Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing has become a promising approach for efficient and versatile genetic engineering in various organisms; however, simple and precise nucleotide modification methods in filamentous fungi have been restricted to double crossover type homologous recombination (HR). In this study, we developed a novel genome editing strategy via single crossover-mediated HR in the model filamentous fungus Pyricularia (Magnaporthe) oryzae. This method includes the CRISPR/Cas9 system and a donor vector harboring a single homology arm with point mutations at the CRISPR/Cas9 cleavage site. Using this strategy, we demonstrated highly efficient and freely programmable base substitutions within the desired genomic locus, and target gene disrupted mutants were also obtained via a shortened (100-1000 bp) single homology arm. We further demonstrated that this method allowed a one-step GFP gene knock-in at the C-terminus of the targeted gene. Since the genomic recombination does not require an intact protospacer-adjacent motif within the donor construct and any additional modifications of host components, this method can be used in various filamentous fungi for CRISPR/Cas9-based basic and applied biological analyses.

Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi.

Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus.

Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA double-strand breaks (DSBs) at a specific site in the desired locus within genome are utilized in a rapidly developing genome editing technology via DSBs repair. However, the use of engineered nucleases in filamentous fungi has not been validated. In this study, we demonstrated that tailor-made transcriptional activator-like effector nucleases (TALENs) system, Platinum-Fungal TALENs (PtFg TALENs), could improve the efficiency of homologous recombination-mediated targeted gene replacement by up to 100% in the rice blast fungus Pyricularia oryzae. This high-efficiency PtFg TALEN has great potential for basic and applied biological applications in filamentous fungi.