Fancl-mutant mice reveal central role of monoubiquitination in Fanconi anemia and a model for therapeutic gene editing.

Fanconi anemia (FA) is a rare genetic disorder causing the progressive loss of hematopoietic stem cells (HSCs) and bone marrow failure. Most cases result from deficient monoubiquitination of FANCD2 by the FA core complex. However, given that additional functions for the complex have been proposed, it remains unclear whether loss of FANCD2 monoubiquitination is the sole cause of all FA phenotypes. Here, we generated a murine allele (FanclTATΔ) that mimics an allele from a patient with FA. This 3-bp deletion removes a catalytic cysteine in the E3 RING ligase domain of the FANCL subunit. Biochemical assays show that the mutant FA core complex retains structural integrity but lacks FANCD2 monoubiquitination activity. Homozygous FanclTATΔ/TATΔ mice phenocopy classical human FA features, including infertility, craniofacial anomalies, DNA damage hypersensitivity, and progressive HSC loss with age. Correcting the mutation using CRISPR-Cas9 or prime editing technology restores FANCD2 monoubiquitination and normal DNA damage resistance in myeloid cells. Collectively, our mouse model demonstrates that loss of RING E3 ubiquitin ligase activity of the FA core complex explains developmental defects and hematopoietic failure in FA and provides a new animal model for testing potentially therapeutic gene editing.