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

Abstract

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.

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Acknowledgments

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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Contributions

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). https://doi.org/10.1038/s41587-019-0065-7

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  • DOI: https://doi.org/10.1038/s41587-019-0065-7

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