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A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice

Nature Plants volume 7, pages 1445–1452 (2021)Cite this article

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Abstract

Structural variations (SVs), such as inversion and duplication, contribute to important agronomic traits in crops1. Pan-genome studies revealed that SVs were a crucial and ubiquitous force driving genetic diversification2,3,4. Although genome editing can effectively create SVs in plants and animals5,6,7,8, the potential of designed SVs in breeding has been overlooked. Here, we show that new genes and traits can be created in rice by designed large-scale genomic inversion or duplication using CRISPR/Cas9. A 911 kb inversion on chromosome 1 resulted in a designed promoter swap between CP12 and PPO1, and a 338 kb duplication between HPPD and Ubiquitin2 on chromosome 2 created a novel gene cassette at the joint, promoterUbiquitin2::HPPD. Since the original CP12 and Ubiquitin2 genes were highly expressed in leaves, the expression of PPO1 and HPPD in edited plants with homozygous SV alleles was increased by tens of folds and conferred sufficient herbicide resistance in field trials without adverse effects on other important agronomic traits. CRISPR/Cas-based genome editing for gene knock-ups has been generally considered very difficult without inserting donor DNA as regulatory elements. Our study challenges this notion by providing a donor-DNA-free strategy, thus greatly expanding the utility of CRISPR/Cas in plant and animal improvements.

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Fig. 1: Temperate-free PPO1 knock-up in rice as a result of CRISPR/Cas9-induced 911-kb inversion.
Fig. 2: T1 plants with inversion alleles showed tolerance to FCD herbicide under field conditions.
Fig. 3: Temperate-free HPPD knock-up in rice as a result of CRISPR/Cas9-induced 338-kb duplication.
Fig. 4: T1 plants with duplication alleles showed tolerance to bipyrazone herbicide under field conditions.

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Data availability

The data that support the findings of this study have been deposited into the NCBI Sequence Read Archive under accession number PRJNA765115 and CNGB Sequence Archive (CNSA)40 of China National GeneBank DataBase (CNGBdb)41 with accession number CNP0001922. Source data are provided with this paper.

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Acknowledgements

This work was supported by funds from Qingdao Kingagroot Compounds Company and grants from National Natural Science Foundation of China (award no. 31872933 to L.J.).

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Author notes

  1. These authors contributed equally: Yu Lu, Jiyao Wang, Bo Chen, Sudong Mo, Lei Lian.

Authors and Affiliations

  1. Department of Plant Biosecurity, Key Laboratory of Pest Monitoring and Green Management, Ministry of Agriculture and Rural Affairs, College of Plant Protection, China Agricultural University, Beijing, China

    Yu Lu, Yating Wang, Zhiting Liu & Linjian Jiang

  2. Qingdao Kingagroot Compounds Co. Ltd, Qingdao, China

    Jiyao Wang, Bo Chen, Sudong Mo, Lei Lian, Yanmin Luo, Dehui Ding, Yanhua Ding, Qing Cao, Yucai Li, Yong Li, Guizhi Liu, Qiqi Hou, Tingting Cheng, Junting Wei, Yanrong Zhang, Guangwu Chen, Chao Song, Qiang Hu & Huarong Li

  3. State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang, China

    Lei Lian & Baoan Song

  4. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Shuai Sun

  5. BGI-Qingdao, BGI-Shenzhen, Qingdao, China

    Shuai Sun & Guangyi Fan

  6. Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Jian-Kang Zhu

Authors
  1. Yu Lu
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Contributions

L.J., H.L., J.-K.Z. and B.S. supervised this project. Y. Lu, J.W., B.C., S.M. and L.L. performed or led key experiments with the help from other authors: Q.C., Yong Li and D.D. for genotyping; Q. Hou and G.L. for protein expression and purification; Yucai Li, Y.D., J.W., Q. Hu, T.C., Y. Luo., Y.W. and Z.L. for vector construction and rice transformation; and C.S., Y.Z. and G.C. for field tests. S.S. and G.F. performed genome SV analysis using NGS deep sequencing data. J.-K.Z., H.L. and L.J. wrote the manuscript with contributions from other co-authors. L.J. conceived the idea.

Corresponding authors

Correspondence to Baoan Song, Jian-Kang Zhu, Huarong Li or Linjian Jiang.

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Competing interests

A patent was filed to patent office in 2019 in China and L.J., H.L., J.W., Y. Lu and S.M. were listed as inventors. All other authors have no competing interests.

Additional information

Peer review information Nature Plants thanks Yaoguang Liu, Sergei Svitashev and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–5.

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Source data

Source Data Fig. 1

Uncropped and unprocessed scans of gels for Fig. 1c.

Source Data Fig. 3

Uncropped and unprocessed scans of gels for Fig. 3d.

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Lu, Y., Wang, J., Chen, B. et al. A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice. Nat. Plants 7, 1445–1452 (2021). https://doi.org/10.1038/s41477-021-01019-4

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