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Design of Guide RNA for CRISPR/Cas Plant Genome Editing

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Abstract

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

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Funding

This work was supported by the Russian Foundation for Basic Research, project nos. 18-04-00118 А, 19-016-00117 А, 19-016-00139 А, and by the governmental grants, nos. АААА-А16-116020350028-4, AAAA-A19-119021190011-0.

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Authors and Affiliations

  1. Institute of Biochemistry and Genetics, Russian Academy of Sciences, 450054, Ufa, Russia

    G. A. Gerashchenkov, N. A. Rozhnova, B. R. Kuluev, O. Yu. Kiryanova, G. R. Gumerova, A. V. Knyazev, Z. R. Vershinina, E. V. Mikhailova, D. A. Chemeris, R. T. Matniyazov, An. Kh. Baimiev, Al. Kh. Baimiev & A. V. Chemeris

  2. Ufa State Petroleum Technological University, 450062, Ufa, Russia

    O. Yu. Kiryanova

  3. Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, 450075, Ufa, Russia

    I. M. Gubaidullin

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  1. G. A. Gerashchenkov
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  2. N. A. Rozhnova
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  3. B. R. Kuluev
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  8. E. V. Mikhailova
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  10. R. T. Matniyazov
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  11. An. Kh. Baimiev
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  14. A. V. Chemeris
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Corresponding author

Correspondence to G. A. Gerashchenkov.

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The present article does not contain any studies with human or animals as objects.

Conflict of interest. The authors declare no conflict of interests.

Additional information

Translated by M. Bibov

Abbreviations: gRNA, guide RNA; Cas9, CRISPR associated protein 9; Cpf1, CRISPR from Prevotella and Francisella 1; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; PAM, Protospacer Adjacent Motif.

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Gerashchenkov, G.A., Rozhnova, N.A., Kuluev, B.R. et al. Design of Guide RNA for CRISPR/Cas Plant Genome Editing. Mol Biol 54, 24–42 (2020). https://doi.org/10.1134/S0026893320010069

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  • DOI: https://doi.org/10.1134/S0026893320010069

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