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CRISPR/Cas9 in rice can induce new mutations in later generations, leading to chimerism and unpredicted segregation of the targeted mutation

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Abstract

CRISPR/Cas9 is a novel tool for targeted mutagenesis and is applicable to plants, including rice. Previous reports on CRISPR/Cas9 in rice have demonstrated that target mutations are transmitted to the next generation in accordance with Mendelian law, but heritability of the target mutation and the role of inherited Cas9 gene have not been fully elucidated. Here, we targeted the rice phytoene desaturase gene, mutants of which exhibit an albino phenotype, by using CRISPR/Cas9 and analyzed segregation of target mutations. Agrobacterium-mediated methods using immature embryos successfully transformed a CRISPR/Cas9 system into five rice cultivars and subsequently induced mutation. Unpredicted segregations, with more mutants than theoretically predicted, were frequently found in T1 plants from monoallelic T0 mutants. Chimeric plants with both biallelic and monoallelic mutated cells were also observed in the T1. Next, we followed segregation of a target mutation in the T2 from monoallelic T1 mutants. When T1 mutants possessed Cas9, unpredicted segregations of the target mutation and chimeric plants were observed again in the T2. When T1 mutants did not possess Cas9, segregation of the target mutations followed Mendelian law and no chimeric plants appeared in the T2. T2 mutants with Cas9 had mutations different from the original ones found in T0. Our results indicated that inherited Cas9 was still active in later generations and could induce new mutations in the progeny, leading to chimerism and unpredicted segregation. We conclude that Cas9 has to be eliminated by segregation in T1 to generate homozygous mutants without chimerism or unpredicted segregation.

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Acknowledgements

I thank the Africa Rice Center for providing the seeds of NERICA1 and the International Center for Tropical Agriculture for providing the seeds of Curinga. This work was supported by grants from the Ministry of Agriculture, Forestry, and Fisheries of Japan. I am grateful for the excellent technical support and valuable suggestions provided by Dr. Yasunari Fujita, Dr. Yukari Nagatosi, Eiko Tamaki, Ryoko Agata, Uday Mitsuyasu, Emiko Kishi, Megumu Yano, and Masami Toyoshima of the Japan International Research Center for Agricultural Sciences.

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  1. Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS), Ishigaki, Okinawa, 907-0002, Japan

    Takuma Ishizaki

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Correspondence to Takuma Ishizaki.

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Ishizaki, T. CRISPR/Cas9 in rice can induce new mutations in later generations, leading to chimerism and unpredicted segregation of the targeted mutation. Mol Breeding 36, 165 (2016). https://doi.org/10.1007/s11032-016-0591-7

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