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

• FanclTAT∆ mice lack ubiquitin ligase activity, and demonstrate central role of monoubiquitination in Fanconi anemia phenotype

• CRISPR-Cas9 correction of FanclTAT restores monoubiquitination activity and serves as a model for testing therapeutic gene editing.

Fanconi anemia (FA) is a rare genetic disorder causing 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, as 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 (FanclTAT∆) that mimics a FA patient allele. This 3bp deletion removes a catalytic cysteine in the RING E3 ligase domain of the FANCL subunit. Biochemical assays show that the mutant FA core complex retains structural integrity but lacks FANCD2 monoubiquitination activity. Homozygous FanclTAT∆/TAT∆ 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.