Yasuhiro Ito
Genome editing is a powerful tool for altering specific target genomic regions, making it promising for crop improvement. However, the vectors used to deliver genome-editing reagents can leave recombinant DNA fragments in the edited plants as foreign DNA insertions. Current regulations for the cultivation of genome-edited crops in open fields differ among countries; however, under some regulatory frameworks, including that of Japan, developers are expected to demonstrate that no detectable residual foreign DNA remains. Mutations in the Ripening inhibitor ( RIN ) locus were previously produced in the tomato ( Solanum lycopersicum L.) genome via genetic transformation using recombinant DNA harboring the CRISPR/Cas9 system. Among the resulting mutants, rinG2 mutant fruits had a significantly extended shelf life, making them potentially useful for tomato breeding and future commercialization. Here, PCR analysis, Southern blot analysis, and high-throughput sequencing followed by k -mer analysis were applied to assess the presence of recombinant DNA in the progeny of T 0 plants carrying the rinG2 mutation. In a selected T 1 candidate plant, none of these three methods detected recombinant DNA, whereas recombinant DNA was detected in its sister plants. The use of parallel analyses based on different principles helps reduce the risk of failing to detect recombinant DNA in genome-edited plants. This case study illustrates the practical evaluation of a rinG2 mutant tomato line within the current regulatory framework for genome-edited crops.