Abstract
F. oxysporum is a notorious filamentous pathogenic fungus that causes serious problems in agriculture and animal/human health. Knowing how the fungus interacts throughout the course of an infection is necessary to propose an effective control strategy, and consequently the manipulation of the F. oxysporum genome is essential to investigate the molecular interplay between the host and fungus. To facilitate assessing protein quantification and subcellular localization, we developed a simple, economical CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). Reporter genes, including GFP and LacZ, can be inserted at the N- or C-terminus of an endogenous gene of interest at the original chromosomal locus, allowing partial characterization of the gene function.
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References
Gordon TR (2017) Fusarium oxysporum and the Fusarium wilt syndrome. Annu Rev Phytopathol 55:23–39
Wang Q, Cobine PA, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes. Fungal Genet Biol 117:21–29
Wang Q, Coleman JJ (2019) CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Fungal Genet Biol 126:17–24
Wang Q, Coleman JJ (2019) Progress and challenges: development and implementation of CRISPR/Cas9 technology in filamentous fungi. Comput Struct Biotechnol J 17:761–769
McCluskey K, Wiest A, Plamann M (2010) The fungal genetics stock center: a repository for 50 years of fungal genetics research. J Biosci 35:119–126
Stevens RB (1974) Mycology guidebook. University of Washington Press, Seattle
Stemmer M, Thumberger T, del Sol KM, Wittbrodt J, Mateo JL (2015) CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10:e0124633
Acknowledgments
Development of this method and publication was partially supported by the Alabama Agricultural Experiment Station, the Hatch program of the National Institute of Food and Agriculture, USDA, and the Special Project of Technological Innovation Guidance in Shaanxi Province (No. 2020QFY12-06).
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Wang, Q., Coleman, J.J. (2022). CRISPR/Cas9 RNP-Mediated Gene Fusion to Assess Protein Quantification and Subcellular Localization in Fusarium oxysporum. In: Coleman, J. (eds) Fusarium wilt. Methods in Molecular Biology, vol 2391. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1795-3_8
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DOI: https://doi.org/10.1007/978-1-0716-1795-3_8
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-1795-3
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