A piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice

Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double‐stranded transfer DNA (dsT‐DNA), with subsequent excision of the transgenes by the re‐transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T‐DNA works to deliver transgenes in rice. Following positive–negative selection to exclude transgenic cells randomly transformed with T‐DNA, piggyBac‐mediated transgenesis from the extrachromosomal dsT‐DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof‐of‐concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9‐induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac‐mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9‐mediated targeted mutagenesis in vegetatively propagated crops.