Development of genome-editing technologies for plant pathogenic fungi
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Development of genome‑editing technologies for plant pathogenic fungi Takayuki Arazoe1 Received: 19 August 2020 / Accepted: 1 September 2020 © The Phytopathological Society of Japan and Springer Japan KK, part of Springer Nature 2020
Abstract In this study, we focused on the DSB repair machinery as a mechanism of asexual pathogenic and genomic evolution in P. oryzae. Using the fungal TALEN system, we demonstrated that DSBs trigger genome rearrangement with deletions, duplications, and translocations of AVR-Pita through somatic and octopic HR between repetitive sequences in the genome. In addition, based on the unique DSB repair machinery of P. oryzae, we developed original genome editing technologies using the fungal CRISPR/Cas9 system. These developed genome editing technologies would accelerate high-throughput functional genomics and reveal genome evolution mechanisms in plant pathogenic fungi. Keywords Genome editing · Pathogenicity evolutiom · Genome evolution · CRISPR/Cas9 · Rice blast fungus · Plant pathogenic fungi
Introduction Rapid and reliable genome manipulation of living organisms is crucial for understanding the fundamental basis of biological systems. Owing to recent progress in the development of programmable nucleases, such as transcription activatorlike effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, genome editing has become a powerful tool for advanced genome manipulation in various research fields (Adli 2018). TALEN comprises a DNA-binding domain and FokI endonuclease domain, which uses protein structure for target DNA recognition. CRISPR/Cas systems, which are RNA-guided programmable nucleases, can recognize target DNA by single-guide (sg) RNA specificity. The CRISPR/Cas9 system from Streptococcus pyogenes has been the most widely used for genome editing because it is This article is an abstract of the paper which was going to be presented by a winner of the Young Scientist Award at the 2020 Annual Meeting of the Phytopathological Society of Japan in Kadoshima. * Takayuki Arazoe [email protected] 1
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Chiba 278‑8510, Japan
simpler and more flexible than other programmable nucleases. TALEN, however, can introduce more specific DNA double-strand breaks (DSBs) than CRISPR/Cas9 because it allows the design of longer target sequences and dimerization of the FokI domain that is required for DNA cleavage. The fundamental strategy for genome editing relies on triggering the host DNA repair mechanisms by site-specific DSBs using artificial programmable nucleases. In eukaryotes, DSBs are mainly repaired by two competing pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Generally, NHEJ-mediated genome editing can induce efficient small insertions and/or deletions at the targeted locus, but the mutation type cannot be controlled. HR-mediated genome editing can be harnessed
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