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Two efficient CRISPR/Cas9 systems for gene editing in soybean

Transgenic Research volume 30, pages 239–249 (2021)Cite this article

Abstract

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

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References

  • Adli M (2018) The CRISPR tool kit for genome editing and beyond. Nat Commun 9:1911

    PubMed  PubMed Central  Google Scholar 

  • Ahmar S, Gill RA, Jung KH, Faheem A, Qasim MU, et al. (2020). Conventional and molecular techniques from simple breeding to speed breeding in crop plants: recent advances and future outlook. Int J Mol Sci. 2020 21(7):2590

  • An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J 4:277–284

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bao A, Chen H, Chen L, Chen S, Hao Q et al (2019) CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean. BMC Plant Biol 19(1):131

    PubMed  PubMed Central  Google Scholar 

  • Barrangou R (2015) Diversity of CRISPR-Cas immune systems and molecular machines. Genome Biol 16:247

    PubMed  PubMed Central  Google Scholar 

  • Cai Y, Chen L, Sun S, Wu C, Yao W, et al. (2018) CRISPR/Cas9-Mediated deletion of large genomic fragments in soybean. Int J Mol Sci 19.

  • Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY et al (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng Q, Dong L, Su T, Li T, Gan Z et al (2019) CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean. BMC Plant Biol 19:562

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chiera JM, Bouchard RA, Dorsey SL, Park EH, Buenrostro-Nava MT, Ling PP, Finer JJ (2007) Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Rep 26:1501–1509

    CAS  PubMed  Google Scholar 

  • Chilcoat D, Liu Z-B, Sander J (2017) Use of CRISPR/Cas9 for Crop Improvement in Maize and Soybean. Prog Mol Biol Transl Sci 149:27–46

    CAS  PubMed  Google Scholar 

  • Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330

    CAS  PubMed  Google Scholar 

  • Dang Y, Jia G, Choi J, Ma H, Anaya E et al (2015) Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol 16:280

    PubMed  PubMed Central  Google Scholar 

  • Davies JP, Kumar S, Sastry-Dent L (2017) Use of Zinc-Finger Nucleases for Crop Improvement. Progress in Molecular Biology and Transl Sci 149:47–63

    CAS  Google Scholar 

  • Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267

    CAS  PubMed  PubMed Central  Google Scholar 

  • Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191

    CAS  PubMed  PubMed Central  Google Scholar 

  • Du H, Zeng X, Zhao M, Cui X, Wang Q et al (2016) Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9. J Biotechnol 217:90–97

    CAS  PubMed  Google Scholar 

  • Fiaz S, Ahmad S, Riaz A, Noor MA, Wang X et al (2019) Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities. Int J Mol Sci 20(4):888

    CAS  PubMed Central  Google Scholar 

  • Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gao Y, Zhang Y, Zhang D, Dai X, Estelle M, Zhao Y (2015) Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development. Proc Natl Acad Sci U S A 112(7):2275–2280

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gao C (2018) The future of CRISPR technologies in agriculture. Nat Rev Mol Cell Biol 19:275–276

    CAS  PubMed  Google Scholar 

  • Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, et al. (2012). Phytozome: a comparative platform for green plant genomics. Nucleic acids research, 40(Database issue), D1178–D1186.

  • Hsu PD, Scott DA, Weinstein J, Ran FA, Konermann S et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA (2015) Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol 15:16

    PubMed  PubMed Central  Google Scholar 

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kim GB, Nam YW (2013) Isolation and characterization of Medicago truncatula U6 promoters for the construction of small hairpin RNA-mediated gene silencing vectors. Plant Mol Biol Report 31(3):581–593

    CAS  Google Scholar 

  • Lee CM, Zhu H, Davis TH, Deshmukh H (2017) Bao G (2017) Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells. Methods Mol Biol 1498:3–21

    CAS  PubMed  Google Scholar 

  • Luttgeharm KD, Wong K-S, Siembieda S (2017) Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines. Biotechniques 62:268–274

    CAS  PubMed  Google Scholar 

  • Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B et al (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284

    CAS  PubMed  Google Scholar 

  • Mali P, Yang LH, Esvelt KM, Aach J, Guell M, DiCarlo JE et al (2013) RNA-Guided human genome engineering via Cas9. Science 339(6121):823–826

    CAS  PubMed  PubMed Central  Google Scholar 

  • Marathe A, Krishnan V, Mahajan MM, Thimmegowda V, Dahuja A, et al, (2018). A. Characterization and molecular modeling of Inositol 1,3,4 tris phosphate 5/6 kinase-2 from Glycine max (L) Merr.: comprehending its evolutionary conservancy at functional level. 3 Biotech. 8 (1):50.

