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.
Keywords: CRISPR/Cas9; Filamentous fungi; Functional genomics; Genome editing; Homologous recombination; Pyricularia (Magnaporthe) oryzae.