CRISPR/Cas12a Ribonucleoprotein Complexes Target Conserved WDV Sequences: An In Vitro Proof-of-Concept for Non-GMO Antiviral Strategy

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 requiring genetic modification of the host plants. A total of 10 WDV isolates from wheat and barley collected across six provinces in Türkiye, along with four reference isolates from Hungary, Sweden, and Iran, were analysed. The study demonstrates selective cleavage of conserved WDV regions by CRISPR/Cas12a RNPs in vitro, representing an early proof-of-concept for non-GMO antiviral approaches rather than a direct field application. 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.