Project idea

Optimization of key components of the CRISPR/Cas9 system and methods of delivery of these components into the nuclear genome, followed by targeted modification of genes responsible for drought tolerance mechanisms in potato.

Genome editing using CRISPR/Cas9 represents a revolutionary technology in plant genetics and breeding. This technology provides a precise and targeted impact on the genome, significantly reducing time costs and allowing the creation of varieties with defined characteristics compared to traditional mutagenesis methods, which are based on random variation. To date, the versatility of CRISPR/Cas9 has been validated, demonstrating its successful application in plant drought tolerance research and the development of new varieties with improved tolerance to this stress.

Relevance

This project will investigate the impact of optimized CRISPR/Cas9 elements on Cas9 specificity in the analysis of potato drought tolerance mechanisms.

The project will be implemented by a team of experts in plant genetic engineering methods, including genomic editing methods, where specialists in the field of molecular biology and plant biotechnology work.

The scientific novelty of the research is that it is planned to improve the efficiency of CRISPR/Cas9 technology by optimizing its elements and methods of delivery of CRISPR/Cas9 components into the nuclear genome of potato. The main emphasis will be placed on the study of potato drought tolerance mechanisms based on high-yielding domestic potato varieties. It is important to note that there are no known other published studies investigating potato drought tolerance mechanisms based on optimization of CRISPR/Cas9 system elements and methods of component delivery to the nuclear genome.

The fundamental importance of this project is demonstrated by the fact that it aims to identify genes that regulate drought tolerance in potato. The results of the study will not only provide a deeper understanding of the molecular mechanisms underlying resistance, but will also provide valuable information for further improvement of genome editing technologies to improve drought tolerance in agricultural crops.

The goal

The goal of the project is to improve the efficiency of CRISPR/Cas9 technology by optimizing system elements, and methods for delivering CRISPR/Cas9 components into the nuclear genome to study mechanisms of drought tolerance in potato.

Expected Results

The main expected outcome of the project is to improve the efficiency of CRISPR/Cas9 technology through optimization of system elements, and methods for delivery of CRISPR/Cas9 components into the nuclear genome to study mechanisms of drought tolerance in potato.

The project will result in:

1. Elements of the CRISPR/Cas9 system will be successfully optimized to modify target genes associated with drought tolerance in potato.
2. Genetically engineered constructs will be created to efficiently deliver these elements into cells.
3. Different methods of delivering CRISPR/Cas9 components into the potato nuclear genome will be analyzed. A preferred method that provides high efficiency of genomic editing will be identified.
4. A novel method for delivery of CRISPR/Cas9 components into the potato nuclear genome using optimized system elements will be developed.
5. A comprehensive evaluation of the developed delivery method including the level of genome editing using optimized CRISPR/Cas9 system elements and drought tolerance will be conducted.
6. Genes and mechanisms associated with drought tolerance in the potato genome will be analyzed. Data from studies of the levels of ROS, proline, MDA, chlorophyll and a complex of enzymes responsible for the formation of antioxidant protection in genome-edited potato plants.
   These results are important for developing resistant potato varieties and increasing productivity under drought conditions. They open up new perspectives in the field of genome editing using CRISPR/Cas9 technology and for agriculture.
7. At least 3 (three) articles and/or reviews will be published in peer-reviewed scientific journals in the scientific area of ​​the project, included in the 1st (first), 2nd (second) and/or 3rd (third) quartiles in the Web of Science database and/or having a CiteScore percentile in the Scopus database of at least 60 (sixty); or at least 2 (two) articles and/or reviews in peer-reviewed scientific journals included in the 1st (first) and/or 2nd (second) quartiles in the Web of Science database and/or having a CiteScore percentile in the Scopus database of at least 70 (seventy); or at least 1 (one) article or review in a peer-reviewed scientific publication with a Journal Citation Reports percentile of at least 90 (ninety) or a CiteScore percentile in the Scopus database of at least 90 (ninety), as well as at least 1 (one) article or review in a peer-reviewed foreign or domestic publication recommended by the CQASHE.

