In this project we will combine two innovative methods (CRISPR/Cas and biolistics) to provide a tool for genome editing in apple trees. The biolistic approach should result in a much broader spectrum of susceptible genotypes while the approach using CRISPR/Cas enables us to specifically modify only targeted genes without integration of foreign DNA. Such systems would prove very useful in research (e.g. identification of gene functions) and commercial breeding (targeted gene editing). We aim to establish a system for transient transformation of plants delivering plasmids carrying CRISPR/Cas constructs by biolistics. Biolistics introduce those constructs into target plant tissues by accelerating DNA-coated microparticles at high velocity. We expect to be able to regenerate plants transiently transformed with CRISPR/Cas-constructs the expression of which should lead to genetically modified plants not containing permanently integrated foreign DNA. Also, we will attempt to modify the described technique in order to further minimize the possibility of introducing foreign DNA into the target genome. In these DNA-free approaches we will prepare ribonucleoproteins (RNPs, consisting of CRISPR/Cas proteins and target specific guide-RNAs). These RNPs shall also be delivered into the target tissues using the biolistic approach. This approach should also lead to genetically edited plants completely eliminating the risk of integrating any foreign DNA into the targets genome. We will also try to achieve DNA-free genome editing by simultaneously introducing guide- and Cas-RNAs without relying on preformed proteins. Thus, we aim to avoid possible restrictions of the biolistic methods due to, for instance, protein size. We aim to modify the genome of apple at specific sites using modern CRISPR/Cas technology by different ways of using CRISPR/Cas reagents (common plasmid-based, modern RNPs and innovative RNA:RNA).

Plant tissue cultures of different apple genotypes will be used. Those genotypes already carry a reporter gene inactivation of which using CRISPR-mediated modification can easily be tracked by the loss of a staining reaction. Different vectors have to be constructed, specific and und effective guide-RNAs have to be designed and in-vitro produced. For a successful development of a biolistic-mediated CRISPR/Cas system many physical parameters have to be considered and optimized. Regenerated plants will be tested for modifications using sequence-related analyses (PCR, Southern Blot, Sanger sequencing, high resolution melting analyses, hetero duplex analyses using capillary electrophoresis).