Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Targeted, efficient sequence insertion and replacement in rice


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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 ( 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 Source data are provided with this paper.


  1. Zhang, H., Zhang, J., Lang, Z., Botella, J. R. & Zhu, J.-K. Genome editing—principles and applications for functional genomics research and crop improvement. Crit. Rev. Plant Sci. 36, 291–309 (2017).

    Article  Google Scholar 

  2. Zhou, H., Liu, B., Weeks, D. P., Spalding, M. H. & Yang, B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res. 42, 10903–10914 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  Google Scholar 

  5. Lu, Y. & Zhu, J. K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 System. Mol. Plant 10, 523–525 (2017).

    Article  CAS  Google Scholar 

  6. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).

    Article  CAS  Google Scholar 

  7. Mao, Y., Botella, J. R., Liu, Y. & Zhu, J.-K. Gene editing in plants: progress and challenges. Natl Sci. Rev. 6, 421–437 (2019).

    Article  CAS  Google Scholar 

  8. Sun, Y. et al. Engineering herbicide-resistant rice plants through crispr/cas9-mediated homologous recombination of acetolactate synthase. Mol. Plant 9, 628–631 (2016).

    Article  CAS  Google Scholar 

  9. Sauer, N. J. et al. Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant Physiol. 170, 1917–1928 (2016).

    Article  CAS  Google Scholar 

  10. Li, J. et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat. Plants 2, 16139 (2016).

    Article  CAS  Google Scholar 

  11. Wang, M. et al. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system. Mol. Plant 10, 1007–1010 (2017).

    Article  CAS  Google Scholar 

  12. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  Google Scholar 

  13. Puchta, H. The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J. Exp. Bot. 56, 1–14 (2005).

    Article  CAS  Google Scholar 

  14. Sugio, T., Satoh, J., Matsuura, H., Shinmyo, A. & Kato, K. The 5′-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells. J. Biosci. Bioeng. 105, 300–302 (2008).

    Article  CAS  Google Scholar 

  15. Ren, Z. H. et al. A rice quantitative trait locus for salt tolerance encodes a sodium transporter. Nat. Genet. 37, 1141–1146 (2005).

    Article  CAS  Google Scholar 

  16. Uga, Y. et al. Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions. Nat. Genet. 45, 1097–1102 (2013).

    Article  CAS  Google Scholar 

  17. Shi, H., Lee, B. H., Wu, S. J. & Zhu, J. K. Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat. Biotechnol. 21, 81–85 (2003).

    Article  CAS  Google Scholar 

  18. Ikeda, A. et al. slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8. Plant Cell 13, 999–1010 (2001).

    Article  CAS  Google Scholar 

  19. Jobling, S. A. & Gehrke, L. Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325, 622–625 (1987).

    Article  CAS  Google Scholar 

  20. Rouached, H., Secco, D., Arpat, B. & Poirier, Y. The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis. BMC Plant Biol. 11, 19 (2011).

    Article  CAS  Google Scholar 

  21. Sahoo, D. K., Sarkar, S., Raha, S., Maiti, I. B. & Dey, N. Comparative analysis of synthetic DNA promoters for high-level gene expression in plants. Planta 240, 855–875 (2014).

    Article  CAS  Google Scholar 

  22. Feng, Z. et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 4632–4637 (2014).

    Article  CAS  Google Scholar 

  23. Li, X. M. et al. Natural alleles of a proteasome alpha2 subunit gene contribute to thermotolerance and adaptation of African rice. Nat. Genet. 47, 827–833 (2015).

    Article  CAS  Google Scholar 

  24. Hu, B. et al. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nat. Genet. 47, 834–838 (2015).

    Article  CAS  Google Scholar 

  25. Lu, Y. et al. Genome-wide targeted mutagenesis in rice using CRISPR/Cas9 system. Mol. Plant 10, 1242–1245 (2017).

    Article  CAS  Google Scholar 

  26. Liu, H. et al. CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol. Plant 10, 530–532 (2017).

    Article  CAS  Google Scholar 

  27. Nishimura, A., Aichi, I. & Matsuoka, M. A protocol for Agrobacterium-mediated transformation in rice. Nat. Protoc. 1, 2796–2802 (2006).

    Article  CAS  Google Scholar 

  28. Liu, W. et al. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol. Plant 8, 1431–1433 (2015).

    Article  CAS  Google Scholar 

Download references


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.).

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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 1–6.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 3

Unprocessed gels and western blots.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lu, Y., Tian, Y., Shen, R. et al. Targeted, efficient sequence insertion and replacement in rice. Nat Biotechnol 38, 1402–1407 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing