Abstract
Fast and flexible genome manipulation is a powerful strategy for an in-depth understanding of molecular mechanisms in biological research. In recent years, CRISPR/Cas9-mediated genome editing has been used as a reliable genome manipulation method in a broad range of biological research including studies of filamentous fungi. The CRISPR/Cas9 system comprises a single-guide RNA (sgRNA) and a Cas9 protein, and the Cas9/sgRNA complex catalyzes a DNA double-strand break at the desired genomic locus. This protocol describes a fundamental CRISPR/Cas9 methodology that includes the design of the target sequence, construction of the CRISPR/Cas9 expression vector, and transformation for genome editing in Pyricularia (Magnaporthe) oryzae. This allows efficient targeted gene disruption, base editing, and reporter gene knock-in without any additional modifications of the host components. This protocol would be suitable for applying other CRISPR/Cas technologies and various functional genomics in P. oryzae.
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Acknowledgments
This work is supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (A) (Grant Number 16H02536) and Grant-in-Aid for Young Scientists (B) (Grant Number 15Â K18647).
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Arazoe, T. (2021). Genome Editing Using CRISPR/Cas9 System in the Rice Blast Fungus . In: Jacob, S. (eds) Magnaporthe oryzae. Methods in Molecular Biology, vol 2356. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1613-0_12
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DOI: https://doi.org/10.1007/978-1-0716-1613-0_12
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