Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9

Genome-editing technologies using CRISPR-Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR-Cas reagent delivery methods may be associated with unanticipated genome changes or damage(1,2), with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis(3). Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR-Cas9 ribonucleoproteins into protoplasts(4)or in vitro fertilized zygotes(5). However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR-Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency(6-9). Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR-Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR-Cas9 gene editing in plants through virus infection. A DNA-free in planta approach for gene editing based on RNA virus infection is developed, allowing delivery of the entire CRISPR-Cas9 cassettes into tobacco host to achieve highly efficient single, multiplex mutagenesis and chromosome deletions.