Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

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 hemophilia 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 four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases. Copyright (c) 2022, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

Automated summary

This study demonstrates the successful application of the HITI strategy for targeted gene integration in a hemophilia B rat model, leading to significant improvement in hemophilia symptoms over a nine-month period.