High-efficiency targeted transgene integration via primed micro-homologues

Cell Discovery

Article

Qingtong Shan¹  Sen Fang²  Guanyi Jiao²  Yangcan Chen²  Xin Wang²  Yanping Hu²  Jing Li²  Qi Zhou³  Guihai Feng³  Na Tang³  Chenxin Wang³  Wei Li³ 

[1] Northeast Agricultural University, Harbin, China;State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China;Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China;University of Chinese Academy of Sciences, Beijing, China;State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China;Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, China;University of Chinese Academy of Sciences, Beijing, China;Bejing Institute for Stem Cell and Regenerative Medicine, Beijing, China;

关键词: ;   

DOI  :  10.1038/s41421-023-00552-0

 received in 2022-10-25, accepted in 2023-04-03,  发布年份 2023

来源: Springer

【 摘 要 】

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

【 授权许可】

CC BY   
© The Author(s) 2023

【 图 表 】

Fig. 1

Fig. 2

Fig. 2

Fig. 11

41116_2023_38_Article_IEq163.gif