CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel haemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level 4 weeks after treatment, and the circulating FIX level is gradually increased up to 52% of the normal level over 9 months even after partial hepatectomy, demonstrating the amelioration of haemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for haemophilia B but also identifies a promising therapeutic approach for rare inherited diseases.