Pre-selection of efficient Cas9 and Cpf1 guides for genome editing in apple

Guide RNAs (gRNA) play a central role for CRISPR-based genome editing (GE) applications using RNA-guided nucleases such as Cas9 or Cpf1. The guide sequence is described to be critical for the efficiency and precision of the nuclease in GE experiments. The selection of efficient gRNAs prior to in vivo GE experiments is a critical factor for success, especially for apple Malus domestica Borkh., which is a species with low transformation rates and long duration of regeneration. In this study, five single guide RNAs (sgRNAs) for Cas9 and three gRNAs for Cpf1 were designed, targeting different exons of the phytoene desaturase gene (PDS) of apple. After synthesis of the guide RNAs, the cleavage efficiency of pre-assembled sgRNA/Cas9 and gRNA/Cpf1 complexes, were determined on DNA substrates originating from the different apple cultivars 'Pinova', 'Golden Delicious', 'Braeburn' and 'Gala'. The observed variance of in vitro cleavage efficiency of the tested sgRNAs for Cas9 was high. Furthermore, it was shown that the prediction and analysis of the secondary sgRNA structure can explain some of the observed differences in efficiency, and may help to exclude non-functional sgRNAs. In contrast, all gRNAs designed for Cpf1 showed a high cleavage efficiency on DNA substrates from all apple genotypes investigated. For downstream in vivo GE experiments on the MdPDS gene of apple, one sgRNA for Cas9 and three gRNAs for Cpf1 can be recommended for various apple cultivars.