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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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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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).
The authors declare no competing financial interests.
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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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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