A decision tree-based toolbox for multiplex CRISPR/Cas to modify complex traits – case studies in potato and chicory

To modify complex traits, such as bitterness in Cichorium or disease resistance in potato, simultaneous expression of CRISPR/Cas combined with multiple gRNAs is needed to obtain multi-gene knockouts.
We designed a toolbox, named GeneBEwise, comprising the complete workflow and providing a user-friendly decision tree to guide users toward specific protocols for multiplex gene editing.
Following the first panel of our toolbox, we identified the candidate target genes involved in bitterness (Cichorium intybus) and Phytophthora infestans susceptibility (Solanum tuberosum). For Cichorium, by integration of genome and transcriptome mining, we studied the gene families of the genes involved in the biosynthesis of bitter sesquiterpene lactones.
A phylogenetic tree was constructed showing gene clades and catalytic active candidate genes were selected for multiplex gene editing.
Furthermore, in silico identification of orthologs of susceptibility genes (S-genes) previously described in Arabidopsis thaliana, led to the selection of 87 putative S-genes in S. tuberosum. The second panel of our toolbox covers the steps for gRNA design and associated mutation screening panels using multiplex amplicon sequencing.
Gene-specific gRNAs and HiPlex amplicon panels were designed using Geneious and the SMAP software package.
The gRNAs were used for multiplex CRISPR/Cas9 editing of protoplasts.
Regenerated calli and shoots were screened for edits using the HiPlex amplicon sequencing assay, according to the tools and workflow described in the third and fourth panels of our toolbox.
Multiplex gene editing resulted in C. intybus mutants with a complete reduction of sesquiterpene lactones.
For S. tuberosum, 33 genes were targeted in a multiplex approach using pools of 11 gRNAs per transfection.
Edited plants were identified containing up to five edited genes in a single mutant plant.
Our toolbox is made open access via a website and will have broad applicability for Cichorium, Solanum, and beyond.

International Symposium on Biotechnological Tools in Horticulture

K. Van Laere, E. Vereecke, T. Eeckhaut, E. Waegneer, T. Ruttink

GeneBEwise, gene editing workflow, Cichorium, Solanum, bitterness, disease resistance

https://doi.org/10.17660/ActaHortic.2026.1454.4