DNA-free CRISPR genome editing in raspberry (Rubus idaeus) protoplast through RNP-mediated transfection

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. DNA-free genome editing followed by protoplast regeneration allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. 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 protoplast. This protocol provides a valuable platform for understanding gene function and facilitates the future development of precision breeding in this important soft fruit crop.