The DNAAF2 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population targeting the DNAAF2 gene. This heterogeneous pool of HAP1 cells harbors various gene disruptions, generating a loss-of-function model of the cytoplasmic preassembly factor essential for outer dynein arm formation. The polyclonal format captures a spectrum of editing outcomes, avoiding clone-specific artifacts and enabling robust functional screening.
The parental HAP1 cell line is a near-haploid human model derived from the CML KBM-7 line, carrying the BCR-ABL1 fusion oncogene and disomy for chromosome 8. This karyotype simplifies genetic studies, and the cells?? ability to form primary cilia upon serum starvation renders them suitable for ciliogenesis research despite their hematopoietic origin.
DNAAF2 acts as a cytoplasmic co-chaperone, collaborating with the R2TP?CHSP90 network and DNAAF1 (LRRC50) to promote assembly of outer dynein arm complexes. It facilitates folding and stabilization of heavy chains (DNAH5, DNAH11), intermediate chains (DNAI1, DNAI2), and light chains before ciliary transport. Transcription of DNAAF2 is regulated by ciliogenic factors including FOXJ1, RFX3, and Multicilin, with influence from Notch signaling. Loss of DNAAF2 disrupts dynein arm preassembly, leading to immotile cilia, impaired mucociliary clearance, and randomized left-right asymmetry, as seen in primary ciliary dyskinesia and Kartagener syndrome.
In the near-haploid HAP1 background, DNAAF2 knockout eliminates functional redundancy, providing clear loss-of-function phenotypes. The BCR-ABL1 signaling context may offer opportunities to explore intersections between oncogenic pathways and ciliary biology. Inducible ciliogenesis by serum starvation allows direct evaluation of ciliary motility, axonemal ultrastructure, and protein localization using high-speed microscopy and electron microscopy.
This model supports diverse applications including PCD modeling, genetic modifier screening, validation of DNAAF2-interacting partners (e.g., DNAAF1, SPAG1, ZMYND10), and high-throughput screening for agents that restore ciliary motility. Recommended analytical readouts include Sanger sequencing, western blotting, RT-qPCR, immunofluorescence for ciliary markers, co-immunoprecipitation of assembly complexes, and ciliary beat frequency assays. For additional information, contact Ascent Research.
