The HERC5 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HAP1 near-haploid human cell line, engineered through CRISPR/Cas9-mediated disruption of the HERC5 gene. This heterogeneous knockout pool provides a robust loss-of-function model for studying HERC5-dependent ISGylation and innate immunity without the biases associated with clonal selection.
HAP1 is a near-haploid human cell line originating from the KBM-7 chronic myeloid leukemia line, extensively utilized in functional genomics owing to its haploid genomic content, which simplifies gene-editing interpretation. Its adherent growth, stable karyotype, and intact interferon signaling machinery render it a versatile host for investigating innate immune pathways and host?Cpathogen interactions.
HERC5 is an interferon-induced E3 ubiquitin-protein ligase that catalyzes ISGylation??the covalent attachment of the ubiquitin-like protein ISG15 to lysine residues on target proteins. Its expression is driven by type I interferons (IFN-??/??) through the JAK-STAT cascade, wherein activated STAT1, STAT2, and IRF9 assemble into the ISGF3 transcriptional complex to induce HERC5. Once produced, HERC5 functions in concert with the E1 enzyme UBE1L and the E2 enzyme UBCH8 to ISGylate a diverse array of substrates, including the signaling intermediates IRF3, JAK1, and STAT1, as well as numerous antiviral effectors. Through ISGylation, HERC5 regulates the stability, activity, and protein?Cprotein interactions of these targets, thereby modulating antiviral signaling strength and specificity. Additionally, HERC5 can exert ISGylation-independent effects by directly binding and influencing signaling proteins.
Knockout of HERC5 in HAP1 cells abolishes interferon-driven ISGylation, creating an ideal system to investigate the contribution of ISG15 conjugation to host antiviral defense and immune regulation. Because HAP1 cells possess a functional interferon response pathway, the knockout model permits side-by-side analysis of wild-type and HERC5-deficient signaling dynamics following viral infection or cytokine stimulation. This tool is particularly useful for dissecting HERC5-dependent regulation of the JAK-STAT?CIRF axis and for identifying novel ISGylation substrates via quantitative proteomics.
Representative research applications include Western blot detection of ISG15 conjugates, RT-qPCR analysis of interferon-stimulated gene expression, viral replication assays (e.g., with influenza virus, RSV, or herpes simplex virus), immunofluorescence microscopy to visualize ISG15 distribution, co-immunoprecipitation?Cmass spectrometry workflows for substrate discovery, RNA-seq transcriptome profiling, and flow cytometry for antiviral surface markers. These polyclonal knockout cells are suitable for functional genomics screening, host-targeted antiviral drug discovery, and mechanistic studies of cancer cell immunity. For further details, please contact Ascent Research.
