NUDT16L1 (TIRR) knockdown increases genome editing efficacy
https://doi.org/10.25557/2073-7998.2021.08.3-10
Abstract
Introduction. Effective precise knock-in is crucial for implementing CRISPR-Cas9 system as an efficient instrument for potential gene therapy. Homology directed repair (HDR) pathway allows correction of all types of existing mutations. However, HDR is not a major repair pathway of the cell that limits its efficiency. In our study, we present for the first time how repair factors NUDT16L1 controls HDR efficiency. Aim: to study an influence of NUDT16L1 knockdown and overexpression on the HDR efficacy. Methods. HEK293T culture was used to perform the research. Plasmid CRISPR-Cas system along with NUDT16L1 overexpression vector were delivered with lipofection. For NUDT16L1 knockdown small interfering RNAs were used. Results. We discovered that knockdown of NUDT16L1 enhances HDR both in the plasmid and genomic loci increasing eGFP signal from 1.8 to 3.6 times in HEK293T cells. Conclusion. NUDT16L1 knockdown could be used for enhancing of the pathogenic mutations correction through genome editing.
About the Authors
A. A. Anuchina
Research Centre for Medical Genetics
Russian Federation
Russian Federation
S. A. Smirnikhina
Research Centre for Medical Genetics
Russian Federation
Russian Federation
M. I. Zaynitdinova
Research Centre for Medical Genetics
Russian Federation
Russian Federation
A. G. Demchenko
Research Centre for Medical Genetics
Russian Federation
Russian Federation
O. S. Mishina
Research Centre for Medical Genetics
Russian Federation
Russian Federation
A. V. Lavrov
Research Centre for Medical Genetics
Russian Federation
Russian Federation
References
1. Liu M., Rehman S., Tang X., Gu K., Fan Q., Chen D., et al. Methodologies for Improving HDR Efficiency. Front Genet [Internet]. 2019 [cited 2021 Jul 15];9(JAN). Available from: https://pubmed.ncbi.nlm.nih.gov/30687381/
2. Scully R., Panday A., Elango R., Willis N.A. DNA double-strand break repair-pathway choice in somatic mammalian cells [Internet]. Vol. 20, Nature Reviews Molecular Cell Biology. Nature Publishing Group; 2019 [cited 2020 Jul 20]. p. 698-714. Available from: https://www.nature.com/articles/s41580-019-0152-0
3. Panier S., Boulton S.J. Double-strand break repair: 53BP1 comes into focus. Nat Rev Mol Cell Biol [Internet]. 2014 Jan 11 [cited 2019 Feb 12];15(1):7-18. Available from: http://www.nature.com/articles/nrm3719
4. Shibata A. Regulation of repair pathway choice at two-ended DNA double-strand breaks. Mutat Res Mol Mech Mutagen [Internet]. 2017 Oct [cited 2019 Feb 7];803-805:51-5. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28781144
5. Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., et al. RNA-Guided Human Genome Engineering via Cas9. Science [Internet]. 2013 Feb 15 [cited 2021 Jul 15];339(6121):823. Available from: /pmc/articles/PMC3712628/
6. He X., Tan C., Wang F., Wang Y., Zhou R., Cui D., et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res [Internet]. 2016 May 19 [cited 2021 Jul 15];44(9):e85. Available from: /pmc/articles/PMC4872082/
7. Cui X., Ji D., Fisher D., Wu Y., Briner D., Weinstein E. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol [Internet]. 2011 Jan [cited 2021 Jul 15];29(1):64-8. Available from: https://pubmed.ncbi.nlm.nih.gov/21151125/
8. Zhu Z., González F., Huangfu D. The iCRISPR platform for rapid genome editing in human Pluripotent Stem Cells. Methods Enzymol [Internet]. 2014 [cited 2021 Jul 15];546(C):215. Available from: /pmc/articles/PMC4418970/
9. Canny M.D., Moatti N., Wan L.C.K., Fradet-Turcotte A., Krasner D., Mateos-Gomez P.A., et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol. 2017 Nov;36(1):95-102.
10. Charpentier M., Khedher A.H.Y, Menoret S., Brion A., Lamribet K., Dardillac E., et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun 2018 91 [Internet]. 2018 Mar 19 [cited 2021 Jul 15];9(1):1-11. Available from: https://www.nature.com/articles/s41467-018-03475-7
11. Hu Z., Shi Z., Guo X., Jiang B., Wang G., Luo D., et al. Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells. Cell Biosci. 2018 Dec;8(1):12.
12. Smirnikhina S.A., Anuchina A.A., Lavrov A.V. Ways of improving precise knock-in by genome-editing technologies. Hum Genet [Internet]. 2019 Jan 2 [cited 2019 Feb 5];138(1):1-19. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30390160
13. Avolio R., Järvelin A.I., Mohammed S., Agliarulo I., Condelli V., Zoppoli P., et al. Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation. Nucleic Acids Res. 2018;46(22):12067-86.
