This program aims to address problems related to increasing crop yields through the implementation of modern CRISPR/Cas technology to improve known crop varieties or create new ones, detect economically important pathogens of vegetable and fruit crops, and develop methods for suppressing viral and viroid infections. This work proposes the development of test systems based on CRISPR/Cas technology for detecting economically important strains/isolates of viruses, viroids, and fungal pathogens of potato, apple, grape, and plum circulating in the country, including for pathogen detection in the field. The development of test systems will be based on whole-genome studies of the above-mentioned pathogens prevalent in different regions of the country. The developed vectors based on plant virus genome for expression of CRISPR/Cas cassettes will be used to improve local crop varieties. The identified mechanisms of maximum viral infection suppression will allow developing strategies for targeted disease control.

The purpose of the program is to use CRISPR/Cas genome editing technology to increase the productivity of economically important cultivated plants through the detection of dangerous phytopathogens and conferring antiviral resistance to plants.

2023

Plant material collection and high-throughput RNA sequencing (HTS) will be conducted for at least 300 samples of apple, grape, and potato, with analysis of complete genome sequences of detected viruses ACLSV, ASPV, ASGV, ApMV, GVA, GFLV, GLRV, GRBD, PPV, PLRV, PVX, PVY. ITS-sequencing of Monilinia fructigena, Venturia inaequalis and the oomycete Phytophthora infestans will be performed. Bioinformatic analysis of genomes and genomic sequences of local isolates will be conducted with available sequences in international databases NCBI and/or EMBL, and specific regions suitable for selective guide RNA selection will be identified. Candidate guide RNAs will be developed and synthesized for selective detection of viruses and fungi. HTS sequencing of double-stranded RNA from potato samples and bioinformatic analysis of PLRV, PVY, and PVM virus genomes and PSTVd viroid will be performed, along with design and cloning of guide RNAs (CRISPR/Cas), sense and antisense sequences (RNA interference) for inactivation of genomic and subgenomic viral and viroid RNAs by targeting genes encoding replication proteins, movement, and RNA interference suppression. Cloning and optimization of transient Cas13 protein expression will be conducted in the model plant Nicothiana benthamiana, as well as in Solanum tuberosum. The dose-dependent effect of P19 protein expression on microRNA binding affinity in planta will be studied. Pre-microRNA expression levels will be evaluated during viral infection with TBSV and its mutants P19, RMJ-1. Experiments will be conducted on N. benthamiana plants. Plants will be inoculated with wtTBSV, P19, RMJ-1, followed by total RNA isolation, cDNA synthesis, and q-rtPCR using primers for pre-microRNA. Endogenous levels of mature microRNA will be evaluated during viral infection with TBSV and its mutants P19, RMJ-1. Experiments will be conducted on N. benthamiana plants. Plants will be inoculated with wtTBSV, followed by total RNA isolation, cDNA synthesis, and q-rtPCR using primers for microRNA. At least three CRISPR/Cas constructs will be developed based on the TBSV viral vector through replacement and/or insertion of heterologous genes, including Cas9 and regulatory sequences for guide RNAs. Cas9 expression analysis in the viral vector will be conducted in N. benthamiana plants.

