Palabras clave
CRISPR-Cas9
Drosophila melanogaster
Genética animal
Biología molecular
Drosophila melanogaster
Genética animal
Biología molecular
Cómo citar
Vargas Sánchez, D., Hernández Vargas, R., Reynaud Garza, E., Dueñas-García, I., & Heres-Pulido, M. (2025). Sistema CRISPR-Cas9, en Drosophila melanogaster. Revista Latinoamericana De Difusión Científica, 7(13), 24-34. https://doi.org/10.5281/zenodo.15832073
Resumen
El descubrimiento del sistema CRISPR-Cas9 hace 13 años provocó un avance tecnológico muy importante para la edición más precisa de los genomas, lo cual se relaciona, entre otras cosas, con la generación de nuevas biomoléculas, la edición de organismos procariotas y eucariotas, y el avance sustancial en el conocimiento de los procesos relacionados con el flujo de la información genética. Realizar esta tecnología en la mosca de la fruta Drosophila melanogaster, que comparte genes con humanos, abre infinitas posibilidades para el estudio de los mecanismos hereditarios de los procesos biológicos normales, y de los alterados por causas internas o externas. Se describen las características particulares de este modelo biológico que es un eucarionte invertebrado, las generalidades del sistema CRISPR-Cas9, sus funciones y componentes, la producción de knockouts, knockins, así como las perspectivas y limitaciones de esta herramienta.
Citas
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Bassett, A., and Liu, J. L. (2014). CRISPR/Cas9 mediated genome engineering in Drosophila. Methods (San Diego, Calif.), 69(2), 128–136. https://doi.org/10.1016/j.ymeth.2014.02.019.
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Bosch, J. A., Knight, S., Kanca, O., Zirin, J., Yang-Zhou, D., Hu, Y., et al. (2020). Use of the CRISPR-Cas9 system in Drosophila cultured cells to introduce fluorescent tags into endogenous genes. Current Protocols in Molecular Biology, 130(1), e112. https://doi.org/10.1002/cpmb.112.
Chávez-Jacobo, Víctor M. (2018). El sistema de edición genética CRISPR/Cas y su uso como antimicrobiano específico. TIP. Revista Especializada en Ciencias Químico- Biológicas, 21(2), e201825. Epub 03 de septiembre de 2020. https://doi.org/10.22201/fesz.23958723e.2018.2.5
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819–823. https://doi.org/10.1126/science.1231143.
Franklin, R. E., and Gosling, R. G. (1953). Molecular configuration in sodium thymonucleate. Nature, 171(4356), 740–741. https://doi.org/10.1038/171740a0.
Fritsch, C., and Sprecher, S. G. (2020). CRISPR/Cas9 genome editing to study nervous system development in Drosophila. Methods in Molecular Biology, 2047:161-189. doi: 10.1007/978-1-4939-9732-9_10. PMID: 31552655.
Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109(39), E2579–E2586. https://doi.org/10.1073/pnas.1208507109.
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Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., and Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429–5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829.
Kanca, O., Zirin, J., Garcia-Marques, J., Knight, S. M., Yang-Zhou, D., Amador, G., et al. Perrimon, N., and Bellen, H. J. (2019). An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms. eLife, 8, e51539. https://doi.org/10.7554/eLife.51539.
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., and Joung, J. K. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587), 490–495. https://doi.org/10.1038/nature16526.
Kohler, R. E. (1993). Drosophila: A life in the laboratory. Journal of the History of Biology, 26(2), 281–310. https://doi.org/10.1007/BF01061971.
Koreman, G. T., Xu, Y., Hu, Q., Zhang, Z., Allen, S. E., Wolfner, M. F., et al. (2021). Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 118(14), e2014255118. https://doi.org/10.1073/pnas.2014255118.
Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhnbashi, O. S., Brouns, S., et al. (2020). Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Reviews. Microbiology, 18(2), 67–83. https://doi.org/10.1038/s41579-019-0299-x.
Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J., and Soria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution, 60(2), 174–182. https://doi.org/10.1007/s00239-004-0046-3.
