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Genome-wide specificity of prime editors in plants

Abstract

Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.

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Fig. 1: Effect of mismatched pegRNA on prime editing in rice protoplasts.
Fig. 2: Prime editing efficiencies at endogenous on-target and off-target sites.
Fig. 3: pegRNA-dependent off-target prime editing events analyses via WGS in rice plants.
Fig. 4: pegRNA-independent off-target analyses of prime editing events via WGS in rice plants.
Fig. 5: Analysis of retrotransposons and telomerase activities in rice.

Data availability

All data supporting the findings of this study are available in the article and supplementary figures and tables or are available from the corresponding author upon reasonable request. For sequence data, rice LOC_Os identifiers (http://rice.plantbiology.msu.edu/) are as follows: LOC_Os03g54790 (OsALS), LOC_Os03g05730 (OsCDC48), LOC_Os08g03290 (OsGAPDH), LOC_Os01g55540 (OsAAT), LOC_Os05g22940 (OsACC), LOC_Os09g26999 (OsDEP1), LOC_Os06g04280 (OsEPSPS), LOC_Os08g39890 (OsIPA1), LOC_Os08g03290 (OsGAPDH) and LOC_Os03g08570 (OsPDS). The NCBI GenBank identifiers are AP005292 and AE017097 (OsTos17). The deep sequencing and genome sequencing data have been deposited in two NCBI BioProject databases (accession codes PRJNA702625 and PRJNA636219). Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31788103 and 31971370), the National Key Research and Development Program of China (2016YFD0100602), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020100), the Chinese Academy of Sciences (QYZDY-SSW-SMC030) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2017140).

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Authors

Contributions

C.G. supervised the project. C.G., S.J. and Y.F.L. designed the experiment. S.J., Q.L., Z.Z., G.L. and K.C. performed the experiments. Y.F.L., S.J. and Y.J.L. performed the bioinformatics analyses. C.G., J.-L.Q., S.J. and Q.L. wrote the manuscript.

Corresponding author

Correspondence to Caixia Gao.

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Peer review information Nature Biotechnology thanks Nicole Gaudelli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Tables 1–16 and Sequences

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Supplementary Table 1

Primers used in this study

Supplementary Data 1

FACS data

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Source Data Supplementary Fig. 1

Source Data for Supplementary Fig. 1b

Source Data Supplementary Fig. 13a

Source Data for Supplementary Fig. 13a

Source Data Supplementary Fig. 13b

Source Data for Supplementary Fig. 13b

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Jin, S., Lin, Q., Luo, Y. et al. Genome-wide specificity of prime editors in plants. Nat Biotechnol 39, 1292–1299 (2021). https://doi.org/10.1038/s41587-021-00891-x

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