Harnessing CRISPR/Cas12a Technology to Combat Wheat Dwarf Virus Without Genetic Modification

ABSTRACT Wheat (Triticum aestivum L.) is a staple crop globally, but its yield and quality are significantly affected by viral diseases, particularly the Wheat Dwarf Virus (WDV). Developing antiviral strategies is crucial for enhancing wheat productivity. This study aims to investigate the use of CRISPR/Cas12a as an antiviral agent against WDV without the need for genetic modification of the host plants. WDV genomic DNAs were isolated from wheat and barley samples collected from various regions in Turkey and other countries. Guide RNAs (gRNAs) targeting conserved regions in the coat protein (CP) gene of WDV were designed and synthesized. The CRISPR/Cas12a ribonucleoprotein (RNP) complex was tested for its ability to induce DNA cleavage in WDV genomes through in vitro assays, and results were analysed using agarose gel electrophoresis. The designed gRNAs effectively induced DNA cleavage in WDV genomes, with gRNA1 showing consistent performance across all tested isolates. gRNA2 exhibited variability, successfully targeting seven out of ten isolates. No off-target effects were observed in control assays, confirming the specificity of the gRNAs. The CRISPR/Cas12a system, guided by specifically designed gRNAs, can effectively target and cleave WDV genomes, providing a potent, non-GMO antiviral strategy. This approach has potential applications for managing WDV infections in wheat and barley, highlighting its versatility and effectiveness. Competing Interest Statement C.T. and B.B. have a patent application within this project's scope with the application number 20770/14 and 2023/006118. The other authors declare no conflict of interest. B.B. and C.T. are inventors of patent applications (pending) including 'Bugday Cuce Virusunu Etkisiz Hale Getirmek Uzere Bir Antiviral Solusyon' (20770/14) and 'PCT/TR2023/051843' (2023/006118) at Turkish Patent and Trademark Office and Patent Cooperation Treaty (PCT). Footnotes Funding: The research was funded, and the investigational products for the study were provided by The Scientific and Technological Research Council of Turkey (TUBITAK) with the grant number 1919B012004554. Conflict of Interest: C.T. and B.B. have a patent application within the scope of this project with the application number 20770/14 and 2023/006118. The other authors declare no conflict of interest. B.B. and C.T. are inventors of patent applications (pending) including “Buğday Cüce Virüsünü Etkisiz Hale Getirmek Üzere Bir Antiviral Solüsyon” (20770/14) and “PCT/TR2023/051843” (2023/006118) at Turkish Patent and Trademark Office and Patent Cooperation Treaty (PCT).