分子对接、CRISPR/Cas9技术筛选并验证DNA ligase Ⅳ抑制剂

Discovery of DNA ligase Ⅳ inhibitor through a docking-based screening and CRISPR/Cas9 study

为了筛选靶向DNA连接酶Ⅳ的抑制剂以实现更有效的基因定点插入,采用分子对接技术筛选出与前期研究中靶向DNA连接酶Ⅳ的抑制剂SCR7作用类似的小分子化合物。筛选到与化合物库一致的nocodazole、Fisetin和Methylene blue 3个小分子化合物,通过用MSTN基因T11位点序列截断的Firefly Luciferase荧光素酶报告载体结合CRISPR/Cas9-gRNA-T11载体共转细胞,并结合不同的小分子化合物处理。结果表明,没有用小分子化合物处理时,萤火虫荧光素酶被截断的位置发生NHEJ修复,不能得到大量有活性的萤火虫荧光素酶;经过小分子化合物处理,抑制了细胞内的NHEJ,提高了HDR效率,能得到大量有活性的萤火虫荧光素酶。使用nocodazole、Methylene blue和Fisetin处理后的萤火虫荧光素酶检测结果65256.3、53713和77058.3分别是SCR7处理后的萤火虫荧光素酶检测结果41905.3的1.6、1.3和1.8倍,是没有SCR7处理后的萤火虫荧光素酶检测结果10120的6.4、5.3和7.6倍。证明所用的筛选策略正确,所筛选出的小分子化合物是具有DNA ligaseⅣ抑制活性的抑制剂,为后续研究奠定了基础。

In order to screen for inhibitors targeting DNA ligaseⅣto 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 ligaseⅣin 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 65256.3,53713,and77058.3,respectively,which were 1.6,1.3 and 1.8 times of the firefly luciferase test result 41905.3 after SCR7 treatment.The results of firefly luciferase after SCR7 treatment were 6.4,5.3 and 7.6 times higher than 10120.The screening strategy used in this study is correct,and the small molecule compounds selected are inhibitors with DNA ligaseⅣinhibitory activity,laying the foundation for subsequent research.