Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9

Nature Plants volume 6, pages 773–779 (2020)Cite this article

Subjects

Abstract

Genome-editing technologies using CRISPR–Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR–Cas reagent delivery methods may be associated with unanticipated genome changes or damage1,2, with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis3. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR–Cas9 ribonucleoproteins into protoplasts4 or in vitro fertilized zygotes5. However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR–Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency6,7,8,9. Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR–Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR–Cas9 gene editing in plants through virus infection.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Engineering SYNV vectors for delivery of CRISPR–Cas9 for N. benthamiana- targeted gene mutagenesis.
Fig. 2: Single and multiplex mutagenesis, and chromosome deletions of endogenous gene loci by SYNV-delivered CRISPR–Cas9.
Fig. 3: Phenotypes and genotypes of M0 plants regenerated from virus-infected leaf tissues.
Fig. 4: Phenotypic segregation patterns of tetra-allelic M0 lines containing non-frameshift PDS homoeoalleles.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are available in the article or its Supplementary Information, or from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Jupe, F. et al. The complex architecture and epigenomic impact of plant T-DNA insertions. PLoS Genet. 15, e1007819 (2019).

    Article  Google Scholar 

  2. Liu, J. et al. Genome-scale sequence disruption following biolistic transformation in rice and maize. Plant Cell 31, 368–383 (2019).

    Article  CAS  Google Scholar 

  3. Voytas, D. F. & Gao, C. Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 12, e1001877 (2014).

    Article  Google Scholar 

  4. Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR–Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

    Article  CAS  Google Scholar 

  5. Toda, E. et al. An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. Nat. Plants 5, 363–368 (2019).

    Article  CAS  Google Scholar 

  6. Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR–Cas9 DNA or RNA. Nat. Commun. 7, 12617 (2016).

    Article  CAS  Google Scholar 

  7. Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K. & Mark Cigan, A. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016).

    Article  CAS  Google Scholar 

  8. Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR–Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).

    Article  CAS  Google Scholar 

  9. Li, S. et al. Precise gene replacement in rice by RNA transcript-templated homologous recombination. Nat. Biotechnol. 37, 445–450 (2019).

    Article  CAS  Google Scholar 

  10. Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 16, 387–399 (2017).

    Article  CAS  Google Scholar 

  11. Honig, A. et al. Transient expression of virally delivered meganuclease in planta generates inherited genomic deletions. Mol. Plant 8, 1292–1294 (2015).

    Article  CAS  Google Scholar 

  12. Marton, I. et al. Nontransgenic genome modification in plant cells. Plant Physiol. 154, 1079–1087 (2010).

    Article  CAS  Google Scholar 

  13. Cody, W. B. & Scholthof, H. B. Plant virus vectors 3.0: transitioning into synthetic genomics. Annu. Rev. Phytopathol. 57, 211–230 (2019).

    Article  CAS  Google Scholar 

  14. Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. & Voytas, D. F. DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).

    Article  CAS  Google Scholar 

  15. Cody, W. B., Scholthof, H. B. & Mirkov, T. E. Multiplexed gene editing and protein overexpression using a tobacco mosaic virus viral vector. Plant Physiol. 175, 23–35 (2017).

    Article  CAS  Google Scholar 

  16. Gao, Q. et al. Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies. New Phytol. 223, 2120–2133 (2019).

    Article  CAS  Google Scholar 

  17. Dietzgen, R. G., Kondo, H., Goodin, M. M., Kurath, G. & Vasilakis, N. The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins. Virus Res. 227, 158–170 (2017).

    Article  CAS  Google Scholar 

  18. Finke, S. & Conzelmann, K. K. Recombinant rhabdoviruses: vectors for vaccine development and gene therapy. Curr. Top. Microbiol. Immunol. 292, 165–200 (2005).

    CAS  PubMed  Google Scholar 

  19. Bukreyev, A., Skiadopoulos, M. H., Murphy, B. R. & Collins, P. L. Nonsegmented negative-strand viruses as vaccine vectors. J. Virol. 80, 10293–10306 (2006).

