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.
CRISPR/Cas9, transgene-free, genome editing, morphogenesis inducer, negative selection marker
We optimized a methodology for generating transgene-free genome-edited plants using CRISPR/Cas9 by combining the transient expression of the morphogenesis inducer ipt with the conditional negative selection marker DAO1. The introduction of DAO1 in tobacco lines allowed us to identify and apply the appropriate selection agents, with 10 mM D-valine effectively reducing the number of vigorously growing shoots. This approach successfully enriched the population of transgene-free shoots, demonstrating the efficiency of the developed selection system.