To be able to adapt to fast changing biotic and abiotic stresses imposed by climate change a method to introduce targeted changes in plant genomes in a relative short time span is highly desirable. A lot is known about the function of individual genes in their potential to increase resistance to stress and to confer agronomic desirable traits such as flower timing and fruit quality. The CRISPR/Cas9 system allows targeted changes in the genome such as introduction of point mutations, gene insertions and deletions. Use of CRISPR/Cas9 is based on the production of transgenic plants expressing a sequence specific guide RNA (gRNA) and the Cas9 nuclease, which obviously does not meet the standards of having stable plant lines that are used in agriculture. Such plants also lack consumer's acceptance, as these are genetic modified organisms (GMO). To meet these concerns, breeders have to eliminate the transgenic background - a task which is time consuming and expensive. To address this, we propose to create CRISPR/Cas9 RNA fusion constructs that move in grafted (chimeric) plants from transgenic stock (lower plant part) to non-transgenic wild-type scion tissues. In grafted plants mobile CRISPR gRNA and Cas9 transcripts can introduce targeted mutations in the wild-type scion plant parts forming flowers. These flowers will produce transgene-free seeds that carry the desired mutation(s). By using this approach - mobile CRIS/Ccas9 transcripts combined with grafting - we can produce transgene free mutants within one generation.

In our previous work on messenger RNA (mRNA) transport between plant parts, we have shown that non-cell autonomous fusion transcripts can be created moving in grafted plants from transgenic plant parts to non-transgenic plant parts. After transport into flowers a mobile mRNA is translated into a fully functional protein (Saplaoura and Kragler, 2016; Zhang et al., 2016). We propose to implement these insights to create mobile gRNA and Cas9 mRNA that is delivered from a transgenic root-stock plant to wild-type scions harboring no genetic modification. The CRISPR/Cas9 fusion transcripts traveling to wild-type tissues will introduce targeted mutations in cells harboring wild-type genomes forming flowers and seeds. A fraction of seeds formed on wild-type plants grafted on CRISPR/Cas9 transgenics should harbor the indented genomic mutation(s) but lack transgenic sequences. These CRISPR/Cas9 mutated tissues and plant lines can be relatively easily identified by their phenotype and confirmed by established genomic screening methods. Wild-type tissue grafted onto transgenic CRISPR/Cas9 root-stock are not GMOs and are indistinguishable from natural occurring mutant plant lines. In addition, this approach will significantly shorten the time needed to establish CRISPR/Cas9 mutated transgene free offspring harboring novel or desired traits.