    Article  CAS  Google Scholar 

  20. Jackson, A. O. & Li, Z. Developments in plant negative-strand RNA virus reverse genetics. Annu. Rev. Phytopathol. 54, 469–498 (2016).

    Article  CAS  Google Scholar 

  21. Wang, Q. et al. Rescue of a plant negative-strand RNA virus from cloned cDNA: Insights into enveloped plant virus movement and morphogenesis. PLoS Pathog. 11, e1005223 (2015).

    Article  Google Scholar 

  22. Xie, K., Minkenberg, B. & Yang, Y. Boosting CRISPR–Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).

    Article  CAS  Google Scholar 

  23. Ruiz, M. T., Voinnet, O. & Baulcombe, D. C. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937–946 (1998).

    Article  CAS  Google Scholar 

  24. Wille, A. C. & Lucas, W. J. Ultrastructural and histochemical studies on guard cells. Planta 160, 129–142 (1984).

    Article  CAS  Google Scholar 

  25. Han, G. Z. & Worobey, M. Homologous recombination in negative sense RNA viruses. Viruses 3, 1358–1373 (2011).

    Article  CAS  Google Scholar 

  26. Mikami, M., Toki, S. & Endo, M. In planta processing of the spCas9–gRNA complex. Plant Cell Physiol. 58, 1857–1867 (2017).

    Article  CAS  Google Scholar 

  27. Bally, J. et al. The rise and rise of Nicotiana benthamiana: a plant for all reasons. Annu. Rev. Phytopathol. 56, 405–426 (2018).

    Article  CAS  Google Scholar 

  28. 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).

    Article  Google Scholar 

  29. Jackson, A. O., Dietzgen, R. G., Goodin, M. M., Bragg, J. N. & Deng, M. Biology of plant rhabdoviruses. Annu. Rev. Phytopathol. 43, 623–660 (2005).

    Article  CAS  Google Scholar 

  30. Zhou, X., Sun, K., Zhou, X., Jackson, A. O. & Li, Z. The matrix protein of a plant rhabdovirus mediates superinfection exclusion by inhibiting viral transcription. J. Virol. 93, e00680-19 (2019).

    Article  Google Scholar 

  31. Ganesan, U. et al. Construction of a sonchus yellow net virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses. J. Virol. 87, 10598–10611 (2013).

    Article  CAS  Google Scholar 

  32. Jackson, A. O. & Christie, S. R. Purification and some physicochemical properties of sonchus yellow net virus. Virology 77, 344–355 (1977).

    Article  CAS  Google Scholar 

  33. Sun, K. et al. Matrix-glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination. Mol. Plant Pathol. 19, 2288–2301 (2018).

    Article  CAS  Google Scholar 

  34. Liu, H. et al. CRISPR–P 2.0: An improved CRISPR–Cas9 tool for genome editing in plants. Mol. Plant 10, 530–532 (2017).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. & Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 691–693 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. O. Jackson and X. Zhou for valuable suggestions and critical reading of the manuscript, and Q.-Y. Shu for assistance in chi-squared test analysis. This work was supported by grants from the National Natural Science Foundation of China (nos. 31870142 and 31671996) and the Natural Science Foundation of Zhejiang Province, China (no. LZ20C140004).

Author information

Authors and Affiliations

  1. State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, China

    Xiaonan Ma, Xiaoyan Zhang, Huimin Liu & Zhenghe Li

  2. Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou, China

    Zhenghe Li

  3. Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Zhejiang University, Hangzhou, China

    Zhenghe Li

Authors
  1. Xiaonan Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huimin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhenghe Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.L. and X.M. conceived the project and designed the experiments. X.M., X.Z. and H.L. performed the experiments. X.M. and Z.L. analysed the data and interpreted the results. Z.L. and X.M. wrote the manuscript.

Corresponding author

Correspondence to Zhenghe Li.

Ethics declarations

Competing interests

Z.L. and X.M. are inventors on a patent application covering the results described in this paper.

