Identification of transgene-free CRISPR edited plants of rice and tomato by monitoring DsRED fluorescence in dry seeds

Abstract

CRISPR/Cas technology for specific and precise modification of genomes has transformed molecular biology. However, quick elimination of the transgene remains a challenge in plant biotechnology after genome edition, especially for crops due to their long life cycle and multiploidy, not only to avoid transgene position effects and to minimize the probability of off-target mutation appearance, but also to deliver end users with edited plants free of the recombinant gene editing machinery. Counter selection based on resistance marker genes are inconvenient in the case of CRISPR/Cas applications because plants lacking the transgene cannot survive the selection, and thus two more generations must be screened to evaluate the presence of the transgene. In the case of some crops, generations can last between 6 months and a few years, and the workload may be a limiting factor because transgene detection by PCR requires germination of seeds, and selected plants must be grown until a new generation can be harvested. The expression of fluorescent proteins as selective marker has been successfully used in Arabidopsis thaliana (Stuitje et al., 2003) as a fast method for transgene presence detection prior to seed germination. Despite this clear advantage, it has not been tested in other species yet, because of the special requirements of in vitro transformation protocols. To overcome the above mentioned inconvenient, we have adapted fluorescence-dependent monitoring of transgene to genome editing approaches in tomato and rice with the goal of obtaining transgene-free homozygous edited crop plants in two generations.