关键词: DNA连接酶IV, 抑制剂, 荧火虫荧光素酶报告载体, CRISPR/Cas9, 小分子化合物

Abstract: In order to screen for inhibitors targeting DNA ligase IV to achieve more effective gene-directed insertion, molecular docking technology was used to screen out small molecule compounds with similar effects to the inhibitor SCR7 targeting DNA ligase IV in the previous study. In this study, three small molecule compounds, nocodazole, Fisetin and Methylene blue, which are consistent with the compounds stored in this unit, were screened. By using the truncated Firefly Luciferase luciferase reporter vector by the T11 site sequence of the MSTN gene and CRISPR/Cas9-gRNA-T11 vector to co-transform cells, combined with different small molecule compound treatments. The results showed that without treatment with small molecule compounds, NHEJ repair occurs at the truncated position of firefly luciferase, and a large amount of active firefly luciferase cannot be obtained; after treatment with small molecule compounds, the intracellular NHEJ is inhibited, and the HDR efficiency was improved. A large amount of active firefly luciferase was obtained. The firefly luciferase test results after treatment with nocodazole, Methylene blue and Fisetin were 65 256.3, 53 713, and 77 058.3, respectively, which were 1.6, 1.3 and 1.8 times of the firefly luciferase test result 41 905.3 after SCR7 treatment. The results of firefly luciferase after SCR7 treatment were 6.4, 5.3 and 7.6 times higher than 10 120. The screening strategy used in this study is correct, and the small molecule compounds selected are inhibitors with DNA ligase IV inhibitory activity, laying the foundation for subsequent research.

Key words: DNA ligase IV, inhibitor, Firefly Luciferase reportor, CRISPR/Cas9, small molecule compound