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

Tohru Yamato, Ai Handa, Takayuki Arazoe, Misa Kuroki, Akihito Nozaka, Takashi Kamakura, Shuichi Ohsato, Tsutomu Arie, Shigeru Kuwata

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number7427
JournalScientific reports
Volume9
Issue number1
DOIs
Publication statusPublished - 1 Dec 2019

Fingerprint

CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Fungi
Nucleotides
Homologous Recombination
Gene Knock-In Techniques
Magnaporthe
Genetic Engineering
Point Mutation
Genetic Recombination
Genes
Oryza

Cite this

Yamato, Tohru ; Handa, Ai ; Arazoe, Takayuki ; Kuroki, Misa ; Nozaka, Akihito ; Kamakura, Takashi ; Ohsato, Shuichi ; Arie, Tsutomu ; Kuwata, Shigeru. / Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus. In: Scientific reports. 2019 ; Vol. 9, No. 1.
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Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus. / Yamato, Tohru; Handa, Ai; Arazoe, Takayuki; Kuroki, Misa; Nozaka, Akihito; Kamakura, Takashi; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru.

In: Scientific reports, Vol. 9, No. 1, 7427, 01.12.2019.

Research output: Contribution to journalArticle

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AU - Nozaka, Akihito

AU - Kamakura, Takashi

AU - Ohsato, Shuichi

AU - Arie, Tsutomu

AU - Kuwata, Shigeru

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