Projeto e caracterização de sgRNAs visando o controle do fitopatógeno Pseudocercospora fijiensis causador da Sigatoka Negra
Palavras-chave:
CRISPR-Cas9, banana, aglomeração, termodinâmica
Resumo
A Sigatoka Negra, causada pelo fungo Pseudocercospora fijiensis (Mycosphaerella fijiensis), é uma doença importante da banana e da banana-da-terra. O desenho de moléculas de sgRNAs para silenciamento de genes oferece um possível controle deste fitopatógeno. sgRNAs são moléculas que se ligam a enzimas para cortar especificamente genes de interesse. O uso dessas moléculas requer o uso de ferramentas de bioinformática para seu estudo. Portanto, o objetivo desta pesquisa foi projetar e caracterizar sgRNAs para silenciar o gene de virulência Fus3 e o gene de crescimento CYP51 em P. fijiensis, por meio da análise de características estruturais, termodinâmicas e funcionais que permitem discriminar sgRNAs candidatos para controlar fitopatógenos. Vários sgRNAs termodinamicamente estáveis foram obtidos com alta especificidade para os genes alvo, bem como sequências facilmente reconhecíveis pela nuclease SpCas9, e com tamanhos que permitem difusão eficiente em citoplasmas eucarióticos. Os resultados sugerem que todos os sgRNAs projetados e caracterizados podem promover o correto silenciamento dos genes selecionados para o controle de P. fijiensis. Além disso, os designs mais ideais foram identificados, com base nas características consideradas neste estudo. Esses resultados, embora necessitem de estudos adicionais para o aprimoramento da tecnologia, são promissores, pois mostram a possibilidade do uso de ferramentas moleculares atóxicas e altamente específicas em biotecnologia vegetal para melhoramento genético, mutagênese dirigida, saneamento vegetal e controle de fitopatógenos.
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Referências
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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
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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
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Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N. J., & Nekrasov, V. (2015). Editing plant genomes with CRISPR/Cas9. Current opinion in biotechnology, 32(1), 76-84. https://doi.org/10.1016/j.copbio.2014.11.007
Campenhout, C. V., Cabochette, P., Veillard, A. C., Laczik, M., Zelisko-Schmidt, A., Sabatel, C., ... & Kruys, V. (2019). Guidelines for optimized gene knockout using CRISPR/Cas9. BioTechniques, 66(6), 295-302. https://doi.org/10.2144/btn-2018-0187
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
Escobar-Tovar, L., Magaña-Ortíz, D., Fernández, F., Guzmán-Quesada, M., Sandoval-Fernández, J. A., Ortíz-Vázquez, E., ... & Gómez-Lim, M. A. (2015). Efficient transformation of Mycosphaerella fijiensis by underwater shock waves. Journal of microbiological methods, 119(1), 98-105. https://doi.org/10.1016/j.mimet.2015.10.006
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
George, D., & Mallery, P. (2016). An Overview of IBM SPSS Statistics. IBM SPSS Statistics 23 Step by Step (14 Edition) Routledge.
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
Publicado
2022-01-03
Como Citar
Moncayo, L., Centanaro, P., Arcos-Jácome, D., Castro, A., Maldonado, C., Vaca, D., González, G., Lossada, C., Perez, A., & González-Paz, L. (2022). Projeto e caracterização de sgRNAs visando o controle do fitopatógeno Pseudocercospora fijiensis causador da Sigatoka Negra. Revista Da Faculdade De Agronomia Da Universidade De Zulia, 39(1), e223909. Obtido de https://mail.produccioncientificaluz.org/index.php/agronomia/article/view/37516
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Produção Vegetal
Direitos de Autor (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.
