CRISPR/Cas9-mediated B2m knockout paves the way for allogeneic basal cell transplantation.

BACKGROUND: Autologous transplantation of basal cells (BCs) has shown promise in treating respiratory diseases, but disease-specific subpopulations among BCs probably diminish the treatment efficacy. An alternative approach involves generating universal and healthy BCs, which offers a potentially more efficient and accessible solution for avoiding using abnormal BCs. However, such hypoimmunogenic BCs have not yet been transplanted into the airways of immunocompetent animals. METHODS: Before producing hypoimmunogenic BCs, the predominant transplantation antigen in BCs was explored through RT-qPCR and flow cytometry to identify the key target of CRISPR/Cas9‐mediated editing. The proliferation and expression of specific markers of BCs were evaluated after gene editing by CCK‐8 and RT‐qPCR, respectively. These gene‐edited BCs and wild‐type (WT) BCs, which were both derived from the same BALB/c mouse, were subsequently allogeneically transplanted into C57BL/6 mice with polidocanol‐induced airway injury to evaluate the differentiation and immune response in the recipient mice via immunehistological staining. RESULTS: In the present study, it was demonstrated that major histocompatibility complex class I (MHC-I) is the predominant transplantation antigen in BCs. The hypoimmunogenic BCs were generated through editing beta‐2 microglobulin (B2m) participating in the encoding of MHC‐I. The knockout of B2m in BCs did not affect their proliferation or the expression of specific markers in vitro. Both WT BCs and B2m‐edited BCs (B2m‾ BCs) successfully differentiated into ciliated and secretory cells in the tracheas following allogeneic transplantation and did not elicit an immune response during the 26‐day observation period in the tracheas. However, WT BCs, compared to B2m‾ BCs, induced severe lung injury by provoking an immune response in the lower airways and alveolar regions, as indicated by increased infiltration of CD45(+) immune cells, epithelial cell shedding in the bronchi, and obvious alveolar hyperemia with collapse. CONCLUSIONS: The hypoimmunogenic BCs generated through CRISPR/Cas9-mediated B2m gene editing retained their ability to differentiate and maintained viability in the allogeneic respiratory system, supporting the potential application in the cell regeneration therapy of airway diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12931-026-03595-5.