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Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System

  • Ahmed Mahas
  • Zahir Ali
  • Manal Tashkandi
  • Magdy M. Mahfouz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.

Key words

CRISPR/Cas9 TRV Nicotiana benthamiana Genome editing Targeted modification Genome engineering RNA viruses 

Notes

Acknowledgments

We would like to thank members of our Laboratory for Genome Engineering for the many helpful discussions. This study was supported by King Abdullah University of Science and Technology (KAUST).

References

  1. 1.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397–405. https://doi.org/10.1016/j.tibtech.2013.04.004CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pennisi E (2010) Sowing the seeds for the ideal crop. Science 327(5967):802–803. https://doi.org/10.1126/science.327.5967.802CrossRefPubMedGoogle Scholar
  4. 4.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355. https://doi.org/10.1038/nbt.2842CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Voytas DF (2013) Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol 64:327–350. https://doi.org/10.1146/annurev-arplant-042811-105552CrossRefGoogle Scholar
  6. 6.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096. https://doi.org/10.1126/science.1258096CrossRefPubMedGoogle Scholar
  7. 7.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/10.1126/science.1225829CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jiang W, Yang B, Weeks DP (2014) Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations. PLoS One 9(6):e99225. https://doi.org/10.1371/journal.pone.0099225CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31(8):688–691. https://doi.org/10.1038/nbt.2654CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Eid A, Ali Z, Mahfouz MM (2016) High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease. Plant Cell Rep 35(7):1555–1558. https://doi.org/10.1007/s00299-016-2000-4CrossRefPubMedGoogle Scholar
  11. 11.
    Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31(8):691–693. https://doi.org/10.1038/nbt.2655CrossRefPubMedGoogle Scholar
  12. 12.
    Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42(17):10903–10914. https://doi.org/10.1093/nar/gku806CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31(8):686–688. https://doi.org/10.1038/nbt.2650CrossRefPubMedGoogle Scholar
  14. 14.
    Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3 3(12):2233–2238. https://doi.org/10.1534/g3.113.008847CrossRefPubMedGoogle Scholar
  15. 15.
    Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9(10):2395–2410. https://doi.org/10.1038/nprot.2014.157CrossRefPubMedGoogle Scholar
  16. 16.
    Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169(2):931–945. https://doi.org/10.1104/pp.15.00793CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Feng C, Yuan J, Wang R, Liu Y, Birchler JA, Han F (2016) Efficient targeted genome modification in maize using CRISPR/Cas9 system. J Genet Genomics 43(1):37–43. https://doi.org/10.1016/j.jgg.2015.10.002CrossRefPubMedGoogle Scholar
  18. 18.
    Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166(3):1292–1297. https://doi.org/10.1104/pp.114.247577CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jia H, Wang N (2014) Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One 9(4):e93806. https://doi.org/10.1371/journal.pone.0093806CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Aouida M, Eid A, Ali Z, Cradick T, Lee C, Deshmukh H, Atef A, AbuSamra D, Gadhoum SZ, Merzaban J, Bao G, Mahfouz M (2015) Efficient fdCas9 synthetic endonuclease with improved specificity for precise genome engineering. PLoS One 10(7):e0133373. https://doi.org/10.1371/journal.pone.0133373CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227–264. https://doi.org/10.1146/annurev-biochem-060815-014607CrossRefPubMedGoogle Scholar
  22. 22.
    Komor AC, Badran AH, Liu DR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168(1–2):20–36. https://doi.org/10.1016/j.cell.2016.10.044CrossRefPubMedGoogle Scholar
  23. 23.
    Ali Z, Ali S, Tashkandi M, Zaidi SS, Mahfouz MM (2016) CRISPR/Cas9-mediated immunity to geminiviruses: differential interference and evasion. Sci Rep 6:26912. https://doi.org/10.1038/srep26912CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM (2015) CRISPR/Cas9-mediated viral interference in plants. Genome Biol 16:238. https://doi.org/10.1186/s13059-015-0799-6CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Baltes NJ (2015) Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system. Nat Plants 1(10):15145CrossRefGoogle Scholar
  26. 26.
    Ji X, Zhang H, Zhang Y, Wang Y, Gao C (2015) Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants. Nat Plants 1:15144. https://doi.org/10.1038/nplants.2015.144CrossRefPubMedGoogle Scholar
  27. 27.
    Pyott DE, Sheehan E, Molnar A (2016) Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants. Mol Plant Pathol 17(8):1276–1288. https://doi.org/10.1111/mpp.12417CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A (2016) Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Mol Plant Pathol 17(7):1140–1153. https://doi.org/10.1111/mpp.12375CrossRefPubMedGoogle Scholar
  29. 29.
    Zaidi SS, Mansoor S, Ali Z, Tashkandi M, Mahfouz MM (2016) Engineering plants for geminivirus resistance with CRISPR/Cas9 system. Trends Plant Sci 21(4):279–281. https://doi.org/10.1016/j.tplants.2016.01.023CrossRefPubMedGoogle Scholar
  30. 30.
    Zaidi SS, Tashkandi M, Mansoor S, Mahfouz MM (2016) Engineering plant immunity: using CRISPR/Cas9 to generate virus resistance. Front Plant Sci 7:1673. https://doi.org/10.3389/fpls.2016.01673CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zaidi SS, Mansoor S (2017) Viral vectors for plant genome engineering. Front Plant Sci 8:539. https://doi.org/10.3389/fpls.2017.00539CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Macfarlane SA (2010) Tobraviruses—plant pathogens and tools for biotechnology. Mol Plant Pathol 11(4):577–583. https://doi.org/10.1111/j.1364-3703.2010.00617.xCrossRefPubMedGoogle Scholar
  33. 33.
    Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31(6):777–786CrossRefGoogle Scholar
  34. 34.
    Senthil-Kumar M, Mysore KS (2014) Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc 9(7):1549–1562. https://doi.org/10.1038/nprot.2014.092CrossRefPubMedGoogle Scholar
  35. 35.
    Senthil-Kumar M, Mysore KS (2011) Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant Biotechnol J 9(7):797–806. https://doi.org/10.1111/j.1467-7652.2011.00589.xCrossRefPubMedGoogle Scholar
  36. 36.
    Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC (2003) Virus-induced gene silencing in plants. Methods 30(4):296–303CrossRefGoogle Scholar
  37. 37.
    Ali Z, Abul-Faraj A, Piatek M, Mahfouz MM (2015) Activity and specificity of TRV-mediated gene editing in plants. Plant Signal Behav 10(10):e1044191. https://doi.org/10.1080/15592324.2015.1044191CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    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(8):1288–1291. https://doi.org/10.1016/j.molp.2015.02.011CrossRefPubMedGoogle Scholar
  39. 39.
    Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7(5):193–195. https://doi.org/10.1016/S1360-1385(02)02251-3CrossRefPubMedGoogle Scholar
  40. 40.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494. https://doi.org/10.1093/mp/ssu044CrossRefPubMedGoogle Scholar
  42. 42.
    Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL (2015) CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10(4):e0124633. https://doi.org/10.1371/journal.pone.0124633CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ahmed Mahas
    • 1
  • Zahir Ali
    • 1
  • Manal Tashkandi
    • 1
  • Magdy M. Mahfouz
    • 1
  1. 1.Laboratory for Genome Engineering, Division of Environmental and Biological Sciences and Engineering4700 King Abdullah University of Science and TechnologyThuwalSaudi Arabia

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