Overall, our CRISPR/Cas9 strategy to obtain FANCA-mutant clones from non-FA HNSCC cell lines gave adequate FA-HNSCC disease models as mutant clones, which (i) are hypersensitive to ICL-agents MMC and cisplatin, (ii) cannot monoubiquitinate FANCD2 and form nuclear foci upon MMC treatment, and (iii) display increased chromosome fragility and G2 arrest when treated with DEB. Here, FANCD2 is linked to head and neck squamous cell carcinoma.