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Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration

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Plant Genome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2653))

Abstract

Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.

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References

  1. Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Mol Plant-Microbe Interact 25(12):1523–1530

    Article  CAS  PubMed  Google Scholar 

  2. Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Li JF, Aach J, Norville JE, 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 31:688–691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  6. Li JF, Zhang D, Sheen J (2015) Targeted plant genome editing via the CRISPR/Cas9 technology. Methods Mol Biol 1284:239–255

    Google Scholar 

  7. Osakabe Y, Osakabe K (2015) Genome editing with engineered nucleases in plants. Plant Cell Physiol 56:389–400

    Article  CAS  PubMed  Google Scholar 

  8. Ma X, Zhu Q, Chen Y, Liu Y (2016) CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974

    Article  CAS  PubMed  Google Scholar 

  9. 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 multi-purpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Malzahn A, Lowder L, Qi Y (2017) Plant genome editing with TALEN and CRISPR. Cell Biosci 7:21

    Article  PubMed  PubMed Central  Google Scholar 

  11. Murovec J, Pirc Z, Yang B (2017) New variants of CRISPR RNA-guided genome editing enzymes. Plant Biotechnol J 15:917–926

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shah T, Andleeb T, Lateef S, Noor MA (2018) Genome editing in plants: advancing crop transformation and overview of tools. Plant Physiol Biochem 131:12–21

    Article  CAS  PubMed  Google Scholar 

  13. Bao A, Burritt DJ, Chen H, Zhou X, Cao D, Tran LP (2019) The CRISPR/Cas9 system and its applications in crop genome editing. Crit Rev Biotechnol 39:1–16

    Article  Google Scholar 

  14. Wang T, Zhang H, Zhu H (2019) CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Hortic Res 6:77

    Article  PubMed  PubMed Central  Google Scholar 

  15. El-Mounadi K, Morales-Floriano ML, Garcia-Ruiz H (2020) Principles, applications, and biosafety of plant genome editing using CRISPR-Cas9. Front Plant Sci 11:56

    Article  PubMed  PubMed Central  Google Scholar 

  16. Tong CG, Wu FH, Yuan YH, Chen YR, Lin CS (2020) High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes. Plant Biotechnol J 18:889–891

    Article  PubMed  Google Scholar 

  17. Yue JJ, Hong CY, Wei PC, Tsai YC, Lin CS (2020) How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis. Rice 13:9

    Article  PubMed  PubMed Central  Google Scholar 

  18. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A (2017) Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol 35:441–443

    Article  CAS  PubMed  Google Scholar 

  19. Wada N, Ueta R, Osakabe Y, Osakabe K (2020) Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering. BMC Plant Biol 20:234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li G, Sretenovic S, Eisenstein E, Coleman G, Qi Y (2021) Highly efficient C-to-T and A-to-G base editing in a Populus hybrid. Plant Biotechnol J 19:1086–1088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hsu CT, Yuan YH, Lin YC, Lin S, Cheng QW, Wu FH, Sheen J, Shih MC, Lin CS (2021) Efficient and economical targeted insertion in plant genomes via protoplast regeneration. CRISPR J 4:752–760

    Article  CAS  PubMed  Google Scholar 

  22. Barone P, Wu E, Lenderts B, Anand A, Gordon-Kamm W, Svitashev S, Kumar S (2020) Efficient gene targeting in maize using inducible CRISPR-Cas9 and marker-free donor template. Mol Plant 13:1219–1227

    Article  CAS  PubMed  Google Scholar 

  23. Dong OX, Yu S, Jain R, Zhang N, Duong PQ, Butler C, Li Y, Lipzen A, Martin JA, Barry KW, Schmutz J, Tian L, Ronald PC (2020) Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9. Nat Commun 11:1178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lu Y, Tian Y, Shen R, Yao Q, Wang M, Chen M, Dong J, Zhang T, Li F, Lei M, Zhu JK (2020) Targeted, efficient sequence insertion and replacement in rice. Nat Biotechnol 38:1402–1407

