Abstract
Characterization of a gene of interest frequently relies on generation of a mutant as a critical component. Transformation to disrupt a gene has been previously accomplished by several methods in Fusarium oxysporum. Here we provide a detailed method to generate a gene mutation mediated by a CRISPR/Cas9 ribonucleoprotein (RNP) complex. The Cas9 RNP cleaves the DNA at the target site, and during DNA repair integration of a dominant selectable marker is incorporated via homologous recombination generating the desired gene disruption.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kistler HC, Benny UK (1988) Genetic transformation of the fungal plant wilt pathogen, Fusarium oxysporum. Curr Genet 13:145–149
Coleman JJ, Wasmann CC, Usami T, White GJ, Temporini ED, McCluskey K, VanEtten HD (2011) Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum. Mol Plant-Microbe Interact 24:1482–1491
Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:173–180
Liang L, Li J, Cheng L, Ling J, Luo Z, Bai M, Xie B (2014) A high efficiency gene disruption strategy using a positive–negative split selection marker and electroporation for Fusarium oxysporum. Microbiol Res 169(11):835–843
Wang Q, Cobine PA, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein compleses. Fungal Genet Biol 117:21–29
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
Schuster M, Kahmann R (2019) CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes. Fungal Genet Biol 130:43–53
Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL (2015) CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10:e0124633
McCluskey K, Wiest A, Plamann M (2010) The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J Biosci 35(1):119–126
Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47(W1):W171–W174. https://doi.org/10.1093/nar/gkz365
Acknowledgments
Development of this method and publication was partially supported by Cotton Inc., the Alabama Agricultural Experiment Station, and the Hatch program of the National Institute of Food and Agriculture, USDA.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Pokhrel, A., Seo, S., Wang, Q., Coleman, J.J. (2022). Targeted Gene Disruption Via CRISPR/Cas9 Ribonucleoprotein Complexes 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_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1795-3_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1794-6
Online ISBN: 978-1-0716-1795-3
eBook Packages: Springer Protocols