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Peculiarities in Creation of Genetic Engineering Constructions for Knock-In Variant of Genome Editing of Arabidopsis thaliana Cell Culture

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Abstract

The peculiarities in the creation of genetic engineering tools for the knock-in variant of genome editing are considered in detail on the example of gfp gene delivery to two target regions (nucleolus organizer region and the region of one of histone H3 genes) of the Arabidopsis thaliana (L.) Heynh. genome using different methods of delivering exogenous DNA (agrobacterium-mediated transformation, bio-ballistics using different vectors and RNP complexes). Differences in the approaches to the creation of donor constructions and Cas9 tools depending on the selected method of delivery are considered. The selected target regions are of interest for further biotechnological studies on the creation of recombinant protein bioproducer lines since they refer to “housekeeping” gene regions and are characterized by a high transcriptional activity. It was established that the selected regions are not equivalent to each other as targets for incorporation of exogenous DNA. A complex compartmentalization of nucleolus, as well as a unique mechanism of “neutralization” of double-strand breaks, acts as barriers preventing the delivery of genetic engineering tools to this region. The second region of “housekeeping” genes (histone Н3.3 gene region) seems accessible and can be used for the knock-in variant of genome editing.

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Funding

This work was supported by the Russian Science Foundation (grant no. 17-14-01099).

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Authors and Affiliations

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

    P. A. Belavin, N. V. Permyakova, A. A. Zagorskaya, T. V. Marenkova, Yu. V. Sidorchuk, E. A. Uvarova, S. M. Rozov & E. V. Deineko

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  1. P. A. Belavin
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  2. N. V. Permyakova
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  3. A. A. Zagorskaya
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  4. T. V. Marenkova
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  5. Yu. V. Sidorchuk
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  6. E. A. Uvarova
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  7. S. M. Rozov
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  8. E. V. Deineko
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Correspondence to E. V. Deineko.

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The authors declare that they have no conflict of interest.

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Translated by A. Barkhash

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Belavin, P.A., Permyakova, N.V., Zagorskaya, A.A. et al. Peculiarities in Creation of Genetic Engineering Constructions for Knock-In Variant of Genome Editing of Arabidopsis thaliana Cell Culture. Russ J Plant Physiol 67, 855–866 (2020). https://doi.org/10.1134/S1021443720040032

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  • DOI: https://doi.org/10.1134/S1021443720040032

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