Design and characterization of sgRNAs aimed at the control of the phytopathogen Pseudocercospora fijiensis that causes Black Sigatoka
Keywords:
CRISPR-Cas9, banana, crowding, thermodynamics
Abstract
Black Sigatoka, caused by the fungus Pseudocercospora fijiensis (Mycosphaerella fijiensis) is an important disease of bananas and plantain. The design of sgRNAs molecules for gene silencing offers the possible control of this phytopathogen. The sgRNAs, are molecules that bind to enzymes to specifically edit genes of interest. The use of these molecules requires the use of bioinformatics tools for their study. Therefore, the objective of this research was to design and characterize sgRNAs to silence the Fus3 virulence gene and CYP51 gene growth in P. fijiensis, through the analysis of structural, thermodynamic and functional characteristics that allow to discriminate the sgRNAs candidates for control of the phytopathogen. Several thermodynamically stable sgRNAs with high specificity for the target genes were achieved, as well as with sequences easily recognizable by the SpCas9 nuclease, and with sizes that allow efficient diffusion in eukaryotic cytoplasms. The results suggest that all the designed and characterized sgRNAs can promote the correct silencing of the genes selected for the control of P. fijiensis. Additionally, the most optimal designs were identified, based on the characteristics considered in this study. These results, although they require additional studies to improve the technology, are promising as they show the possibility of using non-toxic and highly specific molecular tools in plant biotechnology for genetic improvement, directed mutagenesis, plant sanitation and control of phytopathogens.
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References
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Chong, P., Vichou, A. E., Schouten, H. J., Meijer, H. J., Arango Isaza, R. E., & Kema, G. H. (2019). Pfcyp51 exclusively determines reduced sensitivity to 14α-demethylase inhibitor fungicides in the banana black Sigatoka pathogen Pseudocercospora fijiensis. PLOS ONE, 14(10), Article e0223858. https://doi.org/10.1371/journal.pone.0223858
Díaz-Trujillo, C., Kobayashi, A. K., Souza, M., Chong, P., Meijer, H. J., Isaza, R. E. A., & Kema, G. H. (2018). Targeted and random genetic modification of the black Sigatoka pathogen Pseudocercospora fijiensis by Agrobacterium tumefaciens-mediated transformation. Journal of microbiological methods, 148(1), 127-137. https://doi.org/10.1016/j.mimet.2018.03.017
Dupuis, N. F., Holmstrom, E. D., & Nesbitt, D. J. (2014). Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics. Proceedings of the National Academy of Sciences, 111(23), 8464-8469. https://doi.org/10.1073/pnas.1316039111
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Estrela, R., & Cate, J. H. D. (2016). Energy biotechnology in the CRISPR-Cas9 era. Current opinion in biotechnology, 38(1), 79-84. https://doi.org/10.1016/j.copbio.2016.01.005
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Jiang, D., Zhu, W., Wang, Y., Sun, C., Zhang, K. Q., & Yang, J. (2013). Molecular tools for functional genomics in filamentous fungi: recent advances and new strategies. Biotechnology advances, 31(8), 1562-1574. https://doi.org/10.1016/j.biotechadv.2013.08.005
Knight, S. C., Xie, L., Deng, W., Guglielmi, B., Witkowsky, L. B., Bosanac, L., ... & Tjian, R. (2015). Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science, 350(6262), 823-826. https://doi.org/10.1126/science.aac6572
Kocak, D. D., Josephs, E. A., Bhandarkar, V., Adkar, S. S., Kwon, J. B., & Gersbach, C. A. (2019). Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nature biotechnology, 37(6), 657-666. https://doi.org/10.1038/s41587-019-0095-1
Koch, A., Kumar, N., Weber, L., Keller, H., Imani, J., & Kogel, K. H. (2013). Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences, 110(48), 19324-19329. https://doi.org/10.1073/pnas.1306373110