Additional information

Peer review information Nature Plants thanks Lanqin Xia, Jian-Kang Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Stability of SYNV vectors after mechanical transmission.

a, GFP fluorescence images of N. benthamiana 16c plants mechanically inoculated with V-Cas9 or V-gGFP1-Cas9. Photographs were taken under long-wavelength UV illumination at 30 days after systemic infection. b, PCR-RE detection of mutation frequencies in plants mechanically infected by V-gGFP1-Cas9. V-Cas9/U and V-Cas9/D denote a V-Cas9-infected plant without or with restriction digestion. M, 1-kb ladder DNA marker. c, RT-PCR detection of gRNA and Cas9 inserts in upper leaves of plants systemically infected by SYNV derivatives. V-WT, wild-type SYNV infection; d, Western blot analysis of the expression of the viral structural proteins, Cas9 and GFP in infected leaf tissues. The Coomassie Brilliant Blue-stained large subunit of RuBisCO (rbcL) severs as protein loading control.

Source data

Extended Data Fig. 2 Evaluation of the efficiency and precision of the tRNA-mediated gRNA processing by cRT-PCR.

a, Schematic representation of gRNA circularization and terminal sequence mapping. The divergent arrow pairs denote annealing positions of abutting primers used for cRT-PCR. The hypothesized size of each amplified fragment are indicated. A(n), poly(A) tails with undefined base number. b, cRT-PCR analysis of the gRNA processing efficiency. Arrow denotes DNA fragments corresponding in size to mature gRNA, and asterisk labels the unprocessed 5′ UTR-gRNA-3′ UTR fragment. M, 500-bp ladder marker. c, Sanger sequencing of the gRNA-like cRT-PCR products. The linear sequences are aligned with a canonical gRNA (WT). The sequences of gRNA spacer, scaffold, tRNA 5′ leader, and SYNV UTR are shown in blue, black, purple, and red color letters, respectively. x# shown on the right indicate the number of individual colonies with the identical sequence.

Source data

Extended Data Fig. 3 Mutagenesis frequencies induced by tRNA-processed and unprocessed gRNAs.

a, PCR-RE detection of mutagenesis frequency. DNA fragments containing the gGFP2 target site were amplified by PCR from chromosomal DNA extracted from plants infected with SYNV-gRNA-Cas9 or SYNV-tgtRNA-Cas9 and digested by NcoI. Mock/U and Mock/D denote a mock-infected plant without or with restriction digestion. b, Sanger sequencing results of target site mutations induced by SYNV-gRNA-Cas9. Base substitution is italicized, and base deletions are denoted by dashes, with the number of base substitution (s#) and base deletion (d#) indicated on the right of each sequence. Examples of sequencing chromatographs are shown.

Source data

Extended Data Fig. 4 Viral symptoms and mutation phenotypes in N. benthamiana.

Plants were agroinoculated with SYNV-tgtRNA-Cas9 vectors targeting different endogenous genomic sites, as indicated on the top of panels. Infected plants were photographs at 20 days after symptom appearance. Mock, mock inoculation; V-WT, infection with wild-type SYNV. Note: despite high mutagenesis frequencies, target gene mutation phenotypes, such as the photobleaching phenotype anticipated from knock-down of PDS, were not observed in the virus-infected plants. This is likely due to the residual activities of the unaltered and altered but non-frameshift PDS alleles, as shown in Fig. 3 in this study. For the N. benthamiana RDR6 and SGS3 gene targets, previous studies have shown that their transgenic RNAi lines exhibit little developmental abnormalities39,40.

Extended Data Fig. 5 Genotyping of M0 plants regenerated from plant tissues infected with SYNV vector targeting the gR6–4 site.

a, Detection of target mutations and viral vector in independent M0 plants by PCR-RE and RT-PCR assays. The size of undigested DNA fragments (indicated by red arrowhead) as well as digestion products are shown along with a 500-bp ladder DNA marker (M). b, Genotyping of M0 plants by Sanger sequencing. M0 plants carrying tetra-allelic mutations are labeled in blue letters. Mutation type: WT, wild-type sequence with no mutation detected; d#, number of bases deleted from target site; i#, number of bases inserted at target site; s#, number of bases substitution at target site. Asterisks denote that both the M0–1 and -9 plants contain a mutated allele with an 8-base deletion that happens to restore the PvuII site (underlined), thus remaining partially susceptible to PvuII digestion as shown in (a).