  • McCarty NS, Graham AE, Studená L, Ledesma-Amaro R (2020) Multiplexed CRISPR technologies for gene editing and transcriptional regulation. Nat Commun 11(1):1281

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mikami M, Toki S, Endo M (2017) In Planta Processing of the SpCas9-gRNA Complex. Plant Cell Physiol 58:1857–1867

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pellagatti A, Dolatshad H, Valletta S, Boultwood J (2015) Application of CRISPR/Cas9 genome editing to the study and treatment of disease. Arch Toxicol 89:1023–1034

    CAS  PubMed  Google Scholar 

  • Punjabi M, Bharadvaja N, Sachdev A, Krishnan V (2018) Molecular characterization, modeling, and docking analysis of late phytic acid biosynthesis pathway gene, inositol polyphosphate 6-/3-/5-kinase, a potential candidate for developing low phytate crops.8 (8): 344

  • Raza G, Singh MB, Bhalla PL (2020) Somatic Embryogenesis and Plant Regeneration from Commercial Soybean Cultivars. Plants 9(1):38

    CAS  Google Scholar 

  • Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, et al (2018) Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun 9:5416

  • Schardl CL, Byrd AD, Benzion G, Altschuler MA, Hildebrand DA et al (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11

    CAS  PubMed  Google Scholar 

  • Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T et al (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183

    CAS  PubMed  Google Scholar 

  • Shan Q, Wang Y, Li J, Zhang Y, Chen K et al (2013) Targeted genome modification of crop plants using a CRISPR-Cas system: Nat Biotechnol 31(8):686–688

    CAS  PubMed  Google Scholar 

  • Sparvoli F, Cominelli E (2015) Seed Biofortification and Phytic Acid Reduction: A Conflict of Interest for the Plant? Plants 4:728–755

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sun X, Hu Z, Chen R, Jiang Q, Song G, et al. (2015) Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5.

  • Tang X, Zheng X, Qi Y, Zhang D, Cheng Y, Tang A, Voytas DF et al (2016) A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Mol Plant 9:1088–1091

    CAS  PubMed  Google Scholar 

  • Tang X, Ren Q, Yang L, Bao Y, Zhong Z et al (2019) Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17:1431–1445

    CAS  PubMed  PubMed Central  Google Scholar 

  • Torella JP, Lienert F, Boehm CR, Chen JH, Way JC et al (2014) Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications. Nat Protoc 9:2075–2089

    CAS  PubMed  PubMed Central  Google Scholar 

  • Verdaguer B, de Kochko A, Beachy RN et al (1996) Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter. Plant Mol Biol 31:1129–1139. https://doi.org/10.1007/BF00040830

    Article  CAS  PubMed  Google Scholar 

  • Wang M, Mao Y, Lu Y, Wang Z, Tao X, et al. (2018) Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems: J Integr Plant Biol. 2018;60(8):626–631.

  • Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338

    CAS  PubMed  Google Scholar 

  • Wolter F, Schindele P, Puchta H (2019) Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites. BMC Plant Biol 19:176

    PubMed  PubMed Central  Google Scholar 

  • Xie K, Mikenberg B, Yang Y (2014) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112(11):3570–3575

    Google Scholar 

  • Xue LJ, Tsai CJ (2015) AGEseq: Analysis of Genome Editing by Sequencing. Mol Plant 8:1428–1430

    CAS  PubMed  Google Scholar 

  • Yuan FJ, Zhu DH, Tan YY, Dong DK, Fu XJ et al (2012) Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation. Theor Appl Genet 125(7):1413–1423

    CAS  PubMed  Google Scholar 

  • Zhang Z, Mao Y, Ha S, Liu W, Botella JR et al (2016) A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep 35:1519–2153

    CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported in part by Empresa Brasileira de Pesquisa Agropecuária (Embrapa), with funds allocated to the Georgia Agricultural Experiment Stations (Athens, GA, USA) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). J. Carrijo was supported by a fellowship from Cordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

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Authors and Affiliations

  1. Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, PqEB, Av W5 Norte Final 716, Brasília, DF, 70770-917, Brazil

    Jéssica Carrijo, Francisco J. L. Aragão & Giovanni R. Vianna

  2. Department of Molecular Biology, University of Brasilia, Brasília, DF, 70910-900, Brazil

    Jéssica Carrijo, Nathalia Torres, Francisco J. L. Aragão & Giovanni R. Vianna

  3. Center for Applied Genetic Technologies, University of Georgia, Athens, GA, 30602, USA

    Eudald Illa-Berenguer, Peter LaFayette & Wayne Parrott

  4. Department of Crop and Soil Sciences, University of Georgia, Athens, GA, 30602, USA

    Peter LaFayette & Wayne Parrott

Authors
  1. Jéssica Carrijo
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  2. Eudald Illa-Berenguer
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  3. Peter LaFayette
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  4. Nathalia Torres
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  5. Francisco J. L. Aragão
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  6. Wayne Parrott
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  7. Giovanni R. Vianna
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JC, EB and NT. The first draft of the manuscript was written by JC and GRV, and all authors commented on and revised previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Giovanni R. Vianna.

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Carrijo, J., Illa-Berenguer, E., LaFayette, P. et al. Two efficient CRISPR/Cas9 systems for gene editing in soybean. Transgenic Res 30, 239–249 (2021). https://doi.org/10.1007/s11248-021-00246-x

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  • DOI: https://doi.org/10.1007/s11248-021-00246-x

Keywords

  • Genome editing
  • Low phytic acid
  • Single transcriptional unit
  • Two-component transcriptional unit