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CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells

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Abstract

Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cell lines carrying one to three gfp gene copies with a binary vector containing genes coding for Cas9 and guide RNAs targeting the gfp gene. We detected the site-specific mutations by restriction analysis of a gfp amplicon. DNA sequencing of the PCR products confirmed high diversity of insertion-deletion mutations in the cell lines after the editing. We also analyzed gfp mRNA expression by real-time PCR and observed a decrease in gfp transcription after the target site modification. We can conclude that the CRISPR/Cas9 system can be successfully used for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis.

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Acknowledgements

The work was supported by the Russian Science Foundation (Project No. 17-14-01099). The English language was corrected and certified by shevchuk-editing.com.

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Authors and Affiliations

  1. Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, Novosibirsk, Russia, 630090

    Natalya V. Permyakova, Yury V. Sidorchuk, Tatyana V. Marenkova, Sofya A. Khozeeva, Vitaly V. Kuznetsov, Alla A. Zagorskaya, Sergei M. Rozov & Elena V. Deineko

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  1. Natalya V. Permyakova
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  2. Yury V. Sidorchuk
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  5. Vitaly V. Kuznetsov
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  6. Alla A. Zagorskaya
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  7. Sergei M. Rozov
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  8. Elena V. Deineko
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Contributions

NVP and EVD conceived and designed research. NVP, YuVS, TVM, SAK, VVK and AAZ conducted experiments. VVK contributed new reagents or analytical tools. NVP, YuVS and VVK analyzed data. NVP and SMR wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Natalya V. Permyakova.

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Permyakova, N.V., Sidorchuk, Y.V., Marenkova, T.V. et al. CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells. Mol Biol Rep 46, 5735–5743 (2019). https://doi.org/10.1007/s11033-019-05007-y

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