Virus-Mediated System for Simultaneous Gene Silencing and Genome Editing in Cotton

Plant viral vectors are powerful tools for the transient expression of exogenous genes, enabling not only virus-induced gene silencing (VIGS) but also virus-induced genome editing (VIGE). However, technical systems capable of simultaneously achieving gene silencing and gene editing in cotton have been rarely reported to date. Therefore, the development of a virus vector system that can concurrently mediate both gene editing and gene silencing would provide a valuable platform for advancing functional genomics studies and molecular design breeding in cotton. To address this gap, we established a system in cotton that concurrently enables gene silencing and gene editing. This system utilizes cotton Cas9 overexpression (Cas9-OE) as a receptor and CLCrV and TRV as vectors for targeting the GhCLA1 gene, which yields an albino phenotype upon silencing and mutation. Initially, CLCrV and TRV were used independently as vectors for gene editing and gene silencing, respectively. However, our results demonstrated persistent GhCLA1 gene silencing via TRV, but no systemic gene editing via CLCrV, suggesting viral cross-protection may occur between CLCrV and TRV for simultaneous actions. Subsequently, we constructed tandem assemblies of GhCLA1 silencing fragments and sgRNA expression elements in both TRV and CLCrV vectors resulted in successful gene silencing and editing, albeit with low editing efficiency. Further optimization through shortening the gene silencing fragments led to a substantial 2.61 to 3.11-fold increase in editing efficiency, while still maintaining effective GhCLA1 silencing. This refined system provides a robust tool for gene editing in cotton.