TRANSGENE-FREE GENOME EDITING IN TOBACCO MODELS CAN BE FACILITATED BY THE GENE ENCODING D-AMINO ACID OXIDASE, A CONDITIONAL NEGATIVE SELECTION MARKER =

Although CRISPR/Cas9-mediated genome editing has become a common technology in gene manipulation, its application to commercial crop species still needs the development of methodology for generating transgene-free genome-edited plants. The transient expression of morphogenesis inducer, ipt, typically stimulates shoot formation and, thus, serves as a positive selection marker in different plant species. Employing a conditional negative selection maker, DAO1, combined with ipt, sought to streamline the screening of transgene-free genome-edited plants. We first generated transgenic tobacco lines expressing DAO1 encoding D-amino acid oxidase, which reportedly conditions the resistance to a few toxic D-amino acids and the sensitivity to some non-toxic D-amino acids, and chose appropriate selection agents. The negative selection condition optimization with different DAO1 tobacco lines became applicable to test the efficiency of genome editing mediated by the transient expression of CRISPR/Cas9. The negative selection with 10 mM D-valine reduced the number of vigorously growing shoots, resulting in enriching transgene-free shoots. This method allowed for the isolation of transgene-free, genome-edited plants with 1.5 times greater efficiency. Our strategy, which combines ipt-mediated shoot formation with the conditional negative selection marker DAO1, can be functional in other crop species to enhance the precision and applicability of genome editing in various plant species.