Diseño y caracterización de sgRNAs dirigidos al control del fitopatógeno Pseudocercospora fijiensis causante de la Sigatoka Negra
Palabras clave:
CRISPR-Cas9, banano, crowding, termodinámica
Resumen
La Sigatoka Negra, causada por el hongo Pseudocercospora fijiensis (Mycosphaerella fijiensis) es una enfermedad importante del banano y plátano. El diseño de moléculas sgRNAs para el silenciamiento de genes ofrece un posible control de este fitopatógeno. Los sgRNAs son moléculas que se unen a enzimas para cortar de forma específica genes de interés. El aprovechamiento de estas moléculas requiere usar herramientas bioinformáticas para su estudio. Por lo que el objetivo de esta investigación fue diseñar y caracterizar sgRNAs para silenciar el gen de virulencia Fus3 y el gen de crecimiento CYP51 en P. fijiensis, mediante el análisis de características estructurales, termodinámicas y funcionales que permiten discriminar los sgRNAs candidatos a control del fitopatógeno. Se obtuvieron diversos sgRNAs termodinámicamente estables y con alta especificidad para los genes diana, así como con secuencias fácilmente reconocibles por la nucleasa SpCas9, y con tamaños que permiten la difusión eficiente en citoplasmas eucariotas. Los resultados sugieren que todos los sgRNAs diseñados y caracterizados, pueden promover el correcto silenciamiento de los genes seleccionados para el control de P. fijiensis. Adicionalmente, se identificaron los diseños más óptimos, en función de las características consideradas en este estudio. Estos resultados, aunque requieren de estudios adicionales para perfeccionar la tecnología, son prometedores pues muestran la posibilidad de usar herramientas moleculares no tóxicas y de alta especificidad en biotecnología vegetal para el mejoramiento genético, mutagénesis dirigida, saneamiento vegetal y control de fitopatógenos.
Descargas
La descarga de datos todavía no está disponible.
Citas
Bartkowski, B., Theesfeld, I., Pirscher, F., & Timaeus, J. (2018). Snipping around for food: economic, ethical and policy implications of CRISPR/Cas genome editing. Geoforum, 96(1), 172-180. https://doi.org/10.1016/j.geoforum.2018.07.017
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
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
Cómo citar
Moncayo, L., Centanaro, P., Arcos-Jácome, D., Castro, A., Maldonado, C., Vaca, D., González, G., Lossada, C., Perez, A., & González-Paz, L. (2022). Diseño y caracterización de sgRNAs dirigidos al control del fitopatógeno Pseudocercospora fijiensis causante de la Sigatoka Negra. Revista De La Facultad De Agronomía De La Universidad Del Zulia, 39(1), e223909. Recuperado a partir de https://mail.produccioncientificaluz.org/index.php/agronomia/article/view/37516
Número
Sección
Producción Vegetal
Derechos de autor 2022 Luis Moncayo, Paulo Centanaro, Diego Arcos-Jácome, Alex Castro, Cristina Maldonado, Diego Vaca, Gardenia González, Carla Lossada, Aleivi Perez y Lenin González-Paz
Esta obra está bajo licencia internacional Creative Commons Reconocimiento-NoComercial-CompartirIgual 4.0.
