The C9orf40 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population generated from the HAP1 cell line, targeting the C9orf40 gene that encodes the cilia- and flagella-associated protein CFAP96. This polyclonal pool comprises cells with diverse gene disruptions, offering a robust loss-of-function model without single-clone isolation. CRISPR/Cas9-mediated targeting introduces genetic alterations across the population, ensuring a comprehensive assessment of C9orf40 function.
The host HAP1 cell line is a near-haploid human cell line derived from the chronic myeloid leukemia KBM-7 line, exhibiting fibroblast-like adherent morphology. Originating from a male donor, HAP1 cells are BCR-ABL positive and have lost functional p53; however, these characteristics do not impede their widespread use in genetic studies. The haploid status is particularly advantageous for knockout experiments, as disruption of a single allele can unmask recessive phenotypes, eliminating the redundancy present in diploid cells and facilitating clear genotype-phenotype relationships.
C9orf40, also designated CFAP96, functions as a critical structural component of the sperm flagellar axoneme, where it interacts with tubulin, dynein motors, and radial spoke proteins to orchestrate microtubule assembly and stabilization. Its expression is transcriptionally controlled by RFX family transcription factors (RFX2 and RFX3) and FOXJ1 during ciliogenesis, and by CREM in spermatogenesis. Downstream, CFAP96 promotes the formation of outer dynein arms and radial spoke components, integral to the axonemal 9+2 architecture and the nexin-dynein regulatory complex. Disruption of CFAP96 leads to disorganized axonemal structures and severely reduced sperm motility, phenotypic of multiple morphological abnormalities of the sperm flagella (MMAF).
In the HAP1 cellular context, knockout of C9orf40 provides a simplified genetic background to dissect molecular mechanisms underlying ciliary and flagellar assembly. Although HAP1 cells are not constitutively ciliated, serum starvation induces primary cilium formation, enabling visualization of ciliary markers such as acetylated tubulin and Arl13b via immunofluorescence. The haploid nature amplifies phenotypic effects, while the polyclonal composition averages out clonal variability, making this population ideal for genetic modifier screens and pharmacological intervention studies.
Research applications of this model span male infertility mechanism investigation through functional rescue experiments, co-immunoprecipitation to map interactions with CFAP family proteins and dynein subunits, and transcriptomic profiling by RNA-seq to identify downstream targets. Complementary techniques include western blotting, RT-qPCR, and proximity ligation assays for detailed protein analysis. This model aids basic and translational research in ciliopathy and reproductive biology. For further information, please contact Ascent Research.
