Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy.

Transgene-free plant genome editing in the T0 generation is highly desirable but challenging1,2. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.