Abstract
The model filamentous fungus Neurospora crassa has been the focus of functional genomics studies for the past several years. A high-throughput gene knockout procedure has been developed and used to generate mutants for more than two-thirds of the ∼10,000 annotated N. crassa genes. Yeast recombinational cloning was incorporated as an efficient procedure to produce all knockout cassettes. N. crassa strains with the Δmus-51 or Δmus-52 deletion mutations were used as transformation recipients in order to reduce the incidence of ectopic integration and increase homologous recombination of knockout cassettes into the genome. A 96-well format was used for many steps of the procedure, including fungal transformation, isolation of homokaryons, and verification of mutants. In addition, development of software programs for primer design and restriction enzyme selection facilitated the high-throughput aspects of the overall protocol.
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References
Shafran, H., Miyara, I., Eshed, R., Prusky, D., and Sherman, A. (2008) Development of new tools for studying gene function in fungi based on the Gateway system, Fungal Genet Biol 45, 1147–54.
Dunlap, J. C., Borkovich, K. A., Henn, M. R., Turner, G. E., Sachs, M. S., Glass, N. L., McCluskey, K., Plamann, M., Galagan, J. E., Birren, B. W., Weiss, R. L., Townsend, J. P., Loros, J. J., Nelson, M. A., Lambreghts, R., Colot, H. V., Park, G., Collopy, P., Ringelberg, C., Crew, C., Litvinkova, L., DeCaprio, D., Hood, H. M., Curilla, S., Shi, M., Crawford, M., Koerhsen, M., Montgomery, P., Larson, L., Pearson, M., Kasuga, T., Tian, C., Basturkmen, M., Altamirano, L., and Xu, J. (2007) Enabling a community to dissect an organism: overview of the Neurospora functional genomics project. Adv Genet 57, 49–96.
Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, M., M’Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Revuelta, J. L., Riles, L., Roberts, C. J., Ross-MacDonald, P., Scherens, B., Snyder, M., Sookhai-Mahadeo, S., Storms, R. K., Veronneau, S., Voet, M., Volckaert, G., Ward, T. R., Wysocki, R., Yen, G. S., Yu, K., Zimmermann, K., Philippsen, P., Johnston, M., and Davis, R. W. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–6.
Galagan, J. E., Calvo, S. E., Borkovich, K. A., Selker, E. U., Read, N. D., Jaffe, D., FitzHugh, W., Ma, L. J., Smirnov, S., Purcell, S., Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C. B., Butler, J., Endrizzi, M., Qui, D., Ianakiev, P., Bell-Pedersen, D., Nelson, M. A., Werner-Washburne, M., Selitrennikoff, C. P., Kinsey, J. A., Braun, E. L., Zelter, A., Schulte, U., Kothe, G. O., Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis, M., Mauceli, E., Bielke, C., Rudd, S., Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R. L., Perkins, D. D., Kroken, S., Cogoni, C., Macino, G., Catcheside, D., Li, W., Pratt, R. J., Osmani, S. A., DeSouza, C. P., Glass, L., Orbach, M. J., Berglund, J. A., Voelker, R., Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D. O., Alex, L. A., Mannhaupt, G., Ebbole, D. J., Freitag, M., Paulsen, I., Sachs, M. S., Lander, E. S., Nusbaum, C., and Birren, B. (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–68.
Davis, R. H., and Perkins, D. D. (2002) Neurospora: A model of model microbes. Nat Rev Genet 3, 397–403.
Case, M. E., Schweizer, M., Kushner, S. R., and Giles, N. H. (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc. Natl. Acad. Sci. USA 76, 5259–63.
Chakraborty, B. N., Patterson, N. A., and Kapoor, M. (1991) An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Can J Microbiol 37, 858–63.
Davis, R. H., and deSerres, F. J. (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol. 71A, 79–143.
Paietta, J. V., and Marzluf, G. A. (1985) Gene disruption by transformation in Neurospora crassa. Mol Cell Biol 5, 1554–9.
Ninomiya, Y., Suzuki, K., Ishii, C., and Inoue, H. (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101, 12248–53.
Oldenburg, K. R., Vo, K. T., Michaelis, S., and Paddon, C. (1997) Recombination-mediated PCR- directed plasmid construction in vivo in yeast. Nucleic Acids Res 25, 451–2.
Colot, H. V., Park, G., Turner, G. E., Ringelberg, C., Crew, C. M., Litvinkova, L., Weiss, R. L., Borkovich, K. A., and Dunlap, J. C. (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 103, 10352–7.
Hays, S., and Selker, E. (2000) Making the selectable marker bar tighter and more economical. Fungal Genet. Newsl. 47, 107.
Pall, M. L. (1993) The use of Ignite (Basta;glufosinate;phosphinothricin) to select transformants of bar-containing plasmids in Neurospora crassa. Fungal Genet. Newsl. 40, 58.
Avalos, J., Geever, R. F., and Case, M. E. (1989) Bialaphos resistance as a dominant selectable marker in Neurospora crassa. Curr Genet 16, 369–72.
Jones, C. A., Greer-Phillips, S. E., and Borkovich, K. A. (2007) The response regulator RRG-1 functions upstream of a MAPK pathway impacting asexual development, female fertility, osmotic stress and fungicide resistance in Neurospora crassa. Mol Biol Cell 18, 2123–36.
Hoffman, C. S. (2001) Preparation of yeast DNA. Curr Protoc Mol Biol Chapter 13, Unit13 11.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ivey, F. D., Hodge, P. N., Turner, G. E., and Borkovich, K. A. (1996) The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa. Mol. Biol. Cell 7, 1283–97.
Collopy, P. D., Colot, H. V., Park, G., Ringelberg, C., Crew, C. M., Borkovich, K. A. and Dunlap, J. C. (2010) High-throughput construction of gene deletion cassettes for generation of Neurospora crassa knockout strains. Methods in Mol. Biol. 638, 33–40.
Acknowledgments
This work was supported by National Institute of General Medical Sciences grant P01 GM068087. We thank John Jones for software design and implementation of the LIMS and Gloria Turner for helpful comments on the manuscript.
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Park, G. et al. (2011). High-Throughput Production of Gene Replacement Mutants in Neurospora crassa . In: Xu, JR., Bluhm, B. (eds) Fungal Genomics. Methods in Molecular Biology, vol 722. Humana Press. https://doi.org/10.1007/978-1-61779-040-9_13
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DOI: https://doi.org/10.1007/978-1-61779-040-9_13
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