College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China.
Yi Chuan. 2024 Sep;46(9):716-726. doi: 10.16288/j.yczz.24-124.
Targeted precise point editing and knock-in can be achieved by homology-directed repair(HDR) based gene editing strategies in mammalian cells. However, the inefficiency of HDR strategies seriously restricts their application in precision medicine and molecular design breeding. In view of the problem that exogenous donor DNA cannot be efficiently recruited autonomously at double-stranded breaks(DSBs) when using HDR strategies for gene editing, the concept of donor adapting system(DAS) was proposed and the CRISPR/Cas9-Gal4BD DAS was developed previously. Due to the large size of SpCas9 protein, its fusion with the Gal4BD adaptor is inconvenient for protein expression, virus vector packaging and delivery. In this study, two novel CRISPR/Gal4BD-SlugCas9 and CRISPR/Gal4BD-AsCas12a DASs were further developed, using two miniaturized Cas proteins, namely SlugCas9-HF derived from and AsCas12a derived from sp Firstly, the SSA reporter assay was used to assess the targeting activity of different Cas-Gal4BD fusions, and the results showed that the fusion of Gal4BD with SlugCas9 and AsCas12a N-terminals had minimal distraction on their activities. Secondly, the HDR efficiency reporter assay was conducted for the functional verification of the two DASs and the corresponding donor patterns were optimized simultaneously. The results demonstrated that the fusion of the Gal4BD adaptor binding sequence at the 5'-end of intent dsDNA template (BS-dsDNA) was better for the CRISPR/Gal4BD-AsCas12a DAS, while for the CRISPR/Gal4BD-SlugCas9 DAS, the dsDNA-BS donor pattern was recommended. Finally, CRISPR/Gal4BD-SlugCas9 DAS was used to achieve gene editing efficiency of 24%, 37% and 31% respectively for and gene loci in HEK293T cells, which was significantly increased compared with the controls. In conclusion, this study provides a reference for the subsequent optimization of the donor adapting systems, and expands the gene editing technical toolbox for the researches on animal molecular design breeding.
通过同源定向修复（HDR）的基因编辑策略，可在哺乳动物细胞中实现靶向精确点编辑和基因敲入。然而，HDR 策略的低效率严重限制了其在精准医学和分子设计育种中的应用。鉴于在使用 HDR 策略进行基因编辑时，双链断裂（DSB）处无法有效地自主募集外源供体 DNA 的问题，人们提出了供体适应系统（DAS）的概念，并先前开发了 CRISPR/Cas9-Gal4BD DAS。由于 SpCas9 蛋白的体积较大，其与 Gal4BD 接头融合不利于蛋白质表达、病毒载体包装和递送。在本研究中，使用两种小型化的 Cas 蛋白（即源自 和 的 SlugCas9-HF 和 AsCas12a），进一步开发了两种新型的 CRISPR/Gal4BD-SlugCas9 和 CRISPR/Gal4BD-AsCas12a DAS。首先，使用 SSA 报告基因分析评估了不同 Cas-Gal4BD 融合体的靶向活性，结果表明 Gal4BD 与 SlugCas9 和 AsCas12a N 端的融合对其活性的干扰最小。其次，进行 HDR 效率报告基因分析以同时验证这两种 DAS 的功能并对相应的供体模式进行优化。结果表明，Gal4BD 接头结合序列融合到意图双链 DNA 模板（BS-dsDNA）的 5'端更有利于 CRISPR/Gal4BD-AsCas12a DAS，而对于 CRISPR/Gal4BD-SlugCas9 DAS，推荐使用 dsDNA-BS 供体模式。最后，在 HEK293T 细胞中，CRISPR/Gal4BD-SlugCas9 DAS 分别实现了 和 基因座的基因编辑效率为 24%、37%和 31%，与对照组相比显著提高。总之，本研究为随后的供体适应系统优化提供了参考，并为动物分子设计育种的研究扩展了基因编辑技术工具包。
