The strong base for using base editing in plants
- Authors: Lebedeva M.V.1, Razhina O.L.1, Nikanorkina V.V.1, Taranov V.V.1
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Affiliations:
- All-Russia Research Institute of Agricultural Biotechnology
- Issue: Vol 21 (2023): Спецвыпуск
- Pages: 23-23
- Section: Genetically modified organism. The Нistory, Achivements, Social and Environmental Riscs
- URL: https://journals.eco-vector.com/ecolgenet/article/view/567885
- DOI: https://doi.org/10.17816/ecogen567885
- ID: 567885
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Abstract
The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work.
Nicotiana tabacum L. is a plant from Solanaceae family, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant.
In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants.
This work is supported by State Task No. 0431-2022-0004.
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Full Text
The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work.
Nicotiana tabacum L. is a plant from Solanaceae family, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant.
In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants.
This work is supported by State Task No. 0431-2022-0004.
About the authors
Marina V. Lebedeva
All-Russia Research Institute of Agricultural Biotechnology
Email: marilistik@mail.ru
ORCID iD: 0000-0001-5711-8331
SPIN-code: 1681-8890
senior research associate; all-russia research institute of agricultural biotechnology
Russian Federation, MoscowOksana L. Razhina
All-Russia Research Institute of Agricultural Biotechnology
Email: oksana-razhina@yandex.ru
ORCID iD: 0009-0007-2103-1067
SPIN-code: 4365-8902
research laboratory assistant
Russian Federation, MoscowVeronica V. Nikanorkina
All-Russia Research Institute of Agricultural Biotechnology
Email: vnikanorkina@mail.ru
ORCID iD: 0009-0004-1955-668X
SPIN-code: 4552-3576
research laboratory assistant
Russian Federation, MoscowVasiliy V. Taranov
All-Russia Research Institute of Agricultural Biotechnology
Author for correspondence.
Email: v.taranov1@gmail.com
ORCID iD: 0000-0002-0728-0346
SPIN-code: 5008-4691
leading research associate
Russian Federation, Moscow