemia line, offering a relevant context for cancer metabolism and fundamental cell biology studies. This host system positions the CA5B knockout for dissecting mitochondrial carbonic anhydrase contributions in both physiology and disease.
CA5B encodes a mitochondrial carbonic anhydrase that catalyzes CO2 hydration to bicarbonate, critical for matrix pH and metabolic pathways. Its transcription is activated by PPARGC1A, HIF1A, and SP1. CA5B interacts with carbamoyl phosphate synthetase 1 (CPS1) and pyruvate carboxylase (PC), providing bicarbonate for the urea cycle and gluconeogenesis. Disruption impairs CPS1-mediated ammonia detoxification and PC-dependent anaplerosis, causing urea cycle dysfunction, reduced gluconeogenesis, and metabolic acidosis.
In the HAP1 haploid background, loss of CA5B provides a clean platform to study mitochondrial carbonic anhydrase function. The resulting metabolic vulnerabilities in ammonia handling and anaplerosis are relevant to cancer cells dependent on glutamine. The polyclonal population suits high-throughput screening of carbonic anhydrase inhibitors. The leukemia origin also enables investigation of tumor-specific metabolic adaptations.
Applications include characterization of CA5B loss via Western blotting, RT-qPCR, and carbonic anhydrase activity assays. Metabolic consequences can be assessed using Seahorse flux analysis, urea cycle metabolite profiling, and mitochondrial pH measurements, supporting studies of metabolic acidosis, urea cycle disorders, and inhibitor screening. This model also enables cancer metabolism research, probing the role of mitochondrial CO2 hydration in tumor cell proliferation. For further information, custom engineering, or bulk orders, please contact Ascent Research.