14. Kim H., Yoo J., Lee I., Kang Y.J., Cho H.S., Lee W. Crystal structure of syndesmos and its interaction with Syndecan-4 proteoglycan. Biochem Biophys Res Commun [Internet]. 2015 Jul 13 [cited 2020 Apr 3];463(4):762-7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0006291X15300632
15. Baciu P.C., Saoncella S., Lee S.H., Denhez F., Leuthardt D., Goetinck P.F. Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization. J Cell Sci [Internet]. 2000 Jan [cited 2019 Nov 25];113 Pt 2:315-24. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10633082
16. Denhez F., Wilcox-Adelman S.A., Baciu P.C., Saoncella S., Lee S., French B., et al. Syndesmos, a syndecan-4 cytoplasmic domain interactor, binds to the focal adhesion adaptor proteins paxillin and Hic-5. J Biol Chem. 2002 Apr 5;277(14):12270-4.
17. Drané P., Brault M.E., Cui G., Meghani K., Chaubey S., Detappe A., et al. TIRR regulates 53BP1 by masking its histone methyl-lysine binding function. Nature [Internet]. 2017;543(7644):211-6. Available from: http://dx.doi.org/10.1038/nature21358
18. Zhang A., Peng B., Huang P., Chen J., Gong Z. The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response. J Biol Chem [Internet]. 2017 Apr 21 [cited 2019 Feb 6];292(16):6461-7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28213517
19. Drané P., Chowdhury D. TIRR and 53BP1- partners in arms. Cell Cycle [Internet]. 2017;16(13):1235-6. Available from: https://doi.org/10.1080/15384101.2017.1337966
20. Laboratory of virology and genetics EPFL [Internet]. [cited 2021 Jul 15]. Available from: https://www.epfl.ch/labs/tronolab/
21. Cloud-Based Informatics Platform for Life Sciences R&D | Benchling [Internet]. [cited 2021 Jul 15]. Available from: https://www.benchling.com/
22. Yang D., Scavuzzo M.A., Chmielowiec J., Sharp R., Bajic A., Borowiak M. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep [Internet]. 2016 Feb 18 [cited 2020 Oct 14];6(1):1-15. Available from: www.nature.com/scientificreports/
23. Zhang J.-P., Li X.-L., Li G.-H., Chen W., Arakaki.C, Botimer G.D., et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol [Internet]. 2017 Dec 20 [cited 2019 Feb 6];18(1):35. Available from: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1164-8
24. Lin S., Staahl B.T., Alla R.K., Doudna J.A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife. 2014;3:e04766.
25. Gerlach M., Kraft T., Brenner B., Petersen B., Niemann H., Montag J., et al. Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene. Genes (Basel) [Internet]. 2018 Jun 13 [cited 2019 Feb 14];9(6):296. Available from: http://www.mdpi.com/2073-4425/9/6/296
26. Shao S., Ren C., Liu Z., Bai Y., Chen Z., Wei Z., et al. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int J Biochem Cell Biol [Internet]. 2017 Nov 1 [cited 2020 Apr 1];92:43-52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1357272517302388
27. Stazio M. Di, Foschi N., Athanasakis E., Gasparini P., d’Adamo A.P. Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique. PLoS One [Internet]. 2021 Mar 1 [cited 2021 Jul 15];16(3):e0247603. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247603
28. Li G., Wang H., Zhang X., Wu Z., Yang H. A Cas9-transcription factor fusion protein enhances homology-directed repair efficiency. J Biol Chem. 2021 Jan 1;296:100525.
29. Maurissen T.L., Woltjen K. Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation. Nat Commun [Internet]. 2020 Dec 1 [cited 2021 Jul 15];11(1). Available from: /pmc/articles/PMC7280248/
30. Botuyan M.V., Cui G., Drané P., Oliveira C., Detappe A., Brault M.E., et al. Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein. Nat Struct Mol Biol. 2018;1-10.
31. Wang J., Yuan Z., Cui Y., Xie R., Yang G., Kassab M.A., et al. Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR. Nat Commun. 2018;9(1):2689.
32. Anuchina A.A., Lavrov A.V., Smirnikhina S.A. TIRR: a potential front runner in HDR race-hypotheses and perspectives [Internet]. Vol. 47, Molecular Biology Reports. Springer; 2020 [cited 2020 Apr 3]. p. 2371-9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/32036573
Review
For citations:
Anuchina A.A., Smirnikhina S.A., Zaynitdinova M.I., Demchenko A.G., Mishina O.S., Lavrov A.V. NUDT16L1 (TIRR) knockdown increases genome editing efficacy. Medical Genetics. 2021;20(8):3-10. (In Russ.) https://doi.org/10.25557/2073-7998.2021.08.3-10
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