Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks

Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.

Automated summary

Generation of transgene-free plant lines in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors has been achieved by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that facilitate RNA movement between transgenic rootstocks and wild-type scions. The graft-mobile gene editing system has been successfully demonstrated in Arabidopsis thaliana and Brassica rapa, and may have broad applications in breeding programs and crop plants.