CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors

The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterialCRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants thatstably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing ofNicotiana ben-thamianausing two compatible RNA virus vectors derived from tobacco etch virus (TEV; genusPotyvirus) andpotato virus X (PVX; genusPotexvirus), which replicate in the same cells. The TEV and PVX vectors respectivelyexpress a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improvesthe toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breedmore nutritious, resistant, and productive crops.