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

作　　者：肖红卫[1] XIAO Hong-wei(Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding/Institute of Animal Husbandry and Veterinary Research,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)

机构地区：[1]动物胚胎工程与分子育种湖北重点实验室/湖北省农业科学院畜牧兽医研究所,武汉430064

出　　处：《湖北农业科学》2021年第23期177-180,共4页Hubei Agricultural Sciences

基　　金：湖北省农业科技创新中心项目(2019-620-000-001-20);湖北省农业科学院青年拔尖人才项目(Q2018020);湖北省农业科学院领军人才项目(L2018015);国家自然科学基金项目(31772577);湖南创新型省份建设专项(2019RS1068);重点领域研发计划(2020WK2030);重点实验室开放课题(KLAEMB-2020-01);外国青年人才计划项目(QN20200127002);中国科学院战略性先导科技专项(XDA24030204)。

摘　　要：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.

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 

分 类 号：Q819[生物学—生物工程]