Abstract
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.