Relevance

Every year, infectious diseases of agricultural crops lead to significant yield losses. Diagnostics of pathogens using molecular methods in Kazakhstan is not developed or is concentrated in certain large cities, which makes it inaccessible to farms. Timely detection of phytopathogens using modern methods, such as CRISPR/Cas systems, will greatly simplify the identification and availability of diagnostic methods, which will lead to a decrease of crop losses. Effective pathogen diagnostics methods will enable successful pest control and breeding, improve crop yields and ensure food security.

The project’s aim

Development of diagnostics of plant pathogens in agricultural crops based on a modern diagnostic method using previously unexplored Cas12a of CRISPR/Cas systems. The obtained results will broad the range of used endonucleases, allowing the field detection of phytopathogens, which does not require expensive equipment, highly qualified personnel and specialized facilities.

Expected results

A selection of genetic loci widely used for species identification of fungal phytopathogens was carried out. A selection of loci containing a protospacer-adjacent motif (PAM) sequence was performed. The crRNA was designed and synthesized to form a complex with species-specific loci substrate DNA as a detection target.

Project supervisor

Zhumakayev A.R. H-index: 1, ResearcherID: GZG-9689-2022,

ORCID: 0000-0001-9022-0741, Scopus Author ID: 57193544703

Research group members

Abeldenov S.K. H-index – 2, ResearcherID F-5139-2015,

ORCID 0000-0002-6974-9138, Scopus Author ID 56674705400

Turgimbayeva А. М. H-index: 1, ResearcherID N-6857-2017,

ORCID 0000-0001-7263-1643, Scopus Author ID 57202383621

Shaizadinova А. М. Scopus ID 57224822522, ORCID 0000-0002-5911-3064,

ResearcherID: HDM-6767-2022

Amanzholova М.Zh. Scopus ID 58509708700, ORCID 0000-0001-9847-2679,

ResearcherID: JLM-7527-2023

Publications and security documents of the project supervisor and the research group related to the topic of the project

1. Zhumakayev, A.R., Varga, Vörös, Kocsubé, S., Ramteke, Szekeres,, Vágvölgyi, Hatvani, Marik (2022) Characterization of the antagonistic potential of the glyphosate-tolerant Pseudomonas resinovorans SZMC 25872 strain against the plant pathogenic bacterium Agrobacterium tumefaciens.  Frontiers in Plant Science, section Plant Symbiotic Interactions. doi: 10.3389/fpls.2022.1034237
2. Allaga, H., Zhumakayev, A., Büchner., Kocsubé, Szűcs, Vágvölgyi, Kredics, Hatvani (2021) Members of the Trichoderma harzianum species complex with mushroom pathogenic potential. Agronomy, https://doi.org/10.3390/agronomy11122434
3. Zhumakayev, A. R., Vörös, Szekeres, Rakk, Vágvölgyi, Szűcs, Kredics, Škrbić, Hatvani (2021). Comprehensive characterization of stress tolerant bacteria with plant growth-promoting potential isolated from glyphosate-treated environment. World Journal of Microbiology & Biotechnology, https://doi.org/10.1007/s11274-021-03065-8

Achieved results

2023

Amplification and cloning of target sequences into intermediate vectors was performed to use as positive controls. Primers were designed and prepared to amplify target sequences using isothermal methods to amplify selected targets containing a crRNA targeting site. The optimization of the isothermal amplification of loci for production as substrates of the Cas12a/crRNA ribonucleoprotein complex was carried out.

2024

Concentrations of Cas12a, crRNA, and reporter for CRISPR-Cas12a biochemical reactions were optimized. The optimal concentrations of Cas12a were found to be 100 nM, crRNA 100 nM, and reporter 5.0 μM. Isothermal amplification of target genes β-tubulin, Tsr1, noxB, and acl1 was performed using recombinase polymerase reaction, and the resulting products were used to detect Cas12a/crRNA. The cis-activity of Cas12a endonuclease was studied and it was found that substrate cleavage occurred within 5-30 min at Cas12a concentrations of 200 nM and higher. The trans-cleavage activity of Cas12a endonuclease towards ssDNA (FAM-reporter-5-BHQ-1, TTATT) was assessed. Trans-activity was detected within 20-30 min of incubation at 37°C. The detection of Cas12a substrate by immunochromatographic assays using FAM-reporter-10-LFA (TTATT) was assessed.