title: Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.
abstract: 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.
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Arazoe
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Kennosuke
authors.initials: K
authors.lastname: Miyoshi
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yamato
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Ohsato
authors.affiliation: Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-0509, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arie
authors.affiliation: Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan. arazoet@gmail.com, kuwata@meiji.ac.jp.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kuwata
mesh_terms: D064113:CRISPR-Cas Systems; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D059765:Homologous Recombination; D020082:Magnaporthe / Q000201:enzymology / Q000235:genetics*; D012275:Oryza / Q000382:microbiology
keywords: CRISPR/Cas system; engineered nuclease; filamentous fungi; gene targeting; genome editing; rice blast fungus