Prime genome editing in rice and wheat

Abstract

Prime editors, which are CRISPR–Cas9 nickase (H840A)–reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.

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Fig. 1: Frequency of prime editing in rice and wheat protoplasts.
Fig. 2: Optimized prime editors for precise genome editing in rice.

Data availability

All data supporting the findings of this study are available in the article or in Supplementary information files, or are available from the corresponding author upon request. In terms of sequence data, the rice LOC_Os identifiers (http://rice.plantbiology.msu.edu/) are LOC_Os01g55540 (OsAAT), LOC_Os03g54790 (OsALS), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os06g04280 (OsEPSPS), LOC_Os08g03290 (OsGAPDH). The NCBI GenBank identifiers are KJ697755 (TaGW2), KF009556 (TaMLO), KJ000052 (TaGASR7), JF683316 (TaDME), GU167921 (TaLOX2), FJ459808 (TaUbi10). The deep sequencing data have been deposited in an NCBI BioProject database (accession codes PRJNA605069 and PRJNA605074). Plasmids encoding nCas9-PPE, QPM-sgR (for nicking sgRNA construction), and pH-nCas9-PPE (for pH-nCas9-PPE2/3/3b construction) will be made available through Addgene.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31788103), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24000000), and the National Key Research and Development Program of China (2016YFD0101804). A.V.A, A.R., J.L.D., and D.R.L. were supported by US National Institutes of Health (NIH) grants U01AI142756, RM1HG009490, R01EB022376, R35GM118062, and by the Howard Hughes Medical Institute (HHMI).

Author information

Q.L., Y.Z., C.X., Y.W., A.V.A., A.R, J.L.D, D.R.L., and C.G. designed the project; Q.L., Y.Z., C.X., S.W., S.J., and Z.Z. performed the experiments; Q.L., A.V.A., A.R, J.L.D, D.R.L., and C.G. wrote the manuscript.

Correspondence to Caixia Gao.

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Competing interests

The authors have submitted a patent application based on the results reported in this paper. D.R.L. is a consultant for and co-founder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Editas Medicine—companies that use genome editing.

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Supplementary information

Supplementary Figures

Supplementary Figures 1–8, Supplementary Tables 1–7, Supplementary Sequences 1–3, and Supplementary Data 1.

Reporting Summary

Unprocessed gels for Supplementary Fig. 8b

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Lin, Q., Zong, Y., Xue, C. et al. Prime genome editing in rice and wheat. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0455-x

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