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Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9

Nature Plants volume 2, Article number: 16139 (2016) | Download Citation

Abstract

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants1, but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

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References

  1. 1.

    & Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 12, e1001877 (2014).

  2. 2.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  3. 3.

    et al. RNA-Guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  4. 4.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  5. 5.

    & Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

  6. 6.

    & Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res. 25, 4650–4657 (1997).

  7. 7.

    & A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

  8. 8.

    , & Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnol. J. 14, 483–495 (2016).

  9. 9.

    , & Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc. Natl Acad. Sci. USA 93, 5055–5060 (1996).

  10. 10.

    , , & Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

  11. 11.

    , , , & High-frequency, precise modification of the tomato genome. Genome Biol. 16, 232 (2015).

  12. 12.

    et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442–445 (2009).

  13. 13.

    et al. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol. Plant 9, 628–631 (2016).

  14. 14.

    et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459, 437–441 (2009).

  15. 15.

    et al. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol. 169, 931–945 (2015).

  16. 16.

    et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol. 161, 20–27 (2013).

  17. 17.

    , , , & Efficient gene targeting by homologous recombination in rice. Nat. Biotechnol. 20, 1030–1034 (2002).

  18. 18.

    , , , & Positive-negative-selection-mediated gene targeting in rice. Front. Plant Sci. 5, 748–754 (2014).

  19. 19.

    , , , & DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).

  20. 20.

    et al. Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol. 129, 1265–1275 (2002).

  21. 21.

    et al. Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance. Plant Physiol. 167, 1440–1447 (2015).

  22. 22.

    , & Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

  23. 23.

    et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327–338 (2014).

  24. 24.

    & Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 3, 824–834 (2008).

  25. 25.

    et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).

  26. 26.

    Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu. Rev. Plant Biol. 58, 267–294 (2007).

  27. 27.

    , & Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398 (2005).

  28. 28.

    et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol. J. 12, 797–807 (2014).

  29. 29.

    et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).

  30. 30.

    , , & Genome editing in rice and wheat using the CRISPR/Cas system. Nat. Protoc. 9, 2395–2410 (2014).

  31. 31.

    , , , & An improved rice transformation system using the biolistic method. Plant Cell Rep. 12, 250–255 (1993).

Download references

Acknowledgements

The authors thank J.-L. Qiu from Institute of Microbiology, Chinese Academy of Sciences and D.F. Voytas from University of Minnesota for helpful discussions and insightful comments on this manuscript. This work was supported by grants from the Ministry of Science and Technology (2016YFD0101804), the Ministry of Agriculture of China (2016ZX08010002 and 2014ZX0801003B) and the National Natural Science Foundation of China (31420103912, 31271795, 31570369 and 31501376).

Author information

Author notes

    • Jun Li
    •  & Xiangbing Meng

    These authors contributed equally to this work.

Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    • Jun Li
    • , Yuan Zong
    • , Kunling Chen
    • , Huawei Zhang
    • , Jinxing Liu
    •  & Caixia Gao
  2. University of Chinese Academy of Sciences, Beijing 100049, China

    • Jun Li
    •  & Yuan Zong
  3. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    • Xiangbing Meng
    •  & Jiayang Li

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Contributions

C.X.G., J.Y.L. and J.L. designed the experiments; J.L., X.B.M., Y.Z., K.L.C., H.W.Z. and J.X.L. performed the experiments; and C.X.G., J.Y.L. and J.L. wrote the manuscript.

Competing interests

The authors have filed a patent application based on the results reported in this paper.

Corresponding authors

Correspondence to Jiayang Li or Caixia Gao.

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    Supplementary Table of Contents, Supplementary Figures 1-10, Supplementary Table 1-6.

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DOI

https://doi.org/10.1038/nplants.2016.139

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