Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding. However, the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science. Here, we report an efficient, non-transgenic CRISPR/ Cas delivery platform based on the engineered tomato spotted wilt virus (TSWV), an RNA virus with a host range of over 1000 plant species. We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts. The resulting non-insect-transmissible viral vectors enabled effective and stable in planta de-livery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors. In systemically infected plant tissues, the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency. Plants with heritable, bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic se-lection. Moreover, we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100% of regenerated plants and further augmented the recovery of heritable mutations. Because many plants are recalcitrant to stable transformation, the viral delivery system developed in this work pro-vides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

Automated summary

CRISPR/Cas genome-editing tools have revolutionized plant biology research and crop breeding, but the lack of robust delivery methods hinders their widespread adoption in plant science. This study presents an efficient, non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus (TSWV), which can infect over 1000 plant species. By removing viral elements necessary for insect transmission, the TSWV-derived viral vectors enable effective and stable delivery of genome-editing tools in plants. These vectors induced efficient gene mutations and base conversions in multiple crop species, and plants with heritable mutations could be regenerated without antibiotic selection. Additionally, treating the virus-infected tissues with ribavirin during tissue culture cleared the viral vectors and increased the recovery of heritable mutations. This viral delivery system provides a promising tool for overcoming gene delivery bottlenecks in genome editing of various crop species and elite varieties.