Chen Xi, Niu Xuran, Liu Yang, Zheng Rui, Yang Lei, Lu Jian, Yin Shuming, Wei Yu, Pan Jiahao, Sayed Ahmed, Ma Xueyun, Liu Meizhen, Jing Fengxiang, Liu Mingyao, Hu Jiazhi, Wang Liren, Li Dali
Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
J Genet Genomics. 2022 Dec;49(12):1114-1126. doi: 10.1016/j.jgg.2022.06.001. Epub 2022 Jun 9.
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.
CRISPR/Cas9介导的外源基因位点特异性插入具有临床应用潜力。然而，由于同源重组（HR）效率低下，尤其是对于非分裂细胞而言，目前仍不可行。为了克服这一挑战，我们报告了一种不依赖同源性的靶向整合（HITI）策略，该策略用于在一种新型B型血友病（HB）大鼠模型的白蛋白（Alb）基因座上永久整合高特异性活性因子IX变体（F9 Padua，R338L）。通过液滴数字PCR（ddPCR）测定，敲入效率达到3.66%。治疗四周后，凝血时间降至正常水平，即使在部分肝切除术后，循环因子IX（FIX）水平在九个月内也逐渐升高至正常水平的52%，证明了血友病症状得到改善。通过引物延伸介导的测序（PEM-seq），未检测到明显的脱靶效应。本研究不仅为B型血友病提供了一种新型模型，还确定了一种针对罕见遗传性疾病的有前景的治疗方法。