Source data

Extended Data Fig. 6 Genotyping of M0 plants regenerated from plant tissues infected with SYNV vector targeting the gS3–2 site.

a, Detection of target mutations and viral vector in independent M0 plants by PCR-RE and RT-PCR assays. The size of undigested DNA fragments (red arrowhead) and digestion products are labeled, M, 500-bp ladder DNA marker. b, M0 plant genotypes determined by Sanger sequencing. M0 plants with tetra-allelic mutations are labeled in blue letters. Deleted nucleotides are denoted by dash symbols, and inserted nucleotides or substitutions are shown in boldface and italicized letters, respectively. Mutation type: WT, wild-type sequence with no mutation; d#, i#, and s# denote number of bases deleted, inserted, or substituted at target site, respectively.

Source data

Extended Data Fig. 7 Segregation of phenotypes and genotypes in M1 and M2 progeny derived from five representative M0 lines bi-allelic for PDS-A and PDS-B.

For each M0 line, the genotypes of one albino and four normal M1 progeny were determined by Sanger sequencing (Supplementary Figure 5). The seeds produced by the normal M1 plants after self-pollination were germinated on MS media plates and the M2 progeny phenotypes counted. P values were calculated by using a chi-squared test of goodness-of-fit to segregation at a 3:1 or 15:1 ratio. 0.1< P <0.5, in good agreement with theoretical segregation ratios; P >0.5, in very good agreement with theoretical segregation ratios. d# and i# denote # of bp deleted and inserted from the target site, respectively, and the in-frame deletion types (d3, d6, and d9) are highlighted in green letters. Ho, homozygote; Bi, bi-allele; n/a, not applicable.

Extended Data Fig. 8 Genotype inheritance of CRISPR–Cas9-mediated PDS gene loci modifications during the M1 to M2 generation.

Five M1 plants homozygous for PDS-A and PDS-B were selected and the genotypes of 10 M2 progeny for each M1 plants were determined by sequencing. d# and i# denote # of bp deleted and inserted from the target site, respectively, and the in-frame deletion types (d3, d6, and d9) are highlighted in green letters. Ho, homozygote. -, virus-free.

Extended Data Fig. 9 Detection of potential off-target effects.

DNA fragments encompassing the gPDS-1 on-target (PDS-A) and 13 potential off-targets (OT1 to OT13; see Supplementary Table 5) were each amplified from the albino M0–11 plant by PCR with specific primers and then subjected to digestion with T7EI and HinfI (if applicable). Lanes 1, 2, and 3 show samples from a mock-infected plant without or with enzyme digestion, and from the M0–11 plant with enzyme digestion, respectively. M, ladder DNA marker.

Source data

Extended Data Fig. 10 Absence of SYNV vector in M1 progeny derived from various regenerated M0 lines.

Total RNA extracted from four or five individual M1 plants derived from representative M0 lines were analyzed by RT-PCR with SYNV- and actin-specific primer sets. Inf. indicates an RNA sample extracted from an SYNV-infected plant serving as a positive control. M, 1-kb ladder DNA marker.

Source data

Supplementary information

Supplementary Information

Supplementary methods, references, Figs. 1–6 and Tables 1–8.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed imunolots and gels.

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 1

Unprocessed immunolots and gels.

Source Data Extended Data Fig. 2

Unprocessed gel.

Source Data Extended Data Fig. 3

Unprocessed gel.

Source Data Extended Data Fig. 5

Unprocessed gels.

Source Data Extended Data Fig. 6

Unprocessed gels.

Source Data Extended Data Fig. 9

Unprocessed gels.

Source Data Extended Data Fig. 10

Unprocessed gels.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ma, X., Zhang, X., Liu, H. et al. Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9. Nat. Plants 6, 773–779 (2020). https://doi.org/10.1038/s41477-020-0704-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-020-0704-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing