vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2026-71-2-104-115vestib-1009Research ArticleСтатьиРедактирование генома картофеля с помощью системы CRISPR/Cas9 для нокаута генов StDMR6-1 и StCHL1, вовлеченных в устойчивость к фитофторозуPotato genome editing using the CRISPR/Cas9 system to knock out the StDMR6-1 and StCHL1 genes involved in late blight resistanceШишлова-СоколовскаяА. М.ShishlovaA. M.

Шишлова-Соколовская Анастасия Михайловна – ст. науч. сотрудник

ул. Ф. Скорины, 34, 220141, г. Минск

Anastasia M. Shishlova-Sokolovskaya – Senior Researcher

34, F. Skorina Str., 220141, Minsk

s_anastasia78@mail.ruНеборскаяВ. Е.NeborskayaV. E.

Неборская Валерия Евгеньевна – мл. науч. сотрудник

ул. Ф. Скорины, 34, 220141, г. Минск

Valeria E. Neborskaya – Junior Researcher

34, F. Skorina Str., 220141, Minsk

V.Tereshko@igc.byУрбановичО. Ю.UrbanovichO. Yu.

Урбанович Оксана Юрьевна – д-р биол. наук, профессор, заведующий лабораторией

ул. Ф. Скорины, 34, 220141, г. Минск

 

Oksana Yu. Urbanovich – D. Sc. (Biol.), Professor, Head of the Laboratory

34, F. Skorina Str., 220141, Minsk

O.Urbanovich@igc.byИнститут генетики и цитологии Национальной академии наук БеларусиInstitute of Genetics and Cytology of the National Academy of Sciences of Belarus202630042026712104115Copyright © Шишлова-Соколовская А.М., Неборская В.Е., Урбанович О.Ю., 20262026Шишлова-Соколовская А.М., Неборская В.Е., Урбанович О.Ю.Shishlova A.M., Neborskaya V.E., Urbanovich O.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/1009

Методом Agrobacterium-опосредованной трансформации в геном сортов картофеля белорусской селекции Першацвет, Юлия, Красавик были введены векторные генетические конструкции, несущие в своем составе элементы системы CRISPR/Cas9 для нокаута генов регуляторов защитных реакций StDMR6-1 и StCHL1. Нокаут данных генов (S-генов) является перспективным, актуальным и современным подходом для повышения устойчивости картофеля к фитофторозу. Получено 288 трансгенных растений картофеля Т0 поколения, 161 из которых было проанализировано на наличие мутационных событий инсерционно-делеционного типа с использованием метода секвенирования по Сэнгеру. При этом из 161 трансформанта Т0 поколения 84 имели мутантные последовательности генов StCHL1 и StDMR6-1 с частотой встречаемости мутаций от 1 до 97 % при р < 0,001 и 99,2 % при р ≥ 0,001 в зависимости от сорта. В результате эксперимента впервые в Республике Беларусь были получены генетически редактированные растения картофеля сортов белорусской селекции Юлия, Першацвет, Красавик, несущие мутации генов StCHL1 и StDMR6-1, приводящие к сдвигу рамки считывания и, как следствие, к нокауту генов.

Using Agrobacterium-mediated transformation, vector genetic constructs, carrying CRISPR/Cas9 elements in their composition for the knockout of the StDMR6-1 and StCHL1 genes regulating defense responses, were introduced into the genomes of Belarusian potato varieties Pershatsvet, Yuliya and Krasavik. The knockout of these genes (S-genes) is a promising, essential and modern approach for increasing potato resistance to late blight. A total of 288 transgenic T0 potato plants were obtained, and 161 out of them were analyzed for insertion-deletion mutations using Sanger sequencing. Moreover, out of 161 transformants of the T0 generation, 84 had mutant sequences of the StCHL1 and StDMR6-1 genes with a mutation frequency from 1 to 97 % at p < 0.001 and 99.2 % at p ≥ 0.001, depending on the variety. As a result of the experiment, genetically edited potato plants of the varieties of Belarusian selection Yuliya, Pershatsvet, and Krasavik were obtained for the first time in the Republic of Belarus. These plants carry mutations in the StCHL1 and StDMR6-1 genes, leading to a shift in the reading frame and, as a consequence, to gene knockout.