Project Manager

Shuga Askarovna Manabayeva. Hirsch Index 5. https://publons.com/researcher/2449535/shuga-a-manabayeva/; ResearcherID Web of Science: A-2529-2015

Publications and protective documents of the supervisor and members of the research team related to the project topic

1. Abeuova L., Kali B., Tussipkan D., Akhmetollayeva A., Ramankulov Y., Manabayeva Sh. CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato // Transgenic Research (2023). https://doi.org/10.1007/s11248-023-00356-8
2. Tussipkan Dilnur and Manabayeva Shuga A. Employing CRISPR/Cas technology for the improvement of potato and other tuber crops // Frontiers in Plant Science. – 2021, V. 12, p.1-16. doi.org/10.3389/fenvs.2022.828257, IF= 5.753
3. Abeuova L.S., Kali B.R., Rakhimzhanova A.O., Bekkuzhina S.S., Manabayeva Sh.A.  High frequency direct shoot regeneration from Kazakh commercial potato cultivars // PeerJ. –2020. Vol. 2020, Issue 7, 2020, Article number e9447. doi.org/10.7717/peerj.9447 Web of Science Q2, Corresponding author
4. Akhmetollayeva A. and Manabayeva Sh. Creation of an expression vector for multiplex editing of the potato vacuolar invertase gene using the CRISPR/Сas9 system // Eurasian Journal of Applied Biotechnology. – 3, 2024, pp.43–55. //doi.org/10.11134/btp.3.2024.5. [in Russian]
5. Kali B.R., Rakhimzhanova A.O., Manabayeva Sh.A. Indirect regeneration of local potato varieties // BULLETIN OF THE L.N. GUMILYOV EURASIAN NATIONAL UNIVERSITY. BIOSCIENCE SERIES. – 2022. – Vol.2, №139. – P.61-69. [in Kazakh].

Results achieved

2024

To search for genes in the genome of tetraploid potato negatively correlating with drought tolerance, genes-homologs and genes-orthologs negatively correlating with drought tolerance in different crops were analyzed. The efficacy of CRISPR/Cas9 technology for the development of drought tolerant plants was confirmed in Arabidopsis with OST2 mutations that improve stomatal closure. Knockout of SRL1 and SRL2 genes caused leaf curling, enhancing drought tolerance. Analysis of transcription factors such as NPR1 in tomato revealed their important role in the regulation of stomata and drought tolerance-related gene expression. CRISPR mutations of DREB2 and ERF3 factors in wheat and modification of NAC071-A enhanced drought tolerance, and the dCas9-HAT system increased AREB1 expression, enhancing tolerance in rice and rapeseed.

In our studies, microRNA827 (MIR827), which is involved in the negative regulation of drought tolerance at the post-transcriptional level, was selected to investigate the mechanisms of potato drought tolerance using optimized elements of the CRISPR/Cas9 system and improved methods of component delivery to the nuclear genome. The nucleotide sequences of MIR827 (NR_127248.1) were analyzed using bioinformatics software such as https://crispr.dbcls.jp/ and http://crispr.hzau.edu.cn/CRISPR2/. The NGG trinucleotides (PAM) in MIR827 were studied, and the nucleotide composition of 20 nucleotides at the 5′- or 3′-end of the PAM was analyzed for each of them. Three target sequences were selected as potential variable parts of the guide RNA, which were then cloned to the constant part of the guide RNA using BbsI restriction sites. As a result, intermediate vector constructs were obtained: p203mir1, p204mir2, and p205mir3.

The binary expression vector pMR284 (15,567 bp) was selected for editing MIR827 in potato using CRISPR/Cas9, into which intermediate vector constructs were cloned using the Gateway system. Next, the gene encoding the Cas9 protein was optimized by adding a nuclear localization signal using AscI restriction sites and cloned under the control of the PcUbi RNA polymerase II promoter into the pMR284 binary vector. As a result, a vector containing all elements necessary for the modification of MIR827 target sites and functioning of the CRISPR/Cas9 system, as well as for the subsequent study of potato drought tolerance mechanisms was created.