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

• Published in: Biotechnology and bioengineering 2015-12, Vol.112 (12), p.2543-2549
• Main Authors: Arazoe, Takayuki, Miyoshi, Kennosuke, Yamato, Tohru, Ogawa, Tetsuo, Ohsato, Shuichi, Arie, Tsutomu, Kuwata, Shigeru
• Format: Article
• Language: English
• Subjects: CRISPR-Cas Systems; CRISPR/Cas system; Deoxyribonucleic acid; DNA; engineered nuclease; filamentous fungi; Fungi; Fungi - enzymology; Fungi - genetics; Gene expression; Gene sequencing; gene targeting; Gene Targeting - methods; Genes; Genetics, Microbial - methods; genome editing; Genomes; Homologous Recombination; Homology; Magnaporthe - enzymology; Magnaporthe - genetics; Nuclease; Organisms; Oryza - microbiology; Pyricularia oryzae; Ribonucleic acids; rice blast fungus; RNA polymerase
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.25662

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. Biotechnol. Bioeng. 2015;112: 2543–2549. © 2015 Wiley Periodicals, Inc. Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. The authors demonstrated that tailor‐made CRISPR/Cas system, consisting of the endogenous RNA polymerase II promoter for the expression of the single‐guide RNA and the codon‐optimized Cas9 nuclease, could improve the efficiencies of homologous recombination‐mediated gene targeting in the rice blast fungus.