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General guidelines for CRISPR/Cas-based genome editing in plants

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Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.

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Acknowledgements

This manuscript is written by the leadership of the “Plant Science Initiative of Turkey”, a non-governmental organization aiming to bring the scientists together who work in the field of Plant Sciences in Turkey.

Funding

This review received no external funding.

Author information

Author notes

  1. Musa Kavas, Ceyhun Kayihan and Ufuk Demirel are co-second author.

Authors and Affiliations

  1. Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

    Emre Aksoy

  2. Department of Molecular Biology and Genetics, Ondokuz Mayıs University, Samsun, Turkey

    Kubilay Yildirim & İlkay Sevgen

  3. Department of Agricultural Biotechnology, Ondokuz Mayıs University, Samsun, Turkey

    Musa Kavas & Bayram Ali Yerlikaya

  4. Department of Molecular Biology and Genetics, Başkent University, Ankara, Turkey

    Ceyhun Kayihan & Irmak Çalik

  5. Department of Agricultural Genetic Engineering, Niğde Ömer Halisdemir University, Niğde, Turkey

    Ufuk Demirel

Authors
  1. Emre Aksoy
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  2. Kubilay Yildirim
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  3. Musa Kavas
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  4. Ceyhun Kayihan
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  5. Bayram Ali Yerlikaya
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  6. Irmak Çalik
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  7. İlkay Sevgen
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  8. Ufuk Demirel
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Contributions

Conceptualization, EA, KY, MK, CK, UD; investigation, BAY, IÇ, İS; writing-original draft, EA, KY, MK, CK, BAY, IÇ, İS; writing-review and editing, EA, KY, MK, CK, UD; visualization, MK, BAY; supervision, EA, KY. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Emre Aksoy or Kubilay Yildirim.

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Aksoy, E., Yildirim, K., Kavas, M. et al. General guidelines for CRISPR/Cas-based genome editing in plants. Mol Biol Rep 49, 12151–12164 (2022). https://doi.org/10.1007/s11033-022-07773-8

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