2024

Plant material infected with viruses ACLSV, ASPV, ASGV, ApMV, GVA, GFLV, GLRV, GRBD, PPV, PLRV, PVX, PVY will be collected for testing and validation of developed selective guide RNAs. A collection of synthetic control sequences identical to target regions of viruses ACLSV, ASPV, ASGV, ApMV, GVA, GFLV, GLRV, GRBD, PPV, PLRV, PVX, PVY selected for detection will be created. Optimization of virus detection protocols using Cas12 and Cas13 proteins, developed guide RNAs, and synthetic control DNA sequences of pathogens will be conducted. Validation of the CRISPR/Cas-based virus detection method will be performed on infected plant material, comparing its specificity and efficiency with PCR-based methods. Potato plants will be inoculated with viruses and viroid separately and in different combinations. Assembly of cassettes carrying guide RNAs, sense and antisense sequences, Cas13 gene in binary vector, and agroinfiltration of potato plants with different combinations will be performed. Assessment of viral infection levels in potato plants transiently expressing CRISPR/Cas cassettes, sense and antisense will be conducted over time using qPCR. Comparison of infection suppression levels will be made between healthy and infected plants inoculated with the constructs. Design and development of crRNA constructs targeting microRNA and viral RNA will be conducted. Single crRNA constructs and multiplexed crRNA system constructs in pCambia2300 vector will be developed. CrRNA design will be performed on CRISPR-RT platforms, using NJSViewer and mirbase for RNA secondary structure prediction and target selection. CrRNAs will be synthesized and integrated into donor vector using unique BP Gateway sites. LR Gateway recombination will then be performed. Transformation into E. coli cells will be conducted. All crRNAs will be integrated under the 35S promoter. Constructs will be created by inserting viral genetic material (wt TBSV) into pCambia2300 plasmid vector. Viral material will be integrated into pCambia2300 agrobacterial plasmid vector using unique single restriction sites. Design and synthesis of at least three guide RNA sets for PDS/MLO genes will be performed. Vectors containing native suppressor wt P19, P19 atg→ctg, P19 atg→ctg + nucleotide substitution at position 421 a→t and heterologous suppressors will be developed for plant immunity suppression and increased CRISPR/Cas cassette expression efficiency. Expression analysis of heterologous suppressors individually and in different combinations during transient Cas9 expression will be conducted. Suppressor combinations with the best activity will be identified.

2025

A collection of synthetic control sequences identical to target regions of fungi Monilinia fructigena and Venturia inaequalis and oomycete Phytophthora infestans selected for detection will be created. Collection and search for plant material infected with fungi Monilinia fructigena and Venturia inaequalis and oomycete Phytophthora infestans will be conducted for testing and validation of developed selective guide RNAs. Optimization of fungal-like pathogen detection protocols using Cas12 proteins, developed guide RNAs, and synthetic control DNA sequences of pathogens will be performed. Validation of the CRISPR/Cas-based fungal-like pathogen detection method will be conducted on infected plant material, comparing its specificity and efficiency with PCR-based methods. Agroinfiltration with different combinations of guide RNAs, sense and antisense, will be performed on potato varieties infected with different combinations of viruses and viroid. Analysis of infection suppression in multi-viral infection will be conducted over time using qPCR. Sequencing and analysis of total RNA in virus and viroid-infected potato plants transiently expressing CRISPR/Cas cassettes, sense and antisense will be performed to analyze viral RNA, as well as mRNA, mcRNA, miRNA, piRNA and other potato plant RNAs forming immune response. Analysis of viral and viroid infection suppression efficiency will be conducted for RNA interference implemented using sense and antisense sequences, as well as CRISPR/Cas using guide RNAs. The effect of dose-dependent P19 expression during viral infection on targeted CRISPR/Cas13 editing of viral RNA and pre-microRNA will be studied through direct monitoring of virus-delivered GFP expression. Plants will be agroinfiltrated at 25-35 days with GV3101 agrobacteria strain carrying Cas13a, crRNAs targeting P19, GFP, and pre-microRNA sequences. Plants will then be inoculated with viral material. At 7 dpi, RMJ-1 mutant viral titers in plants will be analyzed, immunoblotting will be performed for P19, GFP proteins, and HA-tag insertion. P19-siRNA immunoprecipitation and quantitative PCR for pre-microRNA, microRNA, and AGO1 expression determination will also be conducted. A methodology for conferring stable immunity against viral pathogen through regulated dose-dependent modulation of viral suppressor expression to increase CRISPR/Cas13 gene editing system efficiency will be developed. Analysis of viral vector efficiency will be conducted through PDS and MLO gene knockout in N. benthamiana and targeted sequencing of regenerant genomic regions to determine off-target editing.