CRISPR/Cas9 системаStDMR6-1StCHL1Solanum tuberosumCRISPR/Cas9 systemStDMR6-1StCHL1Solanum tuberosumРабота выполнена в рамках отдельного проекта фундаментальных и прикладных исследований НАН Беларуси «Создать растения картофеля, несущие генетически редактированные с помощью системы CRISPR/Cas9 гены регуляторы защитных реакций StDMR6-1 и StCHL1, для повышения устойчивости к фитофторозу» и при поддержке гранта БРФФИ № Б23-062.The work was carried out within the framework of a separate project of fundamental and applied research of the National Academy of Sciences of Belarus “To create potato plants carrying genetically edited using the CRISPR/Cas9 system genes regulating defense reactions, StDMR6-1 and StCHL1, to increase resistance to late blight” and with the support of the BRFFR grant No. B23-062.ReferencesPotato. Nutrition and Food Security / P. Raigond, B. Singh, S. Dutt, S. K. Chakrabarti – Singapore: Springer, 2020. – 287 p.Raigond P., Singh B., Dutt S., Chakrabarti S. K. Potato. Nutrition and Food Security. Singapore, Springer, 2020. 287 p.The Potato of the Future: Opportunities and Challenges in Sustainable Agri-food Systems / A. Devaux, J.-P. Goffart, P. Kromann [et al.] // Potato Research. – 2021. – Vol. 64, N 4. – P. 681–720. https://doi.org/10.1007/s11540-021-09501-4Devaux A., Goffart J.-P., Kromann P., Andrade-Piedra J., Polar V., Hareau G. The Potato of the Future: Opportunities and Challenges in Sustainable Agri-food. Potato Research, 2021, vol. 64, no. 4, pp. 681–720. https://doi.org/10.1007/s11540021-09501-4Fry, W. Phytophthora infestans: the plant (and R gene) destroyer / W. Fry // Molecular Plant Pathology. – 2008. – Vol. 9, N 3. – P. 385–402. https://doi.org/10.1111/j.1364-3703.2007.00465.xFry W. Phytophthora infestans: the plant (and R gene) destroyer. Molecular Plant Pathology, 2008, vol. 9, no. 3, pp. 385–402. https://doi.org/10.1111/j.1364-3703.2007.00465.xSilencing of six susceptibility genes results in potato late blight resistance / K. Sun, A.-M. A. Wolters, J. H. Vossen [et al.] // Transgenic Research. – 2016. – Vol. 25, N 5. – Р. 731–742. https://doi.org/10.1007/s11248-016-9964-2Sun K., Wolters A.-M. A., Vossen J. H., Rouwet M. E., Loonen A. E. H. M., Jacobsen E. Visser R. G. F., Bai Y. Silencing of six susceptibility genes results in potato late blight resistance. Transgenic Research, 2016, vol. 25, no. 5, pp. 731–742. https://doi.org/10.1007/s11248-016-9964-2Шишлова-Соколовская, А. М. Создание векторных конструкций, несущих CRISPR/Cas9 систему, для направленного мутагенеза генов CHL1 и DMR6-1 Solanum tuberosum / А. М. Шишлова-Соколовская, О. Ю. Урбанович // Молекулярная и прикладная генетика: сб. науч. тр. / Ин-т генетики и цитологии НАН Беларуси; редкол.: А. В. Кильчевский (гл. ред.) [и др.]. – Мн., 2025. – Т. 38. – C. 24–35.Shishlova-Sokolovskaya A. M., Urbanovich O. Yu. Designing vector constructs carrying CRISPR/Cas9 for the directed mutagenesis of the Solanum tuberosum CHL1 and DMR6-1 genes. Molekulyarnaya i prikladnaya genetika: sbornik nauchnykh trudov [Molecular and applied genetics: collection of scientific papers]. Minsk, 2025, vol. 38, pp. 24–35 (in Russian).Jones, J. D. G. The plant immune system / J. D. G. Jones, J. L. Dangl // Nature. – 2006. – Vol. 444, N 7117. – P. 323–329. https://doi.org/10.1038/nature05286Jones J. D. G., Dangl J. L. The plant immune system. Nature, 2006, vol. 444, no. 7117, pp. 323–329. https://doi.org/10.1038/nature05286AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity / D. Turnbull, H. Wang, S. Breen [et al.] // Plant Physiology. – 2019. – Vol. 180, N 1. – P. 571–581. https://doi.org/10.1104/pp.18.01143Turnbull D., Wang H., Breen S., Malec M., Naqvi S., Yang L., Welsh L., Hemsley P., Zhendong T., Brunner F., Gilroy E. M., Birch P. R. J. AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity. Plant Physiology, 