DNA-free CRISPR Genome Editing in Raspberry ( Rubus idaeus ) through RNP-mediated Protoplast Transfection and Comparison of Indel Analysis Techniques

Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression of foreign DNA into plant genomes. This allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. This can be achieved by DNA-free genome editing in protoplasts, followed by regeneration. However, protoplast isolation presents a barrier to the development of advanced breeding technologies in raspberry and no protocol has been published for DNA-free genome editing in the species. Pre-assembled ribonucleoprotein complexes (RNPs) do not require cellular processing and the commercial availability of Cas9 proteins and synthetic guide RNAs has streamlined genome editing protocols. This study presents a novel high-yielding protoplast isolation protocol from raspberry stem cultures and RNP-mediated transfection of protoplast with CRISPR-Cas9. Targeted mutagenesis of the phytoene desaturase gene at two intragenic loci resulted in an editing efficiency of 19%, though estimated efficiency varied depending on the indel analysis technique. Only amplicon sequencing was sensitive enough to confirm genome editing in a low efficiency sample. To our knowledge, this study constitutes the first use of DNA-free genome editing in raspberry. This protocol provides a valuable platform for understanding gene function and facilitates the development of precision breeding in this important soft fruit crop.