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

doi:10.14088/j.cnki.issn0439-8114.2021.23.039

Abstract

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

CLC Number:

Q819

XIAO Hong-wei. Discovery of DNA ligase IV inhibitor through a docking-based screening and CRISPR/Cas9 study[J]. HUBEI AGRICULTURAL SCIENCES, 2021, 60(23): 177-180.

References 12

[1]	肖红卫,毕延震.小分子化合物靶向DNA连接酶IV提高CRISPR/Cas9基因编辑效率的研究进展[J]. 湖北农业科学,2020,59(22):13-19.
[2]	LI G, ZHANG X, ZHONG C, et al.Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells[J]. Sci Rep, 2017, 7: 8943.
[3]	RADHAKRISHNAN S K,JETTE N,LEES-MILLER S P. Non-homologous end joining:Emerging themes and unanswered questions[J]. DNA repair, 2014, 17: 1-7.
[4]	KARRAN P.DNA double strand break repair in mammalian cells[J]. Curr Opin Genet Dev, 2000, 10(2): 144-150.
[5]	SONG J, YANG D S, XU J, et al.RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency[J]. Nat commun, 2016, 7:10548.
[6]	SRIVASTAVA M, NAMBIAR M, SHARMA S, et al.An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression[J]. Cell, 2012, 151: 1474-1487.
[7]	MARUYAMA T, DOUGAN S K, TRUTTMANN M C, et al.Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining[J]. Nat biotechnol, 2015, 33: 538-542.
[8]	VARTAK S V, RAGHAVAN S C.Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing[J]. FEBS J, 2015, 282:4289-4294.
[9]	CHU V T, WEBER T, WEFERS B, et al.Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells[J].Nat biotechnol, 2015, 33(5): 543-548.
[10]	MA Y, CHEN W, ZHANG X, et al.Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein[J], RNA Biol, 2016, 13(7): 605-612.
[11]	HU Z, SHI Z, GUO X, et al.Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells[J], Cell Biosci, 2018, 8: 12.
[12]	LIN C, LI H, HAO M, et al.Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing of HSV-1 virus in human cells[J]. Sci Rep, 2016, 6: 34531.