Evaluation of CRISPR/Cas9 based single and multiplex gRNAs against ChiLCV in tobacco

Chilli leaf curl virus (ChiLCV), a whitefly-transmitted monopartite begomovirus, causes severe yield losses in chilli-growing regions worldwide. This study evaluated the efficiency of single and multiplex CRISPR/Cas9 gRNAs in targeting key coding regions of ChiLCV genome in Nicotiana benthamiana. Four guide RNAs (gRNAs) specific to the CP, C3/C2, C2/C1, and Rep coding regions, were designed and assembled into four single-gRNA expression constructs, each targeting one viral region at a time, and six dual-gRNA constructs, each simultaneously targeting two viral regions. Agroinfiltration assays showed that all constructs reduced viral DNA, with dual-gRNA combinations achieving stronger suppression than single gRNAs. The gRNA1 + gRNA4 dual combination, targeting CP and Rep genes, was the most effective, reducing viral titre by 49.3% at 4 dpi and 47.9% at 20 dpi in systemic leaves. Viral accumulation was quantified using qPCR, disease severity was assessed phenotypically, and gRNA/Cas9 expression was confirmed by qRT-PCR, while HRM analysis verified targeted genome modifications. Overall, the results demonstrate that dual-gRNA constructs efficiently suppress viral replication, reduce disease severity, and induce precise genome edits, underscoring the potential of multiplexed CRISPR/Cas9 for developing durable resistance against ChiLCV.