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PAM-less plant genome editing using a CRISPR–SpRY toolbox


The rapid development of the CRISPR–Cas9, –Cas12a and –Cas12b genome editing systems has greatly fuelled basic and translational plant research1,2,3,4,5,6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox.

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Fig. 1: PAM-less gene editing by SpRY in the protoplasts of rice and the Dahurian larch.
Fig. 2: Comparison of genome editing and self-cleavage by SpRY in stable rice lines.
Fig. 3: PAM-less C-to-T base editing in rice.
Fig. 4: PAM-less A-to-G base editing in rice.

Data availability

Regarding accession codes, the five Gateway-compatible Cas9 entry vectors are available from Addgene: pYPQ166-SpRY (no. 161520, zSpRY), pYPQ266E (no. 161521, SpRY-D01A-PmCDA1-UGI), pYPQ262m (no. 161522, wtTadA-TadA*-zSpCas9-D10A), pYPQ262-ABE8e (no. 161523, TadA8e-zSpCas9-D10A) and pYPQ262B-ABE8e (no. 161524, TadA8e-zSpRY-D10A). The high-throughput sequencing data sets have been submitted to the National Center for Biotechnology information database under Sequence Read Archive Bio Project ID PRJNA665932.


  1. Zhang, Y., Malzahn, A. A., Sretenovic, S. & Qi, Y. The emerging and uncultivated potential of CRISPR technology in plant science. Nat. Plants 5, 778–794 (2019).

    PubMed  Article  Google Scholar 

  2. Tang, X. et al. A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018 (2017).

    CAS  PubMed  Article  Google Scholar 

  3. Ming, M. et al. CRISPR–Cas12b enables efficient plant genome engineering. Nat. Plants 6, 202–208 (2020).

    CAS  PubMed  Article  Google Scholar 

  4. Zhu, H., Li, C. & Gao, C. Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat. Rev. Mol. Cell Biol. 21, 661–677 (2020).

    CAS  PubMed  Article  Google Scholar 

  5. Schindele, A., Dorn, A. & Puchta, H. CRISPR/Cas brings plant biology and breeding into the fast lane. Curr. Opin. Biotechnol. 61, 7–14 (2020).

    CAS  PubMed  Article  Google Scholar 

  6. 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, 84 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  7. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Steinert, J., Schiml, S., Fauser, F. & Puchta, H. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J. 84, 1295–1305 (2015).

    CAS  PubMed  Article  Google Scholar 

  11. Kaya, H., Mikami, M., Endo, A., Endo, M. & Toki, S. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci. Rep. 6, 26871 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  13. Hu, X. et al. Expanding the range of CRISPR/Cas9 genome editing in rice. Mol. Plant 9, 943–945 (2016).

    CAS  PubMed  Article  Google Scholar 

  14. Endo, M. et al. Genome editing in plants by engineered CRISPR–Cas9 recognizing NG PAM. Nat. Plants 5, 14–17 (2019).

    CAS  PubMed  Article  Google Scholar 

  15. Hua, K., Tao, X., Han, P., Wang, R. & Zhu, J. K. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences. Mol. Plant 12, 1003–1014 (2019).

    CAS  PubMed  Article  Google Scholar 

  16. Ren, B. et al. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice. Mol. Plant 12, 1015–1026 (2019).

    CAS  PubMed  Article  Google Scholar 

  17. Zhong, Z. Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol. Plant 12, 1027–1036 (2019).

    CAS  PubMed  Article  Google Scholar 

  18. Sretenovic, S. et al. Expanding plant genome editing scope by an engineered iSpyMacCas9 system targeting the A-rich PAM sequences. Plant Commun. (2020).

  19. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Zhong, Z. et al. Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol. Plant 11, 999–1002 (2018).

    CAS  PubMed  Article  Google Scholar 

  21. Zhou, J. et al. CRISPR–Cas9 based genome editing reveals new insights into microRNA function and regulation in rice. Front. Plant Sci. 8, 1598 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  22. Rodriguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E. & Lippman, Z. B. Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470–480 (2017).

