PT  - JOURNAL ARTICLE
AU  - Ugrappa Nagalakshmi
AU  - Nathan Meier
AU  - Jau-Yi Liu
AU  - Daniel F. Voytas
AU  - Savithramma P Dinesh-Kumar
TI  - High efficiency multiplex biallelic heritable editing in <em>Arabidopsis</em> using an RNA virus
AID  - 10.1101/2022.01.20.477144
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.20.477144
4099  - http://biorxiv.org/content/early/2022/01/22/2022.01.20.477144.short
4100  - http://biorxiv.org/content/early/2022/01/22/2022.01.20.477144.full
AB  - 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 tRNAIleu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis. Inclusion of tRNAIleu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNAIleu 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::tRNAIleu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.Competing Interest StatementThe authors have declared no competing interest.