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Precise gene replacement in rice by RNA transcript-templated homologous recombination


One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.

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Fig. 1: RNA TT-HDR of DSBs generated by the LbCpf1 nuclease.
Fig. 2: Comparison of RNP-mediated HDR efficiencies of various sources of DRTs through ddPCR.
Fig. 3: Generation of stable, precisely edited rice plants through TT-HDR.

Data availability

All data supporting the findings of this study are available in the article and its Supplementary Figures and Tables. Raw Sanger sequencing data are included in Supplementary Data 1.


  1. Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K. & Mark, A. C. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat.Commun. 7, 13274 (2016).

    CAS  Article  Google Scholar 

  2. Puchta, H. Repair of genomic double-strand breaks in somatic plant cells by one-sided invasion of homologous sequences. Plant J. 13, 331–339 (1998).

    CAS  Article  Google Scholar 

  3. Cermak, T., Baltes, N. J., Cegan, R., Zhang, Y. & Voytas, D. F. High-frequency, precise modification of the tomato genome. Genome Biol. 16, 232 (2015).

    Article  Google Scholar 

  4. Gil-Humanes, J. et al. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J. 89, 1251–1262 (2017).

    CAS  Article  Google Scholar 

  5. 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).

    CAS  Article  Google Scholar 

  6. Shi, J. et al. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol. J. 15, 207–216 (2017).

    CAS  Article  Google Scholar 

  7. Sun, Y. et al. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol. Plant 9, 628–631 (2016).

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. Derr, L. K., Strathern, J. N. & Garfinkel, D. J. RNA-mediated recombination in S. cerevisiae. Cell 67, 355–364 (1991).

    CAS  Article  Google Scholar 

  10. Nowacki, M. et al. RNA-mediated epigenetic programming of a genome-rearrangement pathway. Nature 451, 153–158 (2007).

    Article  Google Scholar 

  11. Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A. & Resnick, M. A. RNA-templated DNA repair. Nature 447, 338–341 (2007).

    CAS  Article  Google Scholar 

  12. Keskin, H. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).

    CAS  Article  Google Scholar 

  13. Chien, Y. H. & Davidson, N. RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions. Nucleic Acids Res. 5, 1627–1637 (1978).

    CAS  Article  Google Scholar 

  14. Butt, H. et al. Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide RNA molecule. Front. Plant Sci. 8, 1441 (2017).

    Article  Google Scholar 

  15. Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).

    CAS  Article  Google Scholar 

  16. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-cas system. Cell 163, 759–771 (2015).

    CAS  Article  Google Scholar 

  17. Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

    CAS  Article  Google Scholar 

  18. Kim, H. et al. CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat. Commun. 8, 14406 (2017).

    CAS  Article  Google Scholar 

  19. Tang, X. et al. A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018 (2017).

    CAS  Article  Google Scholar 

  20. Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).

    CAS  Article  Google Scholar 

  21. Mazur, B. J., Chui, C. F. & Smith, J. K. Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiol. 85, 1110–1117 (1987).

    CAS  Article  Google Scholar 

  22. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  Article  Google Scholar 

  23. Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 10, 526–529 (2017).

    CAS  Article  Google Scholar 

  24. Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

    CAS  Article  Google Scholar 

  25. Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

    CAS  Article  Google Scholar 

  26. Paix, A. et al. Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. Proc. Natl Acad. Sci. USA 114, E10745–E10754 (2017).

    CAS  Article  Google Scholar 

  27. Zhang, T., Gao, Y., Wang, R. & Zhao, Y. Production of guide RNAs in vitro and in vivo for CRISPR using ribozymes and RNA polymerase II promoters. Bio Protoc. 7, e2148 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. Li, L., Qu, R., de Kochko, A., Fauquet, C. & Beachy, R. N. An improved rice transformation system using the biolistic method. Plant Cell Rep. 12, 250–255 (1993).

    Article  Google Scholar 

  29. Wang, M., Mao, Y., Lu, Y., Tao, X. & Zhu, J.-k Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol. Plant 10, 1011–1013 (2017).

    CAS  Article  Google Scholar 

  30. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282 (1994).

    CAS  Article  Google Scholar 

  31. 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).

    CAS  Article  Google Scholar 

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We thank J.-K. Zhu for the LbCpf1 plasmid. We thank C.-Y. Wu, whose lab provided the rice transformation service. This work is partly funded by the Ministry of Agriculture and Rural Affairs of China (grant no. 2018ZX0801003B to L.X. and Y.H.), the Ministry of Science and Technology of China (grant no. 2016YFD0100500 to LX), the Ministry of Agriculture of China (grant no. 2016ZX08010003 to L.X.), and the Central Non-Profit Fundamental Research Funding supported by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (S2018QY05 to L.X.).

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Authors and Affiliations



L.X. and Y.Z. conceived the project. S.L., J.L., Y.H., M.X., J.Z., and W.D. performed the experiments. L.X. and Y.Z. wrote the manuscript.

Corresponding authors

Correspondence to Yunde Zhao or Lanqin Xia.

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The authors have filed a patent application based on the system developed in this paper.

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Supplementary information

Supplementary Information

Supplementary Figures 1–5 and Supplementary Tables 1–6

Reporting Summary

Supplementary Data 1

Sanger sequence data

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Li, S., Li, J., He, Y. et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat Biotechnol 37, 445–450 (2019).

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