    Article  CAS  PubMed  Google Scholar 

  25. Peng F, Zhang W, Zeng W, Zhu JK, Miki D (2020) Gene targeting in Arabidopsis via an all-in-one strategy that uses a translational enhancer to aid Cas9 expression. Plant Biotechnol J 18:892–894

    Article  PubMed  Google Scholar 

  26. Dong OX, Ronald PC (2021) Targeted DNA insertion in plants. Proc Natl Acad Sci U S A 118:e2004834117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  CAS  PubMed  Google Scholar 

  29. Wu FH, Shen SC, Lee LY, Le SH, Chan MT, Lin CS (2009) Tape-Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16

    Article  PubMed  PubMed Central  Google Scholar 

  30. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164

    Article  CAS  PubMed  Google Scholar 

  31. Andersson M, Turesson H, Nicolia A, Fält AS, Samuelsson M, Hofvander P (2017) Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep 36:117–128

    Article  CAS  PubMed  Google Scholar 

  32. Lin CS, Hsu CT, Yang LH, Lee LY, Fu JY, Cheng QW, Wu FH, Hsiao HCW, Zhang Y, Zhang R, Chang WJ, Yu CT, Wang W, Liao LJ, Gelvin SB, Shih MC (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16:1295–1310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hsu CT, Cheng YJ, Yuan YH, Hung WF, Cheng QW, Wu FH, Lee LY, Gelvin SB, Lin CS (2019) Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco. Plant Mol Biol 101:355–371

    Article  CAS  PubMed  Google Scholar 

  34. Yu J, Tu L, Subburaj S, Bae S, Lee GJ (2020) Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins. Plant Cell Rep 40:1037–1045

    Article  PubMed  Google Scholar 

  35. Lin CS, Hsu CT, Yuan YH, Zheng PX, Wu FH, Cheng QW, Wu YL, Wu TL, Lin S, Yue JJ, Cheng YH, Lin SI, Shih MC, Sheen J, Lin YC (2022) DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration. Plant Physiol 188(4):1917–1930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife 3:e04766

    Article  PubMed  PubMed Central  Google Scholar 

  37. Huang RS, Shih HA, Lai MC, Chang YJ, Lin S (2020) Enhanced NK-92 cytotoxicity by CRISPR genome engineering using Cas9 ribonucleoproteins. Front Immunol 11:1008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Andersson M, Turesson H, Olsson N, Fält AS, Ohlsson P, Gonzalez MN, Samuelsson M, Hofvander P (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant 164:378–384

    Article  CAS  PubMed  Google Scholar 

  39. 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 7:1494–1496

    Article  CAS  PubMed  Google Scholar 

  40. Liu H, Ding Y, Zhou Y, Jin W, Xie K, Chen LL (2017) CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant 10:530–532

    Article  CAS  PubMed  Google Scholar 

  41. Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL (2008) The Vienna RNA websuite. Nucleic Acids Res 36(Web Server issue):W70–W74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ (2014) Poly peak parser: method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products. Dev Dyn 243:1632–1636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li H, Durbin R (2009) Fast and accurate short read alignment with Bur-rows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30(10):1473–1475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

    Fu-Hui Wu, Chen-Tran Hsu & Choun-Sea Lin

Authors
  1. Fu-Hui Wu
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  2. Chen-Tran Hsu
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  3. Choun-Sea Lin
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Corresponding author

Correspondence to Choun-Sea Lin .

Editor information

Editors and Affiliations

  1. Division of Plant Sciences, University of Missouri, Columbia, MO, USA

    Bing Yang

  2. Department of Crop Genetics, John Innes Centre, Norwich, UK

    Wendy Harwood

  3. Syngenta Crop Protection, LLC., Research Triangle Park, NC, USA

    Qiudeng Que

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Wu, FH., Hsu, CT., Lin, CS. (2023). Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_19

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  • DOI: https://doi.org/10.1007/978-1-0716-3131-7_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3130-0

  • Online ISBN: 978-1-0716-3131-7

  • eBook Packages: Springer Protocols

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