Lv Shihao, Xu Xiaokang, Yang Sijia, Feng Mingjie, Yuan Zhongyu, Liu Xueqing, Jiang Chaoqian, Song Jun, Mu Yanshuang
Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, Northeast Agricultural University, Harbin 150030, China.
College of Life Science, Northeast Agricultural University, Harbin 150030, China.
Biomolecules. 2025 Aug 6;15(8):1132. doi: 10.3390/biom15081132.
The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox's effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway.
CRISPR/Cas9基因组编辑系统已成为一个有效的平台，可通过非同源末端连接（NHEJ）在没有修复模板的情况下产生功能丧失的基因编辑。为了验证小分子是否能提高猪细胞中CRISPR/Cas9介导的NHEJ基因编辑效率，本实验研究了六种小分子化合物，即Repsox、齐多夫定、IOX1、GSK-J4、YU238259和GW843682X，对CRISPR/Cas9介导的NHEJ基因编辑效率的影响。结果显示了这些小分子（包括Repsox、齐多夫定、IOX1、GSK-J4、YU238259和GW843682X）对体外培养的PK15细胞活力的最佳浓度。与对照组相比，在Cas9-sgRNA核糖核蛋白递送系统中，单一小分子Repsox、齐多夫定、GSK-J4和IOX1分别使NHEJ介导的基因编辑效率提高了3.16倍、1.17倍、1.16倍和1.120倍。与对照组相比，使用YU238259和GW843682X没有益处。在CRISPR/Cas9质粒递送系统中，与对照组相比，Repsox、齐多夫定、IOX1和GSK-J4处理分别使NHEJ介导的基因编辑效率提高了1.47倍、1.15倍、1.21倍和1.23倍。Repsox还可以提高基于CRISPR sgRNA-tRNA阵列的NHEJ介导的多基因编辑效率。我们还探讨了Repsox对NHEJ介导的基因编辑效率产生影响的机制。结果表明，Repsox降低了TGF-β途径中SMAD2、SMAD3和SMAD4的表达水平，这表明Repsox可以通过TGF-β途径提高猪细胞中CRISPR NHEJ介导的基因编辑效率。
