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.
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Li, G., Liu, Y. G. & Chen, Y. Genome-editing technologies: the gap between application and policy. Sci. China Life Sci. 62, 1534–1538 (2019).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).
Zhu, H., Li, C. & Gao, C. Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat. Rev. Mol. Cell Bio. 21, 661–677 (2020).
Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).
Bosch, J. A., Birchak, G. & Perrimon, N. Precise genome engineering in Drosophila using prime editing. Proc. Natl Acad. Sci. USA 118, e2021996118 (2021).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Veillet, F. et al. Prime editing is achievable in the tetraploid potato, but needs improvement. Preprint at bioRxiv https://doi.org/10.1101/2020.06.18.159111 (2020).
Jiang, Y. et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes of maize. Genome Biol. 21, 257 (2020).
Wang, L. et al. Spelling changes and fluorescent tagging with prime editing vectors for plants. Preprint at bioRxiv https://doi.org/10.1101/2020.07.16.206276 (2020).
Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Li, J., Hong, S., Chen, W., Zuo, E. & Yang, H. Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing. J. Genet. Genomics 46, 513–521 (2019).
Tang, X. et al. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol. 19, 59 (2018).
Fu et al. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Jin, S. et al. Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol. Cell. 79, 728–740 (2020).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
Wilm, A. et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 40, 11189–11201 (2012).
Kim, S. et al. Strelka2: fast and accurate calling of germline and somatic variants. Nat. Methods 15, 591–594 (2018).
Mitsuhara, I. et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol. 37, 49–59 (1996).
Robinson, J. T. et al. Integrative Genomics Viewer. Nat. Biotechnol. 29, 24–26 (2011).
Gaudelli et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Hirochika, H., Sugimoto, K., Otsuki, Y., Tsugawa, H. & Kanda, M. Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl Acad. Sci. USA 93, 7783–7788 (1996).
Piffanelli, P. et al. Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library. Plant Mol. Biol. 65, 587–601 (2007).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).
Mizuno, H. et al. Characterization of chromosome ends on the basis of the structure of TrsA subtelomeric repeats in rice (Oryza sativa L.). Mol. Genet. Genomics 280, 19–24 (2008).
Demeulemeester, J., De Rijck, J., Gijsbers, R. & Debyser, Z. Retroviral integration: site matters: mechanisms and consequences of retroviral integration site selection. Bioessays 37, 1202–1214 (2015).
Tsuruyama, T., Hiratsuka, T. & Yamada, N. Hotspots of MLV integration in the hematopoietic tumor genome. Oncogene 36, 1169–1175 (2017).
Toki, S. et al. Expression of a maize ubiquitin gene promoter-bar chimeric gene in transgenic rice plants. Plant Physiol. 100, 1503–1507 (1992).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Tycko, J., Myer, V. E. & Hsu, P. D. Methods for optimizing CRISPR–Cas9 genome editing specificity. Mol. Cell 63, 355–370 (2016).
Schmid-Burgk, J. L. et al. Highly parallel profiling of Cas9 variant specificity. Mol. Cell 78, 794–800 (2020).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Chuai, G. H., Wang, Q. L. & Liu, Q. In silico meets in vivo: towards computational CRISPR-based sgRNA design. Trends Biotechnol. 35, 12–21 (2017).
Kim, N. et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat. Biotechnol. 38, 1328–1336 (2020).
Liu, X. et al. SeqCor: correct the effect of gRNA sequences in CRISPR/Cas9 screenings by machine learning algorithm. J. Genet. Genomics https://doi.org/10.1016/j.jgg.2020.10.007 (2020).
Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 (2013).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).
Zhang, T. et al. BIGpre: a quality assessment package for next-generation sequencing data. Genom. Proteom. Bioinf. 9, 238–244 (2011).
Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019).
Su, L., Li, A., Li, H., Chu, C. & Qiu, J. L. Direct modulation of protein level in Arabidopsis. Mol. Plant 6, 1711–1714 (2013).
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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