2019, vol. 180, no. 1, pp. 571–581. https://doi.org/10.1104/pp.18.01143RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity / D. Turnbull, L. Yang, S. Naqvi [et al.] // Plant Physiology. – 2017. – Vol. 174, N 1. – P. 356–369. https://doi.org/10.1104/pp.16.01804Turnbull D., Yang L., Naqvi S., Breen S., Welsh L., Stepchens J., Morris J., Boevink P. C., Hedley P. E., Zhan J., Birch P. R. J., Gilroy E. M. RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity. Plant Physiology, 2017, vol. 174, no. 1, pp. 356–369. https://doi.org/10.1104/pp.16.01804Vlot, A. C. Salicylic Acid, a Multifaceted Hormone to Combat Disease / A. C. Vlot, D. A. Dempsey, D. F. Klessig // Annual Review of Phytopathology. – 2009. – Vol. 47. – P. 177–206. https://doi.org/10.1146/annurev.phyto.050908.135202Vlot A. C., Dempsey D. A., Klessig D. F. Salicylic Acid, a Multifaceted Hormone to Combat Disease. Annual Review of Phytopathology, 2009, vol. 47, pp. 177–206. https://doi.org/10.1146/annurev.phyto.050908.135202Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes / N. P. Kieu, M. Lenman, E. S. Wang [et al.] // Scientific Reports. – 2021. – Vol. 11, N 1. – Art. 4487. https://doi.org/10.1038/s41598-021-83972-wKieu N. P., Lenman M., Wang E. S., Petersen B. L., Andreasson E. Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes. Scientific Reports, 2021, vol. 11, no. 1, art. 4487. https://doi.org/10.1038/s41598-021-83972-wCRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions / M. Karlsson, N. P. Kieu, M. Lenman [et al.] // Horticulture Research. – 2024. – Vol. 11, N 7. – Art. uhae130. https://doi.org/10.1093/hr/uhae130Karlsson M., Kieu N. P., Lenman M., Marttila S., Resjo S., Zahid M. A., Andreasson E. CRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions. Horticulture Research, 2024, vol. 11, no. 7, art. uhae130. https://doi.org/10.1093/hr/uhae130CRISPR/Cas9 editing of Downy mildew resistant 6 (DMR6-1) in grapevine leads to reduced susceptibility to Plasmopara viticola / S. Djennane, S. Gersch, F. Le-Bohec [et al.] // Journal of Experimental Botany. – 2024. – Vol. 75, N 7. – P. 2100–2112. https://doi.org/10.1093/jxb/erad487Djennane S., Gersch S., Le-Bohec F., Piron M.-Ch., Baltenweck R., Lemaire O., Merdinoglu D., Hugueneu Ph., Nogue F., Mestre P. CRISPR/Cas9 editing of Downy mildew resistant 6 (DMR6-1) in grapevine leads to reduced susceptibility to Plasmopara viticola. Journal of Experimental Botany, 2024, vol. 75, no. 7, pp. 2100–2112. https://doi.org/10.1093/jxb/erad487Маренкова, Т. В. Мозаичный характер экспрессии трансгенов у растений / Т. В. Маренкова, Д. Б. Логинова, Е. В. Дейнеко // Генетика. – 2012. – Т. 48, № 3. – С. 293–306.Marenkova T. V., Loginova D. B., Deineko E. V. Mosaic patterns of transgene expression in plants. Russian Journal of Genetics, 2012, vol. 48, no. 3, pp. 249–260. https://doi.org/10.1134/S1022795412030088Маренкова, Т. В. Особенности экспрессии чужеродных генов в сложноорганизованных инсерциях у трансгенных растений табака с мозаичным характером проявления гена nptII / Т. В. Маренкова, В. В. Кузнецов, Е. В. Дейнеко // Генетика. – 2021. – Т. 57, № 3. – С. 321–331.Marenkova T. V., Kuznetsov V. V., Deineko E. V. Features of expression of foreign genes in complex insertions in transgenic tobacco plants with a mosaic pattern of nptII gene expression. Russian Journal of Genetics, 2021, vol. 57, no. 3, pp. 319–328. https://doi.org/10.1134/S1022795421030108Inference of CRISPR Edits from Sanger Trace Data / D. Conant, T. Hsiau, N. Rossi [et al.] // The CRISPR Journal. – 2022. – Vol. 5, N 1. – P. 123–130. https://doi.org/10.1089/crispr.2021.0113Conant D., Hsiau T., Rossi N., Oki J., Maures T., Waite K., Yang J., Joshi S., Kelso R., Holden K., Enzmann B. L., Stoner R. Inference of CRISPR Edits from Sanger Trace Data. The CRISPR Journal, 2022, vol. 5, no. 1, pp. 123–130. https://doi.org/10.1089/crispr.2021.0113Easy quantitative assessment of genome editing by sequence trace decomposition / E. K. Brinkman, T. Chen, M. Amendola, B. Steensel // Nucleic Acids Research. – 2014. – Vol. 42, N 22. – P. e168. https://doi.org/10.1093/nar/gku936Brinkman E. K., Chen T., Amendola M., Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research, 2014, vol. 42, no. 22, p. e168. https://doi.org/10.1093/nar/gku936Brinkman, E. K. Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER / E. K. Brinkman, B. van Steensel // CRISPR Gene Editing. – 2019. – Vol. 1961. – P. 29–44. https://doi.org/10.1007/978-1-4939-9170-9_3Brinkman E. K., van Steensel B. Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER. CRISPR Gene Editing, 2019, vol. 1961, pp. 29–44. https://doi.org/10.1007/978-1-4939-9170-9_3A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants / X. Ma, Q. Zhang, Q. Zhu [et al.] // Molecular Plant. – 2015. – Vol. 8, N 8. – Р. 1274–1284. https://doi.org/10.1016/j.molp.2015.04.007Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B., Yang Z., Li H., Lin Y., Xie Y., Shen R., Chen S., Wang Z., Chen Y., Guo J., Chen L., Zhao X., Dong Z., Liu Y.-G. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 2015, vol. 8, no. 8, pp. 1274–1284. https://doi.org/10.1016/j.molp.2015.04.007Mikami, M. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice / M. Mikami, S. Toki, M. Endo // Plant Cell Reports. – 2015. – Vol. 34, N 10. – Р. 1807–1815. https://doi.org/10.1007/s00299-015-1826-5Mikami M., Toki S., Endo M. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice. Plant Cell Reports, 2015, vol. 34, no. 10, pp. 1807–1815. https://doi.org/10.1007/s00299-015-1826-5Xie, K. RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System / K. Xie, Y. Yang // Molecular Plant. – 2013. – Vol. 6, N 6. – P. 1975–1983. https://doi.org/10.1093/mp/sst119Xie K., Yang Y. RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System. Molecular Plant, 2013, vol. 6, no. 6, pp. 1975–1983. https://doi.org/10.1093/mp/sst119Efficient genome editing in plants using a CRISPR/Cas system / Z. Feng, B. Zhang, W. Ding [et al.] // Cell Research. – 2013. – Vol. 23, N 10. – P. 1229–1232. https://doi.org/10.1038/cr.2013.114Feng Z., Zhang B., Ding W., Lio X., Yang D.-L., Wei P., Cao F., Zhu S., Zhang F., Mao Y., Zhu J.-K. Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 2013, vol. 23, no. 10, pp. 1229–1232. https://doi.org/10.1038/cr.2013.114Schöb, H. Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing / H. Schöb, C. Kunc, F. Meins // Molecular and General Genetics MGG. – 1997. – Vol. 256, N 5. – P. 581–585. https://doi.org/10.1007/s004380050604Schöb H., Kunc C., Meins F. Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Molecular and General Genetics MGG, 1997, vol. 256, no. 5, pp. 581–585. https://doi.org/10.1007/s004380050604Yi, M. Communication Between Filamentous Pathogens and Plants at the Biotrophic Interface / M. Yi, B. Valent // Annual Review of Phytopathology. – 2013. – Vol. 51. – P. 587–611. https://doi.org/10.1146/annurev-phyto-081211-172916Yi M., Valent B. Communication Between Filamentous Pathogens and Plants at the Biotrophic Interface. Annual Review of Phytopathology, 2013, vol. 51, pp. 587–611. https://doi.org/10.1146/annurev-phyto-081211-172916Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism / K. Zhang, R. Halitschke, C. Yin [et al.] // Proceedings of the National Academy of Sciences. – 2013. – Vol. 110, N 36. – P. 14807–14812. https://doi.org/10.1073/pnas.1302702110Zhang K., Halitschke R., Yin C., Lio C.-J., Gan S. S. Salicylic acid 3-hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism. Proceedings of the National Academy of Sciences, 2013, vol. 110, no. 36, pp. 14807–14812. https://doi.org/10.1073/pnas.1302702110

The authors declare that there are no conflicts of interest present.