    CAS  PubMed  Google Scholar 

  23. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 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).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR–Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

    CAS  PubMed  Article  Google Scholar 

  26. Tang, X. et al. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol. J. 17, 1431–1445 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    PubMed  Article  Google Scholar 

  28. Kuang, Y. et al. Base-editing-mediated artificial evolution of OsALS1 in planta to develop novel herbicide-tolerant rice germplasms. Mol. Plant 13, 565–572 (2020).

    CAS  PubMed  Article  Google Scholar 

  29. Zhang, Y. & Qi, Y. CRISPR enables directed evolution in plants. Genome Biol. 20, 83 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  30. Li, C. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat. Biotechnol. 38, 875–882 (2020).

    CAS  PubMed  Article  Google Scholar 

  31. Butt, H. et al. CRISPR-directed evolution of the spliceosome for resistance to splicing inhibitors. Genome Biol. 20, 73 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  32. Lapinaite, A. et al. DNA capture by a CRISPR–Cas9-guided adenine base editor. Science 369, 566–571 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Allen, F. et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat. Biotechnol. 37, 64–72 (2018).

    Article  Google Scholar 

  35. Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning. Cell 182, 463–480 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. Tang, X. et al. A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol. Plant 9, 1088–1091 (2016).

    CAS  PubMed  Article  Google Scholar 

  37. Zhong, Z. et al. Intron-based single transcript unit CRISPR systems for plant genome editing. Rice 13, 8 (2020).

    PubMed  PubMed Central  Article  Google Scholar 

  38. Ren, Q. et al. Bidirectional promoter-based CRISPR-Cas9 systems for plant genome editing. Front Plant Sci. 10, 1173 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  39. Zhou, J. et al. Multiplex QTL editing of grain-related genes improves yield in elite rice varieties. Plant Cell Rep. 38, 475–485 (2019).

    CAS  PubMed  Article  Google Scholar 

  40. Wang, B. et al. Targeted mutagenesis of NAC transcription factor gene, OsNAC041, leading to salt sensitivity in rice. Rice Sci. 26, 98–108 (2019).

    Article  Google Scholar 

  41. You, Q. et al. CRISPRMatch: an automatic calculation and visualization tool for high-throughput CRISPR genome-editing data analysis. Int. J. Biol. Sci. 14, 858–862 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Zheng, X. et al. Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Rep. 35, 1545–1554 (2016).

    CAS  PubMed  Article  Google Scholar 

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This work was supported by the National Transgenic Major Project (award no. 2018ZX08020-003), the National Natural Science Foundation of China (award nos. 31771486, 32072045 and 31960423), the State Key Laboratory of Rice Biology (20200205) and the Science Strength Promotion Program of the University of Electronic Science and Technology of China (UESTC) to Yong Zhang and X.Z. This work was also supported by the National Science Foundation Plant Genome Research Program grants (award nos. IOS-1758745 and IOS-2029889), the US Department of Agriculture Biotechnology Risk Assessment Grant Program competitive grants (award nos. 2018-33522-28789 and 2020-33522-32274) and the Emergency Citrus Disease Research and Extension Program (award no. 2020-70029-33161) to Y.Q. S.S. is a Foundation for Food and Agriculture Research Fellow. Y.C. was supported by a scholarship from China Scholarship Council. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of these funding agencies.

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Authors and Affiliations



Y.Q. and Yong Zhang designed the experiments. S.S., Q.R., S.L., Y.C., D.Y., C.P. and Yingxiao Zhang generated all the constructs. Q.R. and S.L. carried out rice protoplast transformation and data analysis. X.T. performed the data analysis. L.H. performed the Dahurian larch protoplast transformation and analysis. Q.R., S.L., Y.H., L.L. and Y.G. conducted rice stable transformation. Q.R. and S.L. analysed rice transgenic lines. L.L., Z.Z., G.L. and X.Z. helped with rice phenotype and genotype data analysis. W.L., L.Q. and C.L. collected the Dahurian larch material and developed the larch callus culture protocol. Y.Q., Yong Zhang, Q.R. and S.S. wrote the paper with input from other authors. All authors read and approved the final manuscript.

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Correspondence to Yiping Qi or Yong Zhang.

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Peer review information Nature Plants thanks Arjun Khakhar the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Ren, Q., Sretenovic, S., Liu, S. et al. PAM-less plant genome editing using a CRISPR–SpRY toolbox. Nat. Plants 7, 25–33 (2021).

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