Zhang Xiao-Jun, Xu Kun, Shen Jun-Cen, Mu Lu, Qian Hong-Run, Cui Jie-Yu, Ma Bao-Xia, Chen Zhi-Long, Zhang Zhi-Ying, Wei Ze-Hui
College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China.
Yi Chuan. 2022 Aug 20;44(8):708-719. doi: 10.16288/j.yczz.22-118.
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.
快速兴起的源自CRISPR的基因编辑技术已在生命科学、生物医药以及动植物育种领域广泛应用。然而，作为基因敲入和碱基编辑的一项重要策略，同源定向修复（HDR）的效率仍有待提高。在本研究中，我们提出了“供体适配系统”（DAS）这一术语，以概括那些经适配器修饰用于驱动适配体融合供体DNA的CRISPR/Cas9系统。我们的研究设计了一组CRISPR/Cas9-Gal4BD DAS。在该系统中，Gal4 DNA结合结构域（Gal4BD）用作适配器与Cas9蛋白融合，Gal4结合序列（Gal4BS）用作适配体与双链DNA（dsDNA）供体结合，以提高HDR效率。来自HEK293T-HDR.GFP报告细胞系的初步结果表明，当供体同源臂长度在一定范围（100 - 60 bp）时，HDR编辑效率可提高至2 - 4倍。进一步的优化结果表明，融合端口和融合接头的选择会影响Cas9的表达和活性，而带有GGS5接头的Cas9-Gal4BD融合是首选。此外，HDR效率可能取决于适配体-dsDNA供体的设计，建议在目标dsDNA模板的5'端添加单个Gal4BD结合序列（BS）。最后，我们使用CRISPR/Gal4BD-Cas9 DAS在AAVS1和EMX1内源性位点实现了增强的HDR编辑，我们相信该技术未来可应用于促进动物分子设计育种。
