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Two efficient CRISPR/Cas9 systems for gene editing in soybean

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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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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

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  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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