Advancements in Genetic Engineering
Open Access

Synthetic Biology Approaches for Engineering Virus-Resistant Crops Using CRISPR-Cas13 Systems

Maura Aaron^{* *}Correspondence: Maura Aaron, Department of Biotechnology and Bioengineering, University of Geneva, Geneva, Switzerland, Email:

Author info »

Description

Plant viral diseases represent significant threats to global food security, causing billions of dollars in agricultural losses annually. Traditional breeding approaches for viral resistance often require decades of development and may be limited by the availability of natural resistance genes. CRISPR-Cas13 systems, which target Ribonucleic Acid (RNA) molecules rather than Deoxyribonucleic Acid (DNA), offer innovative approaches for engineering broad-spectrum viral resistance in crop plants. This research investigates the application of Cas13a and Cas13d systems for targeting multiple RNA viruses simultaneously in tomato and rice crops.

The experimental framework focused on three economically important viruses: Tomato Yellow Leaf Curl Virus (TYLCV), Rice Stripe Virus (RSV), and Cucumber Mosaic Virus (CMV). Cas13 systems were engineered to target conserved genomic regions across viral strains, with particular emphasis on replicase and coat protein genes essential for viral replication and transmission. Multiple CRISPR RNAs were designed using computational tools to identify optimal target sites with minimal off-target potential against host plant transcripts.

Plant viruses pose a major threat to global agriculture, causing significant yield losses and compromising food security. Traditional methods of managing viral infections in crops-such as breeding for natural resistance, chemical control of vectors, and RNA interference (RNAi)-have met with limited success due to the rapid mutation rates of viruses, narrow-spectrum resistance, and environmental concerns. In response to these challenges, synthetic biology offers powerful new tools to engineer virus-resistant crops with precision, efficiency, and adaptability. Among these tools, the CRISPR-Cas13 system has emerged as a promising RNA-targeting technology for combating viral pathogens in plants.

Unlike the widely used CRISPR-Cas9 system, which targets DNA, Cas13 enzymes specifically cleave single-stranded RNA molecules, making them uniquely suited for targeting RNA viruses that infect crops. By programming Cas13 with guide RNAs complementary to viral RNA genomes, researchers can achieve targeted degradation of viral transcripts, effectively neutralizing infections. This approach provides a flexible and scalable method to engineer broad-spectrum or virus-specific resistance without altering the plant genome, thus reducing potential off-target effects and regulatory hurdles.

Synthetic biology frameworks further enhance the potential of CRISPR-Cas13 systems by enabling the design of modular, inducible, and tissue-specific expression systems to optimize antiviral responses. As a result, this strategy represents a next-generation solution for developing durable and environmentally sustainable virus resistance in crops. This introduction explores the principles, methodologies, and potential applications of CRISPR-Cas13-based synthetic biology approaches in engineering virus-resistant crops, highlighting their impact on future agricultural innovation.

Agrobacterium-mediated transformation was employed to introduce Cas13 expression cassettes into tomato (Solanum lycopersicum) cv. Micro-Tom and rice (Oryza sativa) cv. Nipponbare. Transgenic plants were generated using tissue culture techniques, with molecular confirmation of transgene integration through PCR analysis and Southern blotting. Expression levels of Cas13 proteins were validated through immunoblot analysis using epitope-tagged constructs.

Viral challenge experiments demonstrated significant resistance to target viruses, with Cas13a-expressing tomato plants showing 89% reduction in TYLCV accumulation compared to nontransgenic controls. Rice plants expressing Cas13d exhibited 74% reduction in RSV symptoms and 67% reduction in viral RNA levels. Importantly, the multiplexed crRNA approach enabled simultaneous targeting of multiple viruses, with plants showing resistance to mixed infections. Molecular analysis revealed that Cas13-mediated RNA cleavage effectively disrupted viral replication cycles, preventing systemic infection.

Off-target analysis using RNA sequencing confirmed minimal impact on host plant gene expression, with fewer than 50 genes showing differential expression compared to non-transgenic controls. These changes were primarily related to defense response pathways, consistent with normal plant responses to genetic modification.

Conclusion

CRISPR-Cas13 systems provide effective, broad-spectrum viral resistance in crop plants while maintaining normal plant physiology. The ability to target multiple viruses simultaneously and the minimal off-target effects support the agricultural application of this technology. This work establishes RNAtargeting CRISPR systems as valuable tools for sustainable crop protection against viral pathogens. Importantly, no essential metabolic pathways were significantly affected. Phenotypic analysis revealed normal plant growth and development, with no observable defects in morphology or reproductive capacity.