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Precise genome editing technologies are rapidly emerging as prime tools for improving crop characteristics. The CRISPR-Cas9 system has proved highly versatile and efficient for inducing targeted DNA double-strand breaks (DSBs) in genomes. DNA DSBs in eukaryotes can be repaired using two different pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). Although homology directed repair gene editing (or KI) holds promise for crop breeding, this approach remains more difficult to master.

In the present study, we developed a selection protocol based on the CRISPR-Cas9 system and Agrobacterium-mediated transformation for efficient gene editing of the tomato ALS1 gene through HDR and subsequent recovery of chlorsulfuron-resistant plants that are transgene-free. The efficiency of ALS1 gene editing was high (12.7 % of the transfected events). Moreover, our new strategy to use kanamycin to select for transient expression of the binary vector in regenerating cells allowed us to isolate gene-edited lines that do not contain T-DNA with an efficiency of 4.9%. To the best of our knowledge, this is the first report demonstrating transgene-free HDR-mediated genome editing in one generation for tomato using Agrobacterium CRISPR-Cas9 delivery. This strategy holds promise for genetic improvement in tomato and probably even more so for related species such as potato that are vegetatively propagated.