Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants.

In this study, we describe the establishment of the knockout marker gene for selection of CRISPR/Cas9-edited seedlings and tomato explants in tissue culture. encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also opportunistically transports aminoglycoside antibiotics into these organelles and defects of the gene render plants insensitive to those compounds. Here, we show that mutations of induced by the CRISPR system confer kanamycin-resistance to plants and tomato tissues. is single-copy in a variety of plant species and the corresponding proteins form a distinct phylogenetic clade allowing easy identification of orthologs in different plants. We demonstrate that in multiplexing approaches, where seedlings were selected a CRISPR/Cas9-induced kanamycin resistance mediated by mutation, a mutation in a second target gene was observed with higher frequency than in a control population only selected for the presence of the transgene. This so called co-selection has not been shown before to occur in plants. The technique can be employed to select for edited plants, which might be particularly useful if editing events are rare.