Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA

  • Yuhei Kanazashi
  • Aya Hirose
  • Ippei Takahashi
  • Masafumi Mikami
  • Masaki Endo
  • Sakiko Hirose
  • Seiichi Toki
  • Akito Kaga
  • Ken Naito
  • Masao Ishimoto
  • Jun Abe
  • Tetsuya Yamada
Original Article

Abstract

Key message

Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds.

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

Keywords

CRISPR/Cas9 Generation Glycine max Heritable mutation Null-segregant PEAPOD 

Notes

Acknowledgements

We thank Professor Holger Puchta (University of Karlsruhe) for permission to use plasmid DNAs of pEn-Chimera and pDe-CAS9, and M. Suzuki and Y. Kitsui for general technical assistance. This work was supported by the Cabinet Office, Government of Japan [the Cross-ministerial Strategic Innovation Promotion Program (SIP) for TY].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2018_2251_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1185 KB)

References

  1. Andersson M, Turesson H, Nicolia A, Falt 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 36:117–128CrossRefPubMedGoogle Scholar
  2. Bilyeu KD (2008) Genetics and genomics of soybean. In: Stacey G (ed) Plant genetics/genomics, vol 2. Springer, USA, pp 135–139Google Scholar
  3. Braatz J, Harloff HJ, Mascher M, Stein N, Himmelbach A, Jung C (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus L.). Plant Physiol 174:935–942CrossRefPubMedGoogle Scholar
  4. Cai YP, Chen L, Liu XJ, Sun S, Wu CX, Jiang BJ, Han TF, Hou WS (2015) CRISPR/Cas9-mediated genome editing in soybean hairy roots. Plos One 10:e0136064CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W (2017) CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soybean. Plant Biotechnol J.  https://doi.org/10.1111/pbi.12758PubMedCentralGoogle Scholar
  6. Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, Becraft PW, Meyers BC, Walbot V, Wang K, Yang B (2017) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnol J 15:257–268CrossRefPubMedGoogle Scholar
  7. Curtin SJ, Xiong Y, Michno J-M, Campbell BW, Stec AO, Čermák T, Starker C, Voytas DF, Eamens AL, Stupar RM (2017) CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula. Plant Biotechnol J.  https://doi.org/10.1111/pbi.12857PubMedGoogle Scholar
  8. Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359CrossRefPubMedGoogle Scholar
  9. Feng ZY, Mao YF, Xu NF, Zhang BT, Wei PL, Yang DL, Wang Z, Zhang ZJ, Zheng R, Yang L, Zeng L, Liu XD, Zhu JK (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ge LF, Yu JB, Wang HL, Luth D, Bai GH, Wang K, Chen RJ (2016) Increasing seed size and quality by manipulating BIG SEEDS1 in legume species. Proc Natl Acad Sci USA 113:12414–12419CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gonzalez N, Pauwels L, Baekelandt A, De Milde L, Van Leene J, Besbrugge N, Heyndrickx KS, Perez AC, Durand AN, De Clercq R, Van De Slijke E, Bossche RV, Eeckhout D, Gevaert K, Vandepoele K, De Jaeger G, Goossens A, Inze D (2015) A repressor protein complex regulates leaf growth in Arabidopsis. Plant Cell 27:2273–2287CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hajika M, Igita K, Kitamura K (1991) A line lacking all the seed lipoxygenase isozymes in soybean Glycine max (L.) Merrill induced by gamma-ray irradiation. Jpn J Breed 41:507–509CrossRefGoogle Scholar
  13. Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15:16CrossRefPubMedPubMedCentralGoogle Scholar
  14. Lawrenson T, Shorinola O, Stacey N, Li CD, Ostergaard L, Patron N, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258CrossRefPubMedPubMedCentralGoogle Scholar
  15. Li JF, Norville JE, Aach J, McCormack M, Zhang DD, 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 31:688–691CrossRefPubMedPubMedCentralGoogle Scholar
  16. Li ZS, Liu ZB, Xing AQ, Moon BP, Koellhoffer JP, Huang LX, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970CrossRefPubMedPubMedCentralGoogle Scholar
  17. Liu K (2004) Soybean as functional foods and ingredients. AOCS Press, USA, pp 1–22CrossRefGoogle Scholar
