An accelerated transgene-free genome editing system using microparticle bombardment of sorghum immature embryos

The key factors for genome-editing in plants using CRISPR/Cas9, such as the Cas9 nuclease and guide RNA (gRNA) are typically expressed from a construct that is integrated into the plant genome. However, the presence of foreign DNA in the host genome causes genetic and regulatory concerns, particularly for commercialization. To address this issue, we developed an accelerated pipeline for generating transgene-free genome-edited sorghum (Sorghum bicolor) in the T₀ generation. For proof-of-concept, we selected the Phytoene desaturase (PDS) gene as the target due to its visible phenotype (albinism) upon mutation. Following microprojectile-mediated co-transformation with a maize (Zea mays)-optimized Cas9 vector and a guide RNA (gRNA) cassette with a geneticin (G418) resistance gene, we divided tissue derived from immature embryos into two groups (with and without antibiotic selection) and cultured them separately as parallel experiments. In regenerated plants cultured on medium containing MS basal nutrition (to allow albino plants to survive), we detected higher rates of albinism in the non-selection group, achieving editing rates of 11.1-14.3% compared with 4.2-8.3% in the antibiotic selection group. In the T₀ generation, 22.2-38.1% of albino plants from the non-selection group were identified as transgene-free, whereas only 0-5.9% from the selection group were transgene-free. Therefore, our strategy efficiently produced transgene-free genome-edited plants without the need for self-crossing or outcrossing, demonstrating the feasibility of achieving transgene-free genome-edited sorghum plants within a single generation. These findings pave the way for commercializing transgene-free genome-edited lines, particularly for vegetatively propagated crops like pineapple, sugarcane, and banana.

The online version contains supplementary material available at 10.1007/s42994-025-00204-9.