Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Gene editing in plants typically requires stable transformation, which can be time-consuming and challenging for certain species.
• Developing efficient and accessible methods for plant genome engineering is crucial for crop improvement and research.

Purpose of the Study:

• To develop a transient, in planta gene editing system utilizing RNA viruses for delivering CRISPR-Cas9 components.
• To assess the efficiency and heritability of gene editing mediated by a mobile RNA virus expressing single guide RNAs (sgRNAs).

Main Methods:

• Cas9 transgenic plants were generated and subsequently infected with a modified RNA virus engineered to express single guide RNAs (sgRNAs).
• sgRNAs were designed with sequences to enhance cell-to-cell movement within the plant.
• Mutant progeny were analyzed in the generation following viral infection.

Main Results:

• High frequencies of mutant progeny (65-100%) were recovered, demonstrating the efficacy of the in planta gene editing system.
• Plants infected with a virus expressing three distinct sgRNAs yielded progeny with mutations in up to 30% of the targeted loci.
• The viral delivery system facilitated efficient and heritable gene editing across multiple target sites.

Conclusions:

• This RNA virus-based system provides a rapid and efficient method for in planta gene editing in plants.
• The approach bypasses the need for stable transformation, offering a potentially more accessible tool for plant genetic engineering.
• The high mutation rates and heritability observed highlight the potential of this technology for crop breeding and functional genomics studies.