ARTICLE Tailor-made Talen System for Highly Efficient Targeted Gene Replacement in the Rice Blast Fungus Takayuki Arazoe,1 Tetsuo Ogawa,1 Kennosuke Miyoshi,1 Tohru Yamato,1 Shuichi Ohsato,1 Tetsushi Sakuma,2 Takashi Yamamoto,2 Tsutomu Arie,3 Shigeru Kuwata1 1
Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan; telephone: þ81 44 934 7036; e-mail: [email protected] 2 Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan 3 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
Abstract: Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA doublestrand 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. Biotechnol. Bioeng. 2015;9999: 1–8. ß 2015 Wiley Periodicals, Inc. KEYWORDS: blast fungus; engineered nuclease; filamentous fungi; gene targeting; genome editing; rice; TALEN
Introduction Filamentous fungi are a large group of multicellular eukaryotic microorganisms. This group contains species that play important roles in industry, agriculture, and medicine by producing primary Correspondence to: T. Arazoe Contract grant sponsor: Japan Society for the promotion of Science (JSPS) Grant-inAid for JSPS Fellows Contract grant number: 24.11224 Contract grant sponsor: JSPS Grant-in-Aid for Scientific Research (C) Contract grant number: 24580070 Received 13 January 2015; Revision received 4 February 2015; Accepted 5 February 2015 Accepted manuscript online xx Month 2015; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25559
ß 2015 Wiley Periodicals, Inc.
and secondary metabolites such as organic acids, enzymes, exopolysaccharides, antibiotics, and biofilms (Jiang et. al., 2013). Some species cause important plant and animal diseases including those that affect humans (Jiang et al., 2013; Kuck and Hoff, 2010). Therefore, the genomic engineering of filamentous fungi provides valuable information regarding gene functions and could improve the capacity of fungal species. In eukaryotes, DNA double-strand breaks (DSBs) can be repaired through two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). HR-mediated targeted gene replacement (TGR) is a powerful method for precise gene disruption, modification, or replacement, but its efficiency is low (
Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan; telephone: þ81 44 934 7036; e-mail: [email protected] 2 Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan 3 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
Abstract: Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA doublestrand 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. Biotechnol. Bioeng. 2015;9999: 1–8. ß 2015 Wiley Periodicals, Inc. KEYWORDS: blast fungus; engineered nuclease; filamentous fungi; gene targeting; genome editing; rice; TALEN
Introduction Filamentous fungi are a large group of multicellular eukaryotic microorganisms. This group contains species that play important roles in industry, agriculture, and medicine by producing primary Correspondence to: T. Arazoe Contract grant sponsor: Japan Society for the promotion of Science (JSPS) Grant-inAid for JSPS Fellows Contract grant number: 24.11224 Contract grant sponsor: JSPS Grant-in-Aid for Scientific Research (C) Contract grant number: 24580070 Received 13 January 2015; Revision received 4 February 2015; Accepted 5 February 2015 Accepted manuscript online xx Month 2015; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25559
ß 2015 Wiley Periodicals, Inc.
and secondary metabolites such as organic acids, enzymes, exopolysaccharides, antibiotics, and biofilms (Jiang et. al., 2013). Some species cause important plant and animal diseases including those that affect humans (Jiang et al., 2013; Kuck and Hoff, 2010). Therefore, the genomic engineering of filamentous fungi provides valuable information regarding gene functions and could improve the capacity of fungal species. In eukaryotes, DNA double-strand breaks (DSBs) can be repaired through two main pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). HR-mediated targeted gene replacement (TGR) is a powerful method for precise gene disruption, modification, or replacement, but its efficiency is low (
