Work

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

Year: 2025

Type: article

Abstract: 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 OsEPSPS 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.

Source: Plants

DOI: 10.3390/plants14030477

Authors: Bhabesh Borphukan, Muslima Khatun, Dhirendra Fartyal, Donald James, Malireddy K. Reddy

Institutions: International Centre for Genetic Engineering and Biotechnology, Washington State University, National Institute of Biotechnology, University of Bonn, Kerala Forest Research Institute

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Zero hunger

Open Access status: gold

Estimated APC paid: $3,258