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Virus-Induced Heritable Gene Editing in Plants

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Plant-Virus Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2724))

Abstract

Gene editing using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease is an excellent tool for assessing gene function in plants. However, delivery of CRISPR/Cas-editing components into plant cells is still a major bottleneck and requires tissue culture-based approaches and regeneration of plants. To overcome this limitation, several plant viral vectors have recently been engineered to deliver single-guide RNA (sgRNA) targets into SpCas9-expressing plants. Here, we describe an optimized, step-by-step protocol based on the tobacco rattle virus (TRV)-based vector system to deliver sgRNAs fused to mobile tRNA sequences for efficient heritable editing in Nicotiana benthamiana and Arabidopsis thaliana model systems. The protocol described here could be adopted to study the function of any gene of interest.

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References

  1. Wang JY, Doudna JA (2023) CRISPR technology: a decade of genome editing is only the beginning. Science 379:eadd8643

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Wang M, Gao SL, Zeng WZ, Yang YQ, Ma JF, Wang Y (2020) Plant virology delivers diverse toolsets for biotechnology. Viruses 12:1338

    Article  CAS  Google Scholar 

  4. Cody WB, Scholthof HB (2019) Plant virus vectors 3.0: transitioning into synthetic genomics. Ann Rev Phytopathol 57:211–230

    Article  CAS  Google Scholar 

  5. Ibrahim A, Odon V, Kormelink R (2019) Plant viruses in plant molecular pharming: toward the use of enveloped viruses. Front Plant Sci 10:803

    Article  Google Scholar 

  6. Abrahamian P, Hammond RW, Hammond J (2020) Plant virus-derived vectors: applications in agricultural and medical biotechnology. Annu Rev Virol 7:513–535

    Article  CAS  Google Scholar 

  7. Khakhar A, Voytas DF (2021) RNA viral vectors for accelerating plant synthetic biology. Front Plant Sci 12:668580

    Article  Google Scholar 

  8. Torti S, Schlesier R, Thummler A, Bartels D, Romer P, Koch B, Werner S, Panwar V, Kanyuka K, Wiren NV, Jones JDG, Hause G, Giritch A, Gleba Y (2021) Transient reprogramming of crop plants for agronomic performance. Nat Plants 7:159–171

    Article  CAS  Google Scholar 

  9. Tengzhi X, Nagalakshmi U, Dinesh-Kumar SP (2021) Virus-induced gene silencing (VIGS). In: Bamford DH, Zuckerman M (eds) Encyclopedia of virology, vol 3, 4th edn. Academic Press, Oxford, pp 123–131

    Chapter  Google Scholar 

  10. Rossner C, Lotz D, Becker A (2022) VIGS goes viral: how VIGS transforms our understanding of plant science. Annu Rev Plant Biol 73:703–728

    Article  Google Scholar 

  11. Zaidi SS, Mansoor S (2017) Viral vectors for plant genome engineering. Front Plant Sci 8:539

    Article  Google Scholar 

  12. Zhang C, Liu S, Li X, Zhang R, Li J (2022) Virus-induced gene editing and its applications in plants. Internat J Mol Sci 23:10202

    Article  CAS  Google Scholar 

  13. Cody WB, Scholthof HB, Mirkov TE (2017) Multiplexed gene editing and protein overexpression using a tobacco mosaic virus viral vector. Plant Physiol 175:23–35

    Article  CAS  Google Scholar 

  14. Ariga H, Toki S, Ishibashi K (2020) Potato virus X vector-mediated DNA-free genome editing in plants. Plant Cell Physiol 61:1946–1953

    Article  CAS  Google Scholar 

  15. Uranga M, Aragones V, Selma S, Vazquez-Vilar M, Orzaez D, Daros JA (2021) Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a potato virus X vector. Plant J 106:555–565

    Article  CAS  Google Scholar 

  16. Ali Z, Abul-faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes NJ, Voytas DF, Dinesh-Kumar S, Mahfouz MM (2015) Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8:1288–1291

    Article  CAS  Google Scholar 

  17. Ali Z, Eid A, Ali S, Mahfouz MM (2018) Pea early-browning virus-mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis. Virus Res 244:333–337

    Article  CAS  Google Scholar 

  18. Ellison EE, Nagalakshmi U, Gamo ME, Huang PJ, Dinesh-Kumar S, Voytas DF (2020) Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat Plants 6:620–624

    Article  CAS  Google Scholar 

  19. Nagalakshmi U, Meier N, Liu JY, Voytas DF, Dinesh-Kumar SP (2022) High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus. Plant Physiol 189:1241–1245

    Article  CAS  Google Scholar 

  20. Liu D, Xuan S, Prichard LE, Donahue LI, Pan C, Nagalakshmi U, Ellison EE, Starker CG, Dinesh-Kumar SP, Qi Y, Voytas DF (2022) Heritable base-editing in Arabidopsis using RNA viral vectors. Plant Physiol 189:1920–1924

    Article  CAS  Google Scholar 

  21. Ghoshal B, Vong B, Picard CL, Feng S, Tam JM, Jacobsen SE (2020) A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana. PLoS Genet 16:e1008983

