Generation of NBS1 knockout in Chinese hamster cells revealed ATR role for radiation and etoposide induced DNA damage in absence of NBS1 proteins.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive disorder characterized by chromosomal instability, immunodeficiency, radiosensitivity, and a strong predisposition to lymphoid malignancies. It is caused by mutations in the NBN gene encoding nibrin (NBS1) protein, a core component of the MRE11-RAD50-NBS1 (MRN) complex that senses DNA double-strand breaks (DSBs) and coordinates DNA damage response, including ATM activation. Despite the importance of NBS1, in the Chinese hamster system, which offers significant advantages in radiation biology and toxicology, no mutant lines deficient in the NBS1 gene have been isolated. In this study, we generated two novel NBS1 mutant Chinese hamster cell lines using CRISPR/Cas9, each carrying distinct NBN mutations leading to either null or hypomorphic mutations. These mutants exhibited growth retardation, marked sensitivity to ionizing radiation and various DNA damaging agents and elevated radiation induced chromosomal aberrations, recapitulating key NBS phenotypes. Notably, NBS1 mutant cells displayed pronounced hypersensitivity to ionizing radiation when co-treated with an ATR inhibitor, but not with a DNA-PK inhibitor. The ATR inhibitor also markedly sensitized NBS1 mutants to Etoposide, suggesting that ATR functions as a compensatory pathway in the absence of functional NBS1 during specific types of DNA damage. Collectively, our findings establish valuable NBS1-deficient Chinese hamster cell models that expand understanding of NBS1 function and highlight their utility for investigating DNA repair deficiencies and developing targeted therapeutic approaches for chromosomal instability disorders and cancers with NBS1 mutations.