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

Abstract

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.

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Acknowledgements

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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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). https://doi.org/10.1038/s41477-020-00827-4

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