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

Nature Plants volume 2, Article number: 16139 (2016) Cite this article

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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.

The type II prokaryotic CRISPR–Cas9 system has been used to generate targeted DNA double-strand breaks (DSBs)2–4, which stimulate DNA repair by two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination5. NHEJ is the dominant process; in it the broken DNA ends are simply rejoined6. However, this process is error prone, creating small insertions and/or deletions (indels) at the break, and has been exploited to create targeted gene knockouts in multiple cell types and organisms including plants7,8. Homologous recombination is a less frequent but high-fidelity process, in which targeted gene replacements and insertions can be created using a homologous donor DNA9,10. These changes have been of great value for functional genomics studies in plants and may help create agronomically valuable traits. However, generating precise modifications, such as point mutations, gene replacements and gene knock-ins by homologous recombination remains a serious challenge11. Only a handful of cases have been reported in which endogenous plant genes were accurately modified by homologous recombination, and even the success in these cases generally relied on the use of specifically designed selection schemes11–19. There is a pressing need for simple, universal and efficient methods for replacing or knocking-in DNA fragments to any targeted genes. Since NHEJ is the dominant repair process and minor alterations of intronic sequence may not affect alternative splicing and expression level of the target gene, we chose to modify introns with Cas9 to generate the same results as achieved by homologous recombination. Here, we describe intron-targeting methods using the NHEJ pathway to generate gene replacements and insertions that create glyphosate-resistant rice plants.

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Figure 1: Intron-targeted site-specific gene replacement.
Figure 2: Intron-mediated targeted gene insertions.
Figure 3: EPSPS with TIPS substitutions confers resistance to glyphosate.

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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).

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Author notes

  1. Jun Li and Xiangbing Meng: These authors contributed equally to this work.

Authors and 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, 100049, Beijing, 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

Authors
  1. Jun 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.

Corresponding authors

Correspondence to Jiayang Li or Caixia Gao.

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

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Supplementary Information

Supplementary Table of Contents, Supplementary Figures 1-10, Supplementary Table 1-6. (PDF 1423 kb)

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Li, J., Meng, X., Zong, Y. et al. Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9. Nature Plants 2, 16139 (2016). https://doi.org/10.1038/nplants.2016.139

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