A Gemini Virus-Derived Autonomously Replicating System for HDR-Mediated Genome Editing of the EPSP Synthase Gene in Indica Rice

CRISPR/Cas9-mediated homology-directed repair (HDR) is a powerful tool for precise genome editing in plants, but its efficiency remains low, particularly for targeted amino acid substitutions or gene knock-ins. Successful HDR requires the simultaneous presence of Cas9, guide RNA, and a repair template (RT) in the same cell nucleus. Among these, the timely availability of the RT at the double-strand break (DSB) site is a critical bottleneck. To address this, we developed a sequential transformation strategy incorporating a deconstructed wheat dwarf virus (dWDV)-based autonomously replicating delivery system, effectively simplifying the process into a two-component system. Using this approach, we successfully achieved the targeted editing of the <i>OsEPSPS</i> gene in rice with a 10 percent HDR efficiency, generating three lines (TIPS1, TIPS2, and TIPS3) with amino acid substitutions (T172I and P177S) in the native EPSPS protein. The modifications were confirmed through Sanger sequencing and restriction digestion assays, and the edited lines showed no yield penalties compared to wild-type plants. This study demonstrates the utility of viral replicons in delivering gene-editing tools for precise genome modification, offering a promising approach for efficient HDR in crop improvement programs.