Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus
Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus
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DOI：
10.1038/s41598-019-43913-0
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发表时间：
2019-05
影响因子：
4.6
通讯作者：
Tohru Yamato;Ai Handa;Takayuki Arazoe;Misa Kuroki;Akihito Nozaka;T. Kamakura;S. Ohsato;T. Arie;S. Kuwata
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文献类型：
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作者：
Tohru Yamato;Ai Handa;Takayuki Arazoe;Misa Kuroki;Akihito Nozaka;T. Kamakura;S. Ohsato;T. Arie;S. Kuwata
成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 介导的基因组编辑已成为在各种生物体中进行高效且通用的基因工程的一种有前景的方法；然而，丝状真菌中简单而精确的核苷酸修饰方法仅限于双交换型同源重组（HR）。在这项研究中，我们通过单交叉介导的 HR 在模型丝状真菌稻瘟病菌 (Magnaporthe)oryzae 中开发了一种新的基因组编辑策略。该方法包括 CRISPR/Cas9 系统和具有单个同源臂的供体载体，该同源臂在 CRISPR/Cas9 切割位点具有点突变。使用这种策略，我们证明了在所需基因组位点内高效且可自由编程的碱基替换，并且还通过缩短的（100-1000bp）单同源臂获得了目标基因破坏的突变体。我们进一步证明该方法允许在目标基因的 C 末端进行一步 GFP 基因敲入。由于基因组重组不需要供体结构内完整的原型间隔子相邻基序和宿主组件的任何额外修饰，因此该方法可用于各种丝状真菌中，进行基于 CRISPR/Cas9 的基础和应用生物学分析。
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 fungusPyricularia(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-stepGFPgene 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.