The Plant journal : for cell and molecular biologyJul 6, 2026

Efficient gene editing of plants without added genes using virus-based delivery

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jul 7, 2026

Abstract

Indel rates exceeding 50% were achieved in the Phytoene desaturase (PDS) gene within 6 days using a cymbidium mosaic virus-based genome editing platform.

The novel VIGE platform enables the simultaneous expression of Cas9 and multiple guide RNAs from a single viral RNA.
Over 80% of regenerated plants exhibited targeted mutations, with 82% being transgene- and virus-free.
The system allows for tetra-allelic knockouts directly in the M0 generation.
A Ruby-based visual counterselection marker facilitates identification of transgene-free, edited plants without the need for antibiotics.
The platform demonstrated a 47% indel frequency in the PaPDS gene of Phalaenopsis aphrodite orchids at 20 days post-inoculation.
Multiplex editing was possible due to the expression of multiple guide RNAs from a single CymMV replicon.

Simplified

Virus-induced genome editing (VIGE) has become a useful method by enabling transient delivery of gene-editing reagents; however, many viral systems face limitations in cargo size, host range, or reliance on transgenic Cas9-expressing plants. In this study, we developed a cymbidium mosaic virus (CymMV)-based VIGE platform that enables simultaneous expression of Streptococcus pyogenes Cas9 (SpCas9) and one or more guide RNAs (gRNAs) from a single viral RNA. In Nicotiana benthamiana, this system induced editing in the Phytoene desaturase (PDS) gene, with indel rates exceeding 50% within 6 days after inoculation, outperforming traditional delivery methods by about fivefold. Notably, over 80% of regenerated plants contained targeted mutations, and 82% of these were both transgene- and virus-free, including tetra-allelic knockouts directly in the M0 generation. Adding a Ruby-based visual counterselection marker enabled rapid, reliable identification of transgene-free, edited plants without antibiotic selection. When adapted to Phalaenopsis aphrodite orchids, the platform efficiently edited the PaPDS gene, achieving a 47% indel frequency at 20 days post-inoculation, with visible bleaching in leaf tissue from inoculated protocorm-like bodies. Additionally, expressing multiple gRNAs from a single CymMV replicon enabled multiplex editing in orchid tissues, demonstrating the system's versatility for complex, polyploid crops. Our findings broaden the use of VIGE in orchids and provide a reliable framework for precision plant breeding.

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Competing interests

None of the authors have a conflict of interest to disclose.

PubMed

Efficient gene editing of plants without added genes using virus-based delivery

Abstract

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Funding

Competing interests

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