    Article  CAS  Google Scholar 

  22. Oh Y, Kim SG (2021) RPS5A promoter-driven Cas9 produces heritable virus-induced genome editing in Nicotiana attenuata. Mol Cells 44:911–919

    Article  CAS  Google Scholar 

  23. Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014) DNA replicons for plant genome engineering. Plant Cell 26:151–163

    Article  CAS  Google Scholar 

  24. Butler NM, Atkins PA, Voytas DF, Douches DS (2015) Generation and inheritance of targeted mutations in potato (Solanum tuberosum L.) using the CRISPR/Cas system. PLoS One 10:e0144591

    Article  Google Scholar 

  25. Cermak T, Baltes NJ, Cegan R, Zhang Y, Voytas DF (2015) High-frequency, precise modification of the tomato genome. Genome Biol 16:232

    Article  Google Scholar 

  26. Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217

    Article  CAS  Google Scholar 

  27. Wang M, Lu Y, Botella JR, Mao Y, Hua K, Zhu JK (2017) Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system. Mol Plant 10:1007–1010

    Article  CAS  Google Scholar 

  28. Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sanchez-Leon S, Baltes NJ, Starker C, Barro F, Gao C, Voytas DF (2017) High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J 89:1251–1262

    Article  CAS  Google Scholar 

  29. Dahan-Meir T, Filler-Hayut S, Melamed-Bessudo C, Bocobza S, Czosnek H, Aharoni A, Levy AA (2018) Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. Plant J 95:5–16

    Article  CAS  Google Scholar 

  30. Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y (2015) A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing. Sci Rep 5:14926

    Article  CAS  Google Scholar 

  31. Lei J, Dai P, Li Y, Zhang W, Zhou G, Liu C, Liu X (2021) Heritable gene editing using FT mobile guide RNAs and DNA viruses. Plant Methods 17:20

    Article  CAS  Google Scholar 

  32. Li T, Hu J, Sun Y, Li B, Zhang D, Li W, Liu J, Li D, Gao C, Zhang Y, Wang Y (2021) Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture. Mol Plant 14:1787–1798

    Article  CAS  Google Scholar 

  33. Chen H, Su Z, Tian B, Liu Y, Pang Y, Kavetskyi V, Trick HN, Bai G (2022) Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat. Plant Biotechnol J 20:1018–1020

    Article  CAS  Google Scholar 

  34. Wang W, Yu Z, He F, Bai G, Trick HN, Akhunova A, Akhunov E (2022) Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system. Plant Biotechnol J 20(12):2332–2341

    Article  CAS  Google Scholar 

  35. Hu J, Li S, Li Z, Li H, Song W, Zhao H, Lai J, Xia L, Li D, Zhang Y (2019) A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize. Mol Plant Pathol 20:1463–1474

    Article  CAS  Google Scholar 

  36. Mei Y, Beernink BM, Ellison EE, Konecna E, Neelakandan AK, Voytas DF, Whitham SA (2019) Protein expression and gene editing in monocots using foxtail mosaic virus vectors. Plant Direct 3:e00181

    Article  Google Scholar 

  37. Beernink BM, Lappe RR, Bredow M, Whitham SA (2022) Impacts of RNA mobility signals on virus-induced somatic and germline gene editing. Front Genome Ed 4:925088

    Article  Google Scholar 

  38. Ma X, Zhang X, Liu H, Li Z (2020) Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9. Nat Plants 6:773–779

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Neha Dinesh-Kumar for editing the manuscript. This work was supported by grants from Agricultural Innovation through Gene Editing program grant no. 2020-67013-31544/project accession no. 1022332, USDA National Institute of Food and Agriculture (to S.P.D.-K. and U.N.), USDA-NIFA predoctoral fellowship grant 2021-67034-35187 (to NM), and National Science Foundation (NSF) grant IOS-2139987 (to S.P.D-K).

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Authors and Affiliations

  1. Department of Plant Biology and The Genome Center, College of Biological Sciences, University of California, Davis, Davis, CA, USA

    Ugrappa Nagalakshmi, Nathan Meier & Savithramma P. Dinesh-Kumar

Authors
  1. Ugrappa Nagalakshmi
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  2. Nathan Meier
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  3. Savithramma P. Dinesh-Kumar
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Corresponding authors

Correspondence to Ugrappa Nagalakshmi or Savithramma P. Dinesh-Kumar .

Editor information

Editors and Affiliations

  1. Departamento de Bioquimica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Elizabeth P.B. Fontes

  2. Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

    Kristiina Mäkinen

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Nagalakshmi, U., Meier, N., Dinesh-Kumar, S.P. (2024). Virus-Induced Heritable Gene Editing in Plants. In: Fontes, E.P., Mäkinen, K. (eds) Plant-Virus Interactions. Methods in Molecular Biology, vol 2724. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3485-1_20

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  • DOI: https://doi.org/10.1007/978-1-0716-3485-1_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3484-4

  • Online ISBN: 978-1-0716-3485-1

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