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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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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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.
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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.
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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.
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Supplementary Figs. 1–10 and Tables 1–5.
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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DOI: https://doi.org/10.1038/s41477-021-01019-4
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