Abstract: 【Aim】 To screen and detect the regulation of histone methylation modification in 20-hydroxyecdysone (20E) signaling transduction by CRISPR/Cas9 knockout system in Drosophila melanogaster cells. 【Methods】 Histone methyltransferases of D. melanogaster and their modification sites were analyzed and summarized from Flybase website. After transfection of the constructed knockout vector pAc-sgRNA-Cas9 inserted with the sgRNA of five histone methyltransferase genes ［trr, Mes-4, ash1, Su(var)3-9 and egg］ into Kc cells of D. melanogaster, the gene mutation was detected by TA cloning and sequencing. Taking Trr inducing 20E primary response gene Br-C as a positive control, qRT-PCR and Western blotting were used to test the validity of pAc-sgRNA-Cas9 knockout system. The dual luciferase assay of 20E response element (EcRE) was used to determine whether trr, Mes-4, ash1, Su(var)3-9 and egg participate in 20E signaling transduction. 【Results】 Drosophila histone methylation modification occurs mainly on histone H3 lysine residues and less on H3 arginine residues. Besides, there was less methylation modification on histone H4. After the transfection of its knockout vector pAc-trr-sgRNA-Cas9 in Kc cells, trr was mutated successfully, the tri-methylation level of H3K4 was reduced, and the 20E-induced transcriptional level of its primary response gene Br-C was inhibited, indicating the validity of this knockout system. Besides trr, Mes-4 and egg knockouts also reduced the dual luciferase activity of EcRE. 【Conclusion】 The pAc-sgRNA-Cas9 knockout system inserted with sgRNA of target gene is effective and fast for gene mutation in Drosophila Kc cells. Using this knockout system, in addition to Trr, Mes-4 and egg were found to participate in the regulation of 20E signaling transduction via manipulating the activity of EcRE. This study lays the theoretical basis and work foundation for CRISPR/Cas9-mediated gene knockout in insect cells and further studying histone methylation modification involved in regulating 20E signaling transduction.

Key words: Drosophila; Kc cell, CRISPR-Cas9, histone methylation modification, 20-hydroxyecdysone, Br-C, EcRE