CRISPR-Cas9-mediated homology-directed repair rescues the induced bone marrow failure in Fancc -/- mice.

Fanconi anemia (FA) is a rare recessive genetic disorder resulting from mutations in genes in the FA-DNA repair pathway. Among its subtypes, FA complementation group C (FANCC) is associated with particularly severe hematologic and developmental manifestations. Gene therapy targeting autologous hematopoietic stem/progenitor cells (HSPCs) from FA patients represents a promising curative strategy for FA-associated bone marrow failure (BMF), potentially circumventing the limitations of allogeneic transplantation. Despite substantial progress in developing gene therapy for FA group A (FANCA mutations), therapeutic strategies for FA group C have received comparatively little attention. Although FA-deficient cells are believed to exhibit compromised homologous recombination (HDR), our reporter assay demonstrated that Fancc -/- mouse HSPCs retain HDR activity, supporting the feasibility of precise gene editing. Building on this finding, we established a CRISPR-Cas9/AAV6-HDR platform to integrate Fancc cDNA into its endogenous locus while minimizing off-target effects and AAV integration. Correction of Fancc -/- HSPCs restored Fancc expression, rescued colony-forming capacity, and improved cellular viability. Importantly, transplantation of corrected HSPCs into Fancc -/- mice conferred resistance to mitomycin C-induced BMF, demonstrating durable in vivo functional correction. Collectively, these results establish CRISPR-Cas9-mediated HDR as a viable and potential therapeutic strategy for FA group C.