HO-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on -mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived HO to verify transgene-free genome-edited plants. In transgenic rice containing (), HO content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of HO and malondialdehyde, indicators of oxidative stress, were elevated. Detection of HO by 3,3'-diaminobenzidine (DAB) staining suggested that HO could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an -containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the HO-based method. Moreover, HyB induced overproduction of HO in leaves of , maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and -containing transgenic rice.