Guided genome editing using CRISPR / Cas9 technology has led to significant progress in all areas of life science and is considered one of the most promising in crop production, the advantage of which is the ability to create improved crop plants with resistance to biotic and abiotic stresses. One of the main factors affecting the editing efficiency is the level of Cas9 and sgRNA expression in the cells that can be solved by co-expression of p19.
At present, there are a lot of works that have shown the role of invertase in the accumulation of reducing sugars in potato tubers during prolonged storage in the cold, which leads to the sweetening of the tubers, which is an unacceptable quality in the production of potato products. Currently, potato lines with a low concentration of reducing sugars in tubers have been created using RNA interference and TALEN methods, which are laborious, not always effective, and expensive.
To study the effect of p19 gene expression on the efficiency of targeted mutagenesis, based on CRISPR/Cas9 technology, to minimize reducing sugars in potato tubers under the influence of low temperatures.
1. Expression vectors with the CRISPR / Cas9 system, guide RNA, and p19 gene will be created.
2. Agrobacterium-mediated transformation of potato leaf and stem explants will be carried out.
3. Regenerated plants with a knockout gene will be obtained, which stimulates the accumulation of reducing sugars in potato tubers under the action of low temperatures.
4. The efficiency of CRISPR/Cas9-mediated editing of the target gene will be analyzed using fragment and next-generation sequencing (NGS) analysis.
5. Tubers of the edited potato genomes will be analyzed for the level of reducing sugars and acrylamide.
Manabayeva Shuga. H-index 5. ResearcherID Web of Science: A-2529-2015; https://publons.com/researcher/2449535/shuga-a-manabayeva/ ;
Tussipkan Dilnur, PhD
Akhmetollayeva Ainash, Master of Biological Sciences
Kali Balnur, Master of Science
2021
Main results: The Pain-1 gene encoding vacuolar invertase was selected for reduction of potato reducing sugars using CRISPR/Cas9. Sites of exons 1 and 3 of the Pain-1 gene were identified as potential modification sites and guide oligonucleotides of chimeric guide RNA using the bioinformatics programmes https://crispr.dbcls.jp/ and http://crispr.hzau.edu.cn/CRISPR2/. NGG trinucleotides in the studied exons were analysed and for each trinucleotide the nucleotide composition of 20 nucleotides located at the 5′-end of the RAM was analysed, sequences with the lowest number of non-specific binding sites in the potato genome were selected. The following intermediate vector constructs were obtained as a result of cloning of variable parts to the constant part directing RNA through BbsI sites: p203+T1, p204+T2, p205+T3.
The binary expression vectors pMR284 and pMR287 encoding Cas9 under the PcUbi promoter were selected for editing the potato Pain-1 gene using CRISPR/Cas9. As a result of cloning the expression cassette with chimeric guide RNA under the U6 promoter into the binary vectors pMR284 and pMR287 using the Gateway system, expression binary vectors containing all the elements necessary for modifying the target sites of the Pain-1 gene and functioning of the CRISPR/Cas9 system in potato genome cells were created.
An expression vector with a gene encoding the p19 protein was created to study the effect of co-expression of the p19 gene on the efficiency of targeted mutagenesis for reducing reducing reducing sugars in potato using CRISPR/Cas9.
2022
Main results: Microclonal propagation of in vitro plants of potato cultivar Aksor was carried out monthly by cuttings. For this purpose, potato plants with 5-6 leaflets formed were extracted from tubes under aseptic conditions and divided into segments containing a stem segment with axillary bud and planted in nutrient media MS without hormones. This technique provided the project with potato explants for one year for Agrobacterium-mediated transformation and callusogenesis.
Agrobacterium-mediated transformation of potato leaf and stem explants was performed using genetically engineered constructs pMR287+3T, pMR287+3T+P19 and pMR284+3T. The target gene of directed mutagenesis using CRISPR/Cas9 is the Pain-1 gene encoding potato vacuolar invertase. A total of 925 stem explants and 940 callus of potato cultivar Aksor were transformed. The frequency of regeneration in transformed stem explants was 21.27%, whereas in callus 13.33%, respectively. The DNA of the regenerant plants was tested by PCR for the presence of Cas9 gene. Out of 247 regeneranted plants, 60 regeneranted plants transformed with vector pMR287+3T and 28 plants with vector pMR287+3T+P19 showed positive results. Further, the presence of the mutation in the genome of the regeneranted plants was analysed by fragment analysis. For this purpose, a 356 bp long fragment of Pain-1 gene was amplified by PCR and hydrolysed by ScaI site. Regeneranted plants numbered 135 and 52 putatively mutated at all four loci in the third exon were selected. 18 regeneranted plants were selected with partial hydrolysis at the ScaI site. DNA from all transformants with a putatively edited Pain-1 invertase gene was sequenced and analysed for 3 target sites in two exons.
2023
Main results: In order to reduce the level of reducing sugars in potatoes, the CRISPR/Cas9 method was used, targeting the VInv gene, which encodes vacuolar invertase. After isolating RNA from domestic potato varieties and analyzing potential changes in exons 1 and 3 of the VInv gene, target sequences with a minimum number of nonspecific bindings in the genome were selected. Binary expression vectors were created that included the Cas9 and p19 genes and chimeric guide RNAs. Microclonal propagation of potato plants of the Aksor variety was carried out monthly, ensuring agrobacterial transformation. A total of 1327 stem explants and 1123 calli of the Aksor potato variety were transformed. The regeneration process was successful, reaching a frequency of 21.2% for stem explants and 5.8% for calli. The DNA of the regenerated plants confirmed the presence of the Cas9 and p19 genes. Sequencing revealed a variety of mutations, including substitutions, insertions, and deletions. Analysis of the content of reducing sugars and acrylamide in genome-edited potato tubers confirmed the success of using CRISPR/Cas9 technology to improve potato products.