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Targeted, efficient sequence insertion and replacement in rice

Abstract

CRISPR–Cas9 methods have been applied to generate random insertions and deletions, large deletions, targeted insertions or replacements of short sequences, and precise base changes in plants1,2,3,4,5,6,7. However, versatile methods for targeted insertion or replacement of long sequences and genes, which are needed for functional genomics studies and trait improvement in crops, are few and largely depend on the use of selection markers8,9,10,11. Building on methods developed in mammalian cells12, we used chemically modified donor DNA and CRISPR–Cas9 to insert sequences of up to 2,049 base pairs (bp), including enhancers and promoters, into the rice genome at an efficiency of 25%. We also report a method for gene replacement that relies on homology-directed repair, chemically modified donor DNA and the presence of tandem repeats at target sites, achieving replacement with up to 130-bp sequences at 6.1% efficiency.

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Fig. 1: Optimization of donor DNA for targeted insertion in rice.
Fig. 2: Targeted insertion of ADHE at four endogenous rice loci.
Fig. 3: Precise genome editing in rice using the TR-HDR method.

Data availability

The authors declare that all data supporting the findings of the present study are available in the article and its supplementary figures and tables, or from the corresponding author upon request. For sequence data, rice LOC_Os IDs listed in Supplementary Table 1 are available on the Rice Genome Annotation Project site (http://rice.plantbiology.msu.edu/). The deep sequencing data were deposited with the National Center for Biotechnology Information BioProject database under the accession code PRJNA608130. Source data are provided with this paper.

Code availability

Custom script for analyzing NGS data is available at https://github.com/zhulab-ge/knockin. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by the Chinese Academy of Sciences, including the CAS Strategic Priority Research Program grant no. XDB27040101 to J.-K.Z., and by the Major Project of China on New Varieties of GMO Cultivation (grant no. 2019ZX08010-003 to F.L.).

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Authors and Affiliations

Authors

Contributions

Y.L. and J.-K.Z. designed the experiments. M.C., Y.T., R.S., J.D., F.L. and T.Z. performed the rice transformations. Y.L., Y.T., R.S., Q.Y., M.C., M.W., J.D., T.Z. and M.L. performed all the other experiments. Y.L. and J.-K.Z. wrote the manuscript.

Corresponding author

Correspondence to Jian-Kang Zhu.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–6.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 3

Unprocessed gels and western blots.

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Lu, Y., Tian, Y., Shen, R. et al. Targeted, efficient sequence insertion and replacement in rice. Nat Biotechnol 38, 1402–1407 (2020). https://doi.org/10.1038/s41587-020-0581-5

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