  18. Naito K, Takahashi Y, Chaitieng B, Hirano K, Kaga A, Takagi K, Ogiso-Tanaka E, Thayarasook C, Ishimoto M, Tomooka N (2017) Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mango). Breed Sci 67:151–158CrossRefPubMedPubMedCentralGoogle Scholar
  19. Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693CrossRefPubMedGoogle Scholar
  20. Pan CT, Ye L, Qin L, Liu X, He YJ, Wang J, Chen LF, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:24765CrossRefPubMedPubMedCentralGoogle Scholar
  21. Rahman SM, Takagi Y, Miyamoto K, Kawakita T (1994) Inheritance of low linolenic acid content in soybean mutant line M-5. Breed Sci 44:379–382Google Scholar
  22. Schmutz J, Cannon SB, Schlueter J, Ma JX, Mitros T, Nelson W, Hyten DL, Song QJ, Thelen JJ, Cheng JL, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu SQ, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du JC, Tian ZX, Zhu LC, Gill N, Joshi T, Libault M, Sethuraman A, Zhang XC, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183CrossRefPubMedGoogle Scholar
  23. Sebastian SA, Fader GM, Ulrich JF, Forney DR, Chaleff RS (1989) Semidominant soybean mutation for resistance to sulfonylurea herbicides. Crop Sci 29:1403–1408CrossRefGoogle Scholar
  24. Shan QW, Wang YP, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu JX, Xi JJ, Qiu JL, Gao CX (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefPubMedGoogle Scholar
  25. Sun XJ, Hu Z, Chen R, Jiang QY, Song GH, Zhang H, Xi YJ (2015) Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5:10342CrossRefPubMedPubMedCentralGoogle Scholar
  26. Tsutsui H, Higashiyama T (2017) pKAMA-ITACHI vectors for highly efficient CRISPR/Cas9-mediated gene knockout in Arabidopsis thaliana. Plant Cell Physiol 58:46–56CrossRefPubMedGoogle Scholar
  27. Wang YP, Cheng X, Shan QW, Zhang Y, Liu JX, Gao CX, Qiu JL (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951CrossRefPubMedGoogle Scholar
  28. White DWR (2006) PEAPOD regulates lamina size and curvature in Arabidopsis. Proc Natl Acad Sci USA 103:13238–13243CrossRefPubMedPubMedCentralGoogle Scholar
  29. Xu RF, Li H, Qin RY, Li J, Qiu CH, Yang YC, Ma H, Li L, Wei PC, Yang JB (2015) Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep 5:11491CrossRefPubMedPubMedCentralGoogle Scholar
  30. Yamada T, Watanabe S, Arai M, Harada K, Kitamura K (2010) Cotyledonary node pre-wounding with a micro-brush increased frequency of Agrobacterium-mediated transformation in soybean. Plant Biotechnol 27:217–220CrossRefGoogle Scholar
  31. Yamada T, Mori Y, Yasue K, Maruyama N, Kitamura K, Abe J (2014) Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds. Plant Cell Rep 33:1963–1976CrossRefPubMedGoogle Scholar
  32. Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D (2017) Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant J 91:714–724CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yuhei Kanazashi
    • 1
  • Aya Hirose
    • 1
  • Ippei Takahashi
    • 1
  • Masafumi Mikami
    • 2
    • 3
  • Masaki Endo
    • 2
  • Sakiko Hirose
    • 2
  • Seiichi Toki
    • 2
    • 3
    • 4
  • Akito Kaga
    • 5
  • Ken Naito
    • 6
  • Masao Ishimoto
    • 7
  • Jun Abe
    • 1
  • Tetsuya Yamada
    • 1
  1. 1.Graduate School of AgricultureHokkaido UniversitySapporoJapan
  2. 2.Plant Genome Engineering Research Unit, Institute of Agrobiological SciencesNational Agricultural and Food Research OrganizationTsukubaJapan
  3. 3.Graduate School of NanobioscienceYokohama City UniversityYokohamaJapan
  4. 4.Kihara Institute for Biological ResearchYokohama City UniversityYokohamaJapan
  5. 5.Soybean and Field Crop Applied Genomics Research Unit, Institute of Crop ScienceNational Agricultural and Food Research OrganizationTsukubaJapan
  6. 6.Plant Diversity Research Team, Genetic Resources CenterNational Agricultural and Food Research OrganizationTsukubaJapan
  7. 7.Division of Basic Research, Institute of Crop ScienceNational Agricultural and Food Research OrganizationTsukubaJapan

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