Multigene CRISPR/Cas9 genome editing of S-genes in potato.

In this project, Susceptibility genes (S-genes) are knocked out to make a Phytophthora infestans resistant potato cultivar. S-genes are innate plant genes required by pathogens for successful infection, and loss-of-function mutations in S-genes can lead to lower disease susceptibility. Here, 33 candidate S-genes were selected for CRISPR/Cas9 genome editing in tetraploid potato cultivars Bintje and Spunta. Highly multiplex amplicon sequencing (HiPlex) primers flanking gRNA binding sites were designed and validated on wild type Bintje and Spunta genomic DNA. A total of 44 gRNAs targeting 33 genes were selected and cloned into separate Cas9/gRNA expression vectors. Four vector pools containing 11 Cas9/gRNA expression vectors each were assembled, and single or multiple vector pools were used for co-transfection in protoplasts to generate a range of combinatorial mutation profiles. HiPlex sequencing of all target loci showed that single calli were edited to various degree, ranging from one to four alleles per locus, and in one or more genes (up to 11 genes in Spunta). Spunta calli were regenerated into shoots (15-20% regeneration efficiency), and will be used for detached leaf assays to test for susceptibility to Phytophthora infestans. These results show that multiple genes in different combinations can be edited using a multiplex CRISPR/Cas9 approach, and provides a valid strategy to engineer Phytophthora infestans resistance in potato.