Kuan, P. F., Powers, S., He, S., Li, K., Zhao, X., & Huang, B. (2017). A systematic evaluation of nucleotide properties for CRISPR sgRNA design. Bmc Bioinformatics, 18(1), 1-9. https://doi.org/10.1186/s12859-017-1697-6
Li, J., Sun, Y., Du, J., Zhao, Y., & Xia, L. (2017). Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Molecular plant, 10(3), 526-529. http://dx.doi.org/10.1111/pbi.12611
Liang, X., Potter, J., Kumar, S., Ravinder, N., & Chesnut, J. D. (2017). Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. Journal of biotechnology, 241(1), 136-146. https://doi.org/10.1016/j.jbiotec.2016.11.011
Ma, B., & Tredway, L. P. (2013). Induced overexpression of cytochrome P450 sterol 14 α‐demethylase gene (CYP51) correlates with sensitivity to demethylation inhibitors (DMIs) in Sclerotinia homoeocarpa. Pest management science, 69(12), 1369-1378. https://doi.org/10.1002/ps.3513
Mumbanza, F. M., Kiggundu, A., Tusiime, G., Tushemereirwe, W. K., Niblett, C., & Bailey, A. (2013). In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis. Pest management science, 69(10), 1155-1162. https://doi.org/10.1002/ps.3480
Onyilo, F., Tusiime, G., Tripathi, J. N., Chen, L. H., Falk, B., Stergiopoulos, I., ... & Tripathi, L. (2018). Silencing of the mitogen-activated protein kinases (MAPK) Fus3 and Slt2 in Pseudocercospora fijiensis reduces growth and virulence on host plants. Frontiers in plant science, 9(291), 1-12. https://doi.org/10.3389/fpls.2018.00291
Podust, L. M., Poulos, T. L., & Waterman, M. R. (2001). Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proceedings of the National Academy of Sciences, 98(6), 3068-3073. https://doi.org/10.1073/pnas.061562898
Regan, K., Dotterweich, R., Ricketts, S., & Robertson-Anderson, R. M. (2018). Diffusion and conformational dynamics of single DNA molecules crowded by cytoskeletal proteins. Journal of Undergraduate Reports in Physics, 28(1), 100001-100005. https://doi.org/10.1063/1.5109559
Ren, X., Yang, Z., Xu, J., Sun, J., Mao, D., Hu, Y., ... & Ni, J. Q. (2014). Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell reports, 9(3), 1151-1162. https://doi.org/10.1016/j.celrep.2014.09.044
Scott, D. A., & Zhang, F. (2017). Implications of human genetic variation in CRISPR-based therapeutic genome editing. Nature medicine, 23(9), 1095–1101. https://doi.org/10.1038/nm.4377
Tripathi, J. N., Ntui, V. O., Ron, M., Muiruri, S. K., Britt, A., & Tripathi, L. (2019). CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding. Communications biology, 2(1), 1-11. https://doi.org/10.1038/s42003-019-0288-7
Xu, J. R. (2000). MAP kinases in fungal pathogens. Fungal Genetics and Biology, 31(3), 137-152. https://doi.org/10.1006/fgbi.2000.1237
Zaynab, M., Sharif, Y., Fatima, M., Afzal, M. Z., Aslam, M. M., Raza, M. F., ... & Li, S. (2020). CRISPR/Cas9 to generate plant immunity against pathogen. Microbial pathogenesis, 141(1), Article 103996. https://doi.org/10.1016/j.micpath.2020.103996
Zhang, X. H., Tee, L. Y., Wang, X. G., Huang, Q. S., & Yang, S. H. (2015). Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy-Nucleic Acids, 4, Article e264. https://doi.org/10.1038/mtna.2015.37
Published
2022-01-03
How to Cite
Moncayo, L., Centanaro, P., Arcos-Jácome, D., Castro, A., Maldonado, C., Vaca, D., González, G., Lossada, C., Perez, A., & González-Paz, L. (2022). Design and characterization of sgRNAs aimed at the control of the phytopathogen Pseudocercospora fijiensis that causes Black Sigatoka. Revista De La Facultad De Agronomía De La Universidad Del Zulia, 39(1), e223909. Retrieved from https://produccioncientificaluz.org/index.php/agronomia/article/view/37516
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Crop Production
Copyright (c) 2022 Luis Moncayo, Paulo Centanaro, Diego Arcos-Jácome, Alex Castro, Cristina Maldonado, Diego Vaca, Gardenia González, Carla Lossada, Aleivi Perez y Lenin González-Paz
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
