2. Academic Validation
  3. Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion

Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion

  • Trends Biotechnol. 2025 Jul;43(7):1743-1764. doi: 10.1016/j.tibtech.2025.02.020.
Yafang Lu 1,  Jialu Wang 1,  Yilun Xu 2,  Mengli Xu 3,  Borui Li 1,  Zhan Fan 3,  Jinxin Liu 1,  Xinlin Li 1,  Zhenzhen Cai 2,  Yuanzhe Zheng 2,  Wenjing Wang 1,  Jie Yang 1,  Zhihong Zhang 4,  Zheng Liu 5
Affiliations

Affiliations

  • 1 Britton Chance Center and MoE Key Laboratory for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, Hubei 430074, China.
  • 2 State Key Laboratory of Digital Medical Engineering, School of Biomedical Engineering, Hainan University, Haikou, Hainan 570228, China.
  • 3 School of Life and Health Sciences, Key Laboratory of One Health of Hainan Province, Collaborative Innovation Center of One Health, Hainan University, Haikou, Hainan 570228, China.
  • 4 Britton Chance Center and MoE Key Laboratory for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; State Key Laboratory of Digital Medical Engineering, School of Biomedical Engineering, Hainan University, Haikou, Hainan 570228, China; School of Life and Health Sciences, Key Laboratory of One Health of Hainan Province, Collaborative Innovation Center of One Health, Hainan University, Haikou, Hainan 570228, China. Electronic address: zhzhang@hainanu.edu.cn.
  • 5 State Key Laboratory of Digital Medical Engineering, School of Biomedical Engineering, Hainan University, Haikou, Hainan 570228, China; School of Life and Health Sciences, Key Laboratory of One Health of Hainan Province, Collaborative Innovation Center of One Health, Hainan University, Haikou, Hainan 570228, China. Electronic address: liu-zheng@hainanu.edu.cn.
PMID: 40199626 DOI: 10.1016/j.tibtech.2025.02.020
Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-based targeted insertion of DNA fragments holds great promise for gene therapy. However, designing highly efficient and precise integration of large DNA segments in somatic cells while avoiding unpredictable products remains challenging. Here, we devised a novel long-offset paired nicking target integration (LOTI) strategy, which enhances the capacity of Cas9 nickase (Cas9n) in targeted gene integration in somatic cells, yielding higher knock-in (KI) efficiency compared with classical nickase-based approaches. The underlying repair mechanism involves the DNA repair proteins RAD51 and Rad52, and Ligase I/III. Moreover, we achieved efficient KI of at least 1.5-kb gene fragments in hepatocytes and recovery 55% FIX activity in a hemophilia B mouse model using only one-dose plasmid DNA delivery. Compared with the Cas9-based strategy, LOTI reduces off-target activity and restricts the formulation of unwanted insertions and deletions (indels) at the target site. Thus, LOTI provides a precise and efficient strategy for gene integration in somatic cells in vivo.

Keywords

Cas9 nickase; gene editing; in vivo; precise.

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