CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

• Published in: EBioMedicine 2017-06, Vol.20 (C), p.19-26
• Main Authors: Yao, Xuan, Wang, Xing, Liu, Junlai, Hu, Xinde, Shi, Linyu, Shen, Xiaowen, Ying, Wenqin, Sun, Xinyao, Wang, Xin, Huang, Pengyu, Yang, Hui
• Format: Article
• Language: English
• Subjects: Animals; Biomarkers; Cell Line; CRISPR-Cas Systems; CRISPR/Cas9; Fah−/− mice; Female; Gene Targeting; Gene therapy; Gene Transfer Techniques; Genetic Engineering; Genetic Therapy; Genetic Vectors - genetics; Genotype; Hepatocytes - metabolism; Humans; Hydrolases - genetics; In vivo targeted integration; Male; Mice; Mice, Knockout; MMEJ; Neurons - metabolism; Phenotype; Recombinational DNA Repair; Research Paper
• ISSN: 2352-3964; 2352-3964
• DOI: 10.1016/j.ebiom.2017.05.015

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah−/− liver failure mice, offering an efficient approach for precisely targeted gene therapies. •MMEJ-based strategy showed much higher knock-in (up to 10 fold) efficiencies than HDR-based strategy in adult mouse tissues.•MMEJ-based strategy proved the efficacy in correction of Fah mutation and rescue of Fah−/− liver failure mice.