CRISPR/Cas9-mediated Targeted Gene Editing in Tetraploid Potato to Reduce the Accumulation of Toxic Steroidal Glycoalkaloids

Potato (Solanum tuberosum L.) is one of the most important food crops globally, consumed by over a billion people worldwide and hence considered a major global food security crop. Potato contains SGAs (α-solanine and α-chaconine) which are antinutritional compounds that provide defence against pests and pathogens. However, at high concentrations, tuber SGAs can become highly toxic to humans following ingestion and thus constitute a major food safety concern globally. Breeding/genetic improvement has long been recognised as the most promising approach to control tuber SGA levels. To this end, in the current study, three key SGA-related genes: SGT2, SGT3 and GAME9 were targeted for knockout mutagenesis using the CRISPR/Cas9 genome editing system towards developing low/zero-SGA genotypes. Following Agrobacterium-mediated stable transformation of potato stem explants, a total of seventeen CRISPR/Cas9-transgenic, gene-edited, healthy-looking plants were obtained, comprising six, three and eight mutant lines of genes SGT2, SGT3 and GAME9, respectively. Amplicon NGS analysis revealed the presence of variably sized CRISPR-induced indel mutations within the respective target genes. Indel frequency and size distribution corresponded with the spacing of multiplexed-sgRNAs across individual target genes: the widely spaced sgRNAs of the SGT2 gene resulted in a high frequency of large deletions whereas the closely spaced/overlapping sgRNAs of the GAME9 gene produced smaller indels but with very high indel frequencies ranging 95-100% in all eight GAME9-mutant lines. Besides spacing, sgRNA gene copy number appears to have influenced mutation frequencies and in general, the observed in vivo mutation efficiencies for individual sgRNAs strongly reflected sgRNA performance in vitro. Overall, mutation analysis revealed a highly efficient multiallelic CRISPR-editing of the SGA-related genes: SGT2, SGT3 and GAME9, with a high frequency of frameshift indels expected to disrupt gene function towards a significant reduction/elimination of SGAs in the respective transgenic lines. Additionally, in line with objectives to further develop transgene-free, CRISPR-edited, SGA-free potato genotypes, a PEG-mediated protoplast transformation and regeneration protocol was successfully established. The current study therefore demonstrates the utility of the CRISPR/Cas editing system and sets the stage for developing transgene-free, SGA-free potato genotypes.