遗传 ›› 2022, Vol. 44 ›› Issue (8): 708-719.doi: 10.16288/j.yczz.22-118

• 研究报告 • 上一篇    下一篇

一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统

张潇筠(), 徐坤(), 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉()   

  1. 西北农林科技大学动物科技学院,杨凌 712100
  • 收稿日期:2022-04-05 修回日期:2022-05-11 出版日期:2022-08-20 发布日期:2022-05-23
  • 通讯作者: 徐坤,魏泽辉 E-mail:mshn15@163.com;weizehui7848@163.com;xukunas@nwafu.edu.cn
  • 作者简介:张潇筠, 在读硕士研究生,专业方向:动物生物技术。E-mail: mshn15@163.com
  • 基金资助:
    国家自然科学基金项目编号(32172736);,陕西省重点研发计划项目编号(2021NY-027)

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair

Zhang Xiaojun(), Xu Kun(), Shen Juncen, Mu Lu, Qian Hongrun, Cui Jieyu, Ma Baoxia, Chen Zhilong, Zhang Zhiying, Wei Zehui()   

  1. College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China
  • Received:2022-04-05 Revised:2022-05-11 Online:2022-08-20 Published:2022-05-23
  • Contact: Xu Kun,Wei Zehui E-mail:mshn15@163.com;weizehui7848@163.com;xukunas@nwafu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(32172736);the Shaanxi Key R&D Program(2021NY-027)

摘要:

近年来,CRISPR基因编辑及衍生技术迅速发展,在生命科学、生物医学研究以及动植物育种领域得到了广泛应用。基于DNA双链断裂(double-stranded break, DSB)同源指导修复(homology-directed repair, HDR)机制的基因敲入和点编辑是基因编辑的重要策略,但效率偏低亟待提高。本文提出了驱动供体DNA富集至DSB处以提高HDR效率的新策略,并设计了一套CRISPR/Cas9-Gal4BD 供体适配基因编辑系统(donor adapting system, DAS)。该系统主要利用Gal4 DNA结合域(Gal4 binding domain, Gal4BD)作为配体蛋白与Cas9融合表达,将Gal4BD结合序列(Gal4 binding sequence, Gal4BS)作为受体序列与双链DNA (double-stranded DNA, dsDNA)供体结合,以期提高HDR效率。使用HEK293T-HDR.GFP报告细胞系的初步研究结果表明当dsDNA供体同源臂在一定长度(100~60 bp)时该系统能够提高HDR效率2~4倍。进一步的优化研究表明,融合端口和融合使用连接子(linker)的选择会影响Cas9表达效果及活性,而GGS5作为Cas9-Gal4BD融合的连接子则影响较小。同时,本研究还发现Gal4BS-dsDNA供体的差异化设计也会影响HDR效率,将Gal4BS添加到dsDNA供体5′-端的效果最佳。综上所述,本研究利用CRISPR/Cas9-Gal4BD DAS在AAVS1EMX1位点上实现了HDR编辑效率的提高,为进一步利用该系统进行动物分子设计育种研究提供了参考和借鉴。

关键词: CRISPR/Cas9, 基因编辑, 供体DNA, 供体适配, 同源指导修复

Abstract:

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5′-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Key words: CRISPR/Cas9, gene editing, donor DNA, donor adapting, homology-directed repai