Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy

Transgene-free genome editing of plants in the T0 generation is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Here, we investigated the co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in tomato, tobacco, potato, and citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. The co-editing strategy is efficient for generating transgene-free, genome-edited plants in the T0 generation, thus being a potent tool for plant genetic improvement.