Neville, M. C., Eastwood, A., Allen, A. M., de Haan, A., Nojima, T., and Goodwin, S. F. (2021). Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster. Journal of Neurogenetics, 35(3), 285–294. https://doi.org/10.1080/01677063.2021.1931179.
Pandey, U.B. and Nichols C.D. (2011) Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacological Reviews, 63,411-436. https://pubmed.ncbi.nlm.nih.gov/21415126/
Port, F., and Bullock, S. L. (2016a). Creating heritable mutations in Drosophila with CRISPR-Cas9. Methods in Molecular Biology, 1478, 145–160. https://doi.org/10.1007/978-1-4939-6371-3_7.
Port, F., and Bullock, S. L. (2016b). Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nature Methods, 13(10), 852–854. https://doi.org/10.1038/nmeth.3972.
Rubin, G. M., Yandell, M. D., Wortman, J. R., Gabor Miklos, G. L., Nelson, C. R., Hariharan, I. K., et al. (2000). Comparative genomics of the eukaryotes. Science, 287(5461), 2204–2215. https://doi.org/10.1126/science.287.5461.2204.
Ugur, B., Chen, K., and Bellen, H. J. (2016). Drosophila tools and assays for the study of human diseases. Disease Models and Mechanisms, 9(3), 235–244. https://doi.org/10.1242/dmm.023762.
van Kampen, S. J., and van Rooij, E. (2019). CRISPR craze to transform cardiac biology. Trends in Molecular Medicine, 25(9), 791–802. https://doi.org/10.1016/j.molmed.2019.06.008.
Watson, J. D., and Crick, F. H. (1953). Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature, 171(4356), 737–738. https://doi.org/10.1038/171737a0.
Yang, H., Ren, S., Yu, S., Pan, H., Li, T., Ge, S., et al. (2020). Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. International Journal of Molecular Sciences, 21(18), 6461. https://doi.org/10.3390/ijms21186461.
Yamaguchi, M., and Yoshida, H. (2018). Drosophila as a model organism. Advances in Experimental Medicine and Biology, 1076, 1–10. https://doi.org/10.1007/978-981-13- 0529-0_1
Zirin J, Bosch J, Viswanatha R, Mohr SE, and Perrimon N. (2021). State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila. Trends in Genetic, 38(5):437-453. https://pubmed.ncbi.nlm.nih.gov/34933779/
Arnoult, N., Correia, A., Ma, J., Merlo, A., Garcia-Gomez, S., Maric, M., et al. (2017). Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN. Nature, 549(7673), 548–552. https://doi.org/10.1038/nature24023.
Bassett, A., and Liu, J. L. (2014). CRISPR/Cas9 mediated genome engineering in Drosophila. Methods (San Diego, Calif.), 69(2), 128–136. https://doi.org/10.1016/j.ymeth.2014.02.019.
Bassett, A. R., Tibbit, C., Ponting, C. P., and Liu, J. L. (2013). Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell reports, 4(1), 220–228. https://doi.org/10.1016/j.celrep.2013.06.020.
Bosch, J. A., Knight, S., Kanca, O., Zirin, J., Yang-Zhou, D., Hu, Y., et al. (2020). Use of the CRISPR-Cas9 system in Drosophila cultured cells to introduce fluorescent tags into endogenous genes. Current Protocols in Molecular Biology, 130(1), e112. https://doi.org/10.1002/cpmb.112.
Chávez-Jacobo, Víctor M. (2018). El sistema de edición genética CRISPR/Cas y su uso como antimicrobiano específico. TIP. Revista Especializada en Ciencias Químico- Biológicas, 21(2), e201825. Epub 03 de septiembre de 2020. https://doi.org/10.22201/fesz.23958723e.2018.2.5
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819–823. https://doi.org/10.1126/science.1231143.
Franklin, R. E., and Gosling, R. G. (1953). Molecular configuration in sodium thymonucleate. Nature, 171(4356), 740–741. https://doi.org/10.1038/171740a0.
Fritsch, C., and Sprecher, S. G. (2020). CRISPR/Cas9 genome editing to study nervous system development in Drosophila. Methods in Molecular Biology, 2047:161-189. doi: 10.1007/978-1-4939-9732-9_10. PMID: 31552655.
Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109(39), E2579–E2586. https://doi.org/10.1073/pnas.1208507109.
Gaj, T., Gersbach, C. A., and Barbas, C. F., 3rd (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31(7), 397–405. https://doi.org/10.1016/j.tibtech.2013.04.004.
Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., and Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429–5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829.
Kanca, O., Zirin, J., Garcia-Marques, J., Knight, S. M., Yang-Zhou, D., Amador, G., et al. Perrimon, N., and Bellen, H. J. (2019). An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms. eLife, 8, e51539. https://doi.org/10.7554/eLife.51539.
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., and Joung, J. K. (2016). High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587), 490–495. https://doi.org/10.1038/nature16526.
Kohler, R. E. (1993). Drosophila: A life in the laboratory. Journal of the History of Biology, 26(2), 281–310. https://doi.org/10.1007/BF01061971.
Koreman, G. T., Xu, Y., Hu, Q., Zhang, Z., Allen, S. E., Wolfner, M. F., et al. (2021). Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 118(14), e2014255118. https://doi.org/10.1073/pnas.2014255118.
Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhnbashi, O. S., Brouns, S., et al. (2020). Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Reviews. Microbiology, 18(2), 67–83. https://doi.org/10.1038/s41579-019-0299-x.
Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J., and Soria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution, 60(2), 174–182. https://doi.org/10.1007/s00239-004-0046-3.
Neville, M. C., Eastwood, A., Allen, A. M., de Haan, A., Nojima, T., and Goodwin, S. F. (2021). Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster. Journal of Neurogenetics, 35(3), 285–294. https://doi.org/10.1080/01677063.2021.1931179.
Pandey, U.B. and Nichols C.D. (2011) Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery. Pharmacological Reviews, 63,411-436. https://pubmed.ncbi.nlm.nih.gov/21415126/
Port, F., and Bullock, S. L. (2016a). Creating heritable mutations in Drosophila with CRISPR-Cas9. Methods in Molecular Biology, 1478, 145–160. https://doi.org/10.1007/978-1-4939-6371-3_7.
Port, F., and Bullock, S. L. (2016b). Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nature Methods, 13(10), 852–854. https://doi.org/10.1038/nmeth.3972.
Rubin, G. M., Yandell, M. D., Wortman, J. R., Gabor Miklos, G. L., Nelson, C. R., Hariharan, I. K., et al. (2000). Comparative genomics of the eukaryotes. Science, 287(5461), 2204–2215. https://doi.org/10.1126/science.287.5461.2204.
Ugur, B., Chen, K., and Bellen, H. J. (2016). Drosophila tools and assays for the study of human diseases. Disease Models and Mechanisms, 9(3), 235–244. https://doi.org/10.1242/dmm.023762.
van Kampen, S. J., and van Rooij, E. (2019). CRISPR craze to transform cardiac biology. Trends in Molecular Medicine, 25(9), 791–802. https://doi.org/10.1016/j.molmed.2019.06.008.
Watson, J. D., and Crick, F. H. (1953). Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature, 171(4356), 737–738. https://doi.org/10.1038/171737a0.
Yang, H., Ren, S., Yu, S., Pan, H., Li, T., Ge, S., et al. (2020). Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. International Journal of Molecular Sciences, 21(18), 6461. https://doi.org/10.3390/ijms21186461.
Yamaguchi, M., and Yoshida, H. (2018). Drosophila as a model organism. Advances in Experimental Medicine and Biology, 1076, 1–10. https://doi.org/10.1007/978-981-13- 0529-0_1
Zirin J, Bosch J, Viswanatha R, Mohr SE, and Perrimon N. (2021). State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila. Trends in Genetic, 38(5):437-453. https://pubmed.ncbi.nlm.nih.gov/34933779/
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