Genome editing in cells of apple cultivar ‘Fuji’ using geminivirus-derived replicons for transient expression of CRISPR/Cas9 components

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple (Malus × domestica). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Streptococcus pyogenes Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via Agrobacterium-mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.