High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus

Delivery of gene editing components such as the Cas nuclease and single guide RNAs (sgRNAs) into plant cells is commonly accomplished by agrobacterium-mediated transformation. Although Arabidopsis is easy to transform, generation of biallelic edited plants requires screening a large number of plants in subsequent generations. Here, we describe optimization of the Tobacco rattle virus (TRV) for in planta delivery of sgRNAs fused to a tRNA Ileu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis . Inclusion of tRNA Ileu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNA Ileu infected Arabidopsis , which allows for the uncovering of lethal phenotypes. Mutant progeny are recovered in the next generation (M1) at frequencies ranging from 30-60%, with 100% mutant recovery in the following (M2) generation. TRV::